pa po va virus hpv molecular oncogenesis
Post on 14-Apr-2022
3 Views
Preview:
TRANSCRIPT
HPV
Molecular
Oncogenesis
Pa Po Va virus
Nam Hoon Cho MD
Yonsei Univ Coll Med Dept of Pathology
Rudolf Virchow Father of Pathology
G Nikolas Pappanicaulau ndashPAP test
James Ewing ndashtumor metastasis theory
Arias-Stella- AS reaction
Azzopardi JG- One sees and recognizes only those features which have already been described
Arthur Purdy Stout- Atlas of Tumor Pathology
Stanley L Robbins- 1957 textbook 1st ed
Lesions do not arise in cadevars But the study of morphology is only one facet of pathology Pathology contributes much to clinical medicine The pathologist is interested not only in the recognition of structural alterations but also in their significance ie the effects of these changes on cellular and tissue function and ultimately the effect of these changes on the patient It is not a discipline isolated from the living patient but rather a basic approach to a better understanding of disease
and therefore a foundation of sound clinical medicine
Virchowrsquos triad
PIONEERS of PATHOLOGY
윤일선 교수 (1896-1987) 한국 최초의 (일본 연수) 병리학자1929-1945 세브란스 의전 교수-초대 주임교수
최동 교수 한국 최초의 (순수국내) 연세의대 병리학 및법의학교수- 한국 최초의 종양등록 문헌 발표
서재필 1929 병리학 전문의 취득하여 한국 최초의 병리전문의
Pioneers in Korean Pathologists
백순명 연세의대 졸업-미국 피츠버그대-유방암 조직은행-현 삼성병원 암연구소소장
Alois Alzheimer
Von Reklinghausen
인간은 생을 살려고 태어난 것이지생을 준비하려고 태어난 것은 아니다
하얀 거탑
Presentation Flow
HPV genome new highlight-
E6-E6AP-PDZ binding
E1^E4 and E2^E5
Overall roles of genomes
Controversial issue in cytopathology
What is phenotype with ASCUS
CIN 2 from the viewpoint of HPV
HPV testing guideline
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV 58 PPV for CIN 3+= 549
(An HJ et al Cancer 2003 971672)
HPV frequency 16185839525651
(Cho et al Am J Obstet Gynecol 2003 18856)
HPV with no CIN 372
(Hwang et al Gynecol Oncol 2003 9051)
HPV in Korean commercial sex workers47 (16 51)
(Choi et al J Med Virol 2003 71440)
HPV multiple infection in 91 and OR 318 folds for CIN 3+
(Lee et al Cancer Lett 2003198187)
Time Line of Cervical HPV
Infections And Progression
to Cervical Cancer
15 yo 30 yo 45 yo
HPV
PrecancerCancer
Age
Rate
Adapted from Schiffman amp Castle New Eng J Med 3532101-4 2005
Lifetime incidence of genital HPV infection gt80 in US
Most infections are asymptomatic and clear spontaneously
eliminating cancer risk for that infection
Persistent infection with a high-risk HPV especially HPV16
or 18 is the single most important risk factor for
progression to precancer and cancer
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Rudolf Virchow Father of Pathology
G Nikolas Pappanicaulau ndashPAP test
James Ewing ndashtumor metastasis theory
Arias-Stella- AS reaction
Azzopardi JG- One sees and recognizes only those features which have already been described
Arthur Purdy Stout- Atlas of Tumor Pathology
Stanley L Robbins- 1957 textbook 1st ed
Lesions do not arise in cadevars But the study of morphology is only one facet of pathology Pathology contributes much to clinical medicine The pathologist is interested not only in the recognition of structural alterations but also in their significance ie the effects of these changes on cellular and tissue function and ultimately the effect of these changes on the patient It is not a discipline isolated from the living patient but rather a basic approach to a better understanding of disease
and therefore a foundation of sound clinical medicine
Virchowrsquos triad
PIONEERS of PATHOLOGY
윤일선 교수 (1896-1987) 한국 최초의 (일본 연수) 병리학자1929-1945 세브란스 의전 교수-초대 주임교수
최동 교수 한국 최초의 (순수국내) 연세의대 병리학 및법의학교수- 한국 최초의 종양등록 문헌 발표
서재필 1929 병리학 전문의 취득하여 한국 최초의 병리전문의
Pioneers in Korean Pathologists
백순명 연세의대 졸업-미국 피츠버그대-유방암 조직은행-현 삼성병원 암연구소소장
Alois Alzheimer
Von Reklinghausen
인간은 생을 살려고 태어난 것이지생을 준비하려고 태어난 것은 아니다
하얀 거탑
Presentation Flow
HPV genome new highlight-
E6-E6AP-PDZ binding
E1^E4 and E2^E5
Overall roles of genomes
Controversial issue in cytopathology
What is phenotype with ASCUS
CIN 2 from the viewpoint of HPV
HPV testing guideline
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV 58 PPV for CIN 3+= 549
(An HJ et al Cancer 2003 971672)
HPV frequency 16185839525651
(Cho et al Am J Obstet Gynecol 2003 18856)
HPV with no CIN 372
(Hwang et al Gynecol Oncol 2003 9051)
HPV in Korean commercial sex workers47 (16 51)
(Choi et al J Med Virol 2003 71440)
HPV multiple infection in 91 and OR 318 folds for CIN 3+
(Lee et al Cancer Lett 2003198187)
Time Line of Cervical HPV
Infections And Progression
to Cervical Cancer
15 yo 30 yo 45 yo
HPV
PrecancerCancer
Age
Rate
Adapted from Schiffman amp Castle New Eng J Med 3532101-4 2005
Lifetime incidence of genital HPV infection gt80 in US
Most infections are asymptomatic and clear spontaneously
eliminating cancer risk for that infection
Persistent infection with a high-risk HPV especially HPV16
or 18 is the single most important risk factor for
progression to precancer and cancer
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
윤일선 교수 (1896-1987) 한국 최초의 (일본 연수) 병리학자1929-1945 세브란스 의전 교수-초대 주임교수
최동 교수 한국 최초의 (순수국내) 연세의대 병리학 및법의학교수- 한국 최초의 종양등록 문헌 발표
서재필 1929 병리학 전문의 취득하여 한국 최초의 병리전문의
Pioneers in Korean Pathologists
백순명 연세의대 졸업-미국 피츠버그대-유방암 조직은행-현 삼성병원 암연구소소장
Alois Alzheimer
Von Reklinghausen
인간은 생을 살려고 태어난 것이지생을 준비하려고 태어난 것은 아니다
하얀 거탑
Presentation Flow
HPV genome new highlight-
E6-E6AP-PDZ binding
E1^E4 and E2^E5
Overall roles of genomes
Controversial issue in cytopathology
What is phenotype with ASCUS
CIN 2 from the viewpoint of HPV
HPV testing guideline
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV 58 PPV for CIN 3+= 549
(An HJ et al Cancer 2003 971672)
HPV frequency 16185839525651
(Cho et al Am J Obstet Gynecol 2003 18856)
HPV with no CIN 372
(Hwang et al Gynecol Oncol 2003 9051)
HPV in Korean commercial sex workers47 (16 51)
(Choi et al J Med Virol 2003 71440)
HPV multiple infection in 91 and OR 318 folds for CIN 3+
(Lee et al Cancer Lett 2003198187)
Time Line of Cervical HPV
Infections And Progression
to Cervical Cancer
15 yo 30 yo 45 yo
HPV
PrecancerCancer
Age
Rate
Adapted from Schiffman amp Castle New Eng J Med 3532101-4 2005
Lifetime incidence of genital HPV infection gt80 in US
Most infections are asymptomatic and clear spontaneously
eliminating cancer risk for that infection
Persistent infection with a high-risk HPV especially HPV16
or 18 is the single most important risk factor for
progression to precancer and cancer
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Alois Alzheimer
Von Reklinghausen
인간은 생을 살려고 태어난 것이지생을 준비하려고 태어난 것은 아니다
하얀 거탑
Presentation Flow
HPV genome new highlight-
E6-E6AP-PDZ binding
E1^E4 and E2^E5
Overall roles of genomes
Controversial issue in cytopathology
What is phenotype with ASCUS
CIN 2 from the viewpoint of HPV
HPV testing guideline
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV 58 PPV for CIN 3+= 549
(An HJ et al Cancer 2003 971672)
HPV frequency 16185839525651
(Cho et al Am J Obstet Gynecol 2003 18856)
HPV with no CIN 372
(Hwang et al Gynecol Oncol 2003 9051)
HPV in Korean commercial sex workers47 (16 51)
(Choi et al J Med Virol 2003 71440)
HPV multiple infection in 91 and OR 318 folds for CIN 3+
(Lee et al Cancer Lett 2003198187)
Time Line of Cervical HPV
Infections And Progression
to Cervical Cancer
15 yo 30 yo 45 yo
HPV
PrecancerCancer
Age
Rate
Adapted from Schiffman amp Castle New Eng J Med 3532101-4 2005
Lifetime incidence of genital HPV infection gt80 in US
Most infections are asymptomatic and clear spontaneously
eliminating cancer risk for that infection
Persistent infection with a high-risk HPV especially HPV16
or 18 is the single most important risk factor for
progression to precancer and cancer
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Presentation Flow
HPV genome new highlight-
E6-E6AP-PDZ binding
E1^E4 and E2^E5
Overall roles of genomes
Controversial issue in cytopathology
What is phenotype with ASCUS
CIN 2 from the viewpoint of HPV
HPV testing guideline
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV 58 PPV for CIN 3+= 549
(An HJ et al Cancer 2003 971672)
HPV frequency 16185839525651
(Cho et al Am J Obstet Gynecol 2003 18856)
HPV with no CIN 372
(Hwang et al Gynecol Oncol 2003 9051)
HPV in Korean commercial sex workers47 (16 51)
(Choi et al J Med Virol 2003 71440)
HPV multiple infection in 91 and OR 318 folds for CIN 3+
(Lee et al Cancer Lett 2003198187)
Time Line of Cervical HPV
Infections And Progression
to Cervical Cancer
15 yo 30 yo 45 yo
HPV
PrecancerCancer
Age
Rate
Adapted from Schiffman amp Castle New Eng J Med 3532101-4 2005
Lifetime incidence of genital HPV infection gt80 in US
Most infections are asymptomatic and clear spontaneously
eliminating cancer risk for that infection
Persistent infection with a high-risk HPV especially HPV16
or 18 is the single most important risk factor for
progression to precancer and cancer
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV 58 PPV for CIN 3+= 549
(An HJ et al Cancer 2003 971672)
HPV frequency 16185839525651
(Cho et al Am J Obstet Gynecol 2003 18856)
HPV with no CIN 372
(Hwang et al Gynecol Oncol 2003 9051)
HPV in Korean commercial sex workers47 (16 51)
(Choi et al J Med Virol 2003 71440)
HPV multiple infection in 91 and OR 318 folds for CIN 3+
(Lee et al Cancer Lett 2003198187)
Time Line of Cervical HPV
Infections And Progression
to Cervical Cancer
15 yo 30 yo 45 yo
HPV
PrecancerCancer
Age
Rate
Adapted from Schiffman amp Castle New Eng J Med 3532101-4 2005
Lifetime incidence of genital HPV infection gt80 in US
Most infections are asymptomatic and clear spontaneously
eliminating cancer risk for that infection
Persistent infection with a high-risk HPV especially HPV16
or 18 is the single most important risk factor for
progression to precancer and cancer
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
HPV types assessed by the IARC
Monograph Working group
α β1 16 Most potent type
1 1831333539455152565859
Sufficient carcinogen
2A 68 Probably carcinogenic but limited evidence
2B 26536667707382 Possibly carcinogenic
2B 3034698597 Limited evidence
3 611
2B 58 Limited
3 Other type
Lancet Oncol 200910321-2
HPV 58 PPV for CIN 3+= 549
(An HJ et al Cancer 2003 971672)
HPV frequency 16185839525651
(Cho et al Am J Obstet Gynecol 2003 18856)
HPV with no CIN 372
(Hwang et al Gynecol Oncol 2003 9051)
HPV in Korean commercial sex workers47 (16 51)
(Choi et al J Med Virol 2003 71440)
HPV multiple infection in 91 and OR 318 folds for CIN 3+
(Lee et al Cancer Lett 2003198187)
Time Line of Cervical HPV
Infections And Progression
to Cervical Cancer
15 yo 30 yo 45 yo
HPV
PrecancerCancer
Age
Rate
Adapted from Schiffman amp Castle New Eng J Med 3532101-4 2005
Lifetime incidence of genital HPV infection gt80 in US
Most infections are asymptomatic and clear spontaneously
eliminating cancer risk for that infection
Persistent infection with a high-risk HPV especially HPV16
or 18 is the single most important risk factor for
progression to precancer and cancer
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Time Line of Cervical HPV
Infections And Progression
to Cervical Cancer
15 yo 30 yo 45 yo
HPV
PrecancerCancer
Age
Rate
Adapted from Schiffman amp Castle New Eng J Med 3532101-4 2005
Lifetime incidence of genital HPV infection gt80 in US
Most infections are asymptomatic and clear spontaneously
eliminating cancer risk for that infection
Persistent infection with a high-risk HPV especially HPV16
or 18 is the single most important risk factor for
progression to precancer and cancer
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Life Cycle of HPV Infection
HPV Infection is usually transient8-14 months
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Life Cycle of HPV Infection
Basement
membrane
Dermis
Stratified
squamous
epithelium Virion
Virion
Supra-
basal
cells
Basal
cellsHPV
DNA
HPV DNAreplication
AssembledVirus
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
HPV16 Binds
the Basement Membrane
HPV-16 Laminin-5
Mouse vaginal tract 2 hours after exposure to HPV16
(8 hours after exposure to nonoxynol-9)
Kines Thompson Lowy Schiller and Day PNAS 106 20458-63 2009
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Neutralizing
L1 Antibodies
(in red)
Bound to
Papillomavirus
Particle
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
VLP Vaccination Induces High
Titer Antibodies that Prevent
Basement Membrane Binding
Day et al Cell Host Microbe 8 260-70 2010
Basement
membrane
Dermis
Stratified
squamous
epithelium
Virion
Virion
No Infection
STOP
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
bull Episomal replication E1E2
bull Host integration E1E2 ORF disruption
E2-DNA dimer strong
E1-DNA hexamer weak
E2E6 real-time PCR
1episomal
gtor lt1mixed
0 integration
0 1000 2000 3000 4000 5000 6000 7000 7905 bp
E6
E1E7
E2
E4 L2E5
L1
My 0911
gp5+6+
HPV16-1
HPV16-2
HPV-induced tumorigenesis
1 integration pathway
2 episomal pathway
E1E2 promote genomic
instability through aberrant
replication of integrated
sequence
Genomic Organization of HPV
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
HR vs LR HPV
From Infection to Tumor InitiationIntegration 16 gt50 18 most
vs LR seldom to never
Transforming activityHR E7 immortalize at a low frequency- CDK2hg p21 p27low
vs LR E7- decreased p21 abrogation
HR E6 no transforming activity growth arrest abrogation by p53-
E6-E6AP trimeric complex vs LR E6- E6AP-p53 binding but no p53 degradation
HR E6+E7 highly efficient immortalizing proliferation and avoid
apoptosis but not tumorigenicv-rasv-fos coexpression tumorigenic
E6 telomerase ex PDZ domain [ X-ST-X-VLI]-containing protein
(p53-independent target) TNF modulation vs LR no PDZ domain
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
HPV E7
pRB functional inactivation
= hyperphosphorylation
=TF release
=TF activation
=G1-S restriction point entry
HPV E6
- p53 Proteolysis
- No mutation of p53
Both TSG are
impaired by
HPV E6E7
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
E6 and PDZ domain
PDZ-domain protein scaffolding protein
cell polarity cell junction
E6 HRE6AP-PDZ binding induce loss of cell polarity EMT
and carcinogenesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
E2 and CK13 expression
is reverse to P16CK14ki67
Xue Y et al Cancer Res 2010705316-5325
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
E1^E4 (amino-terminal E1 ORF splicing to E4 ORF)
Most divergent domain in sequence according
to HPV types
E4
L1
N terminal Binding to cytokeratin and concomitant
destabilization of cytokeratin network
Body cell cycle arrest at G2M checkpoint
L1 protein encoding
Long-acting multitasking
role of E4 genome
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
HPV E4
functions
cytokeratin
disruption
Virions
L1 Capsid cross-linking in oxidizing
environment of corneum
The more L1 capsid the less progressive
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
E5 Genomic Organization of HPV
E2
L2E56 Cys residue
bull Localized to Endoplasmic Reticulum (ER)-trafficking of cytopl memb protein
bull Weak transforming activity alone but strong in BPV E5
ndash enhancing transforming activity of E6E7 as oncogenic potential
bull EGFR (ErbB1) activity alteration through binding to vacuolar ATPase-
endosomal PH alteration- EGFR turnover alteration
bull PDGFR ligand-independent complex during tissue repair after HPV entry
bull Interfere with gap junction and alter caveolin-1
bull Anchorage-independent growth (anoikis) stimulation (HPV release)
bull Inducer of koilocytosis as cofactor with E6 in vitro (Am J Pathol2008 173682)
E2^E5 (HPV 83 aa BPV 86 aa)
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Koilocytosis by E5
Cytoplasmic vaculation-unclear reason
but contributing to fragility to make it easy
to release viral particlesAm J Pathol 2008 173682-8
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
E7 inhibits koilocytosis
E5 and E6
induce
koilocyte
E7 inhibits
koilocyte
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Sequence of events
of HPV genome
E1 E2 E4 E5 E6 E7 L1 L2
Location Cyto-Nc
shuttling
Cyto-
NcCyto Cyto-ER Cyto-Nc Cyto-Nc Cyto-
Nc
Cyto-Nc
Main
action
Replication Replicat
ion
CK-disruption EGFRPDGFR
Anoikis-
resistance
P53
disruption
pRb mut
P16 ovex
Ki67 ovex
Major
capsid
Minor
capsid
Sequence
of events
5 6 () 2
(92a-a)
1
(16kDa83a-a)
4
(18kDa
150a-a)
3
(13 kDa
98 a-a)
7 8
Cell cycle G2 arrest S-phase
entry
Oncogeni
c
suggested Key role
(HR vs LR)
Key role
(HR vs LR)
Prime
goal
Replication Control Most abundant
viral product
Lateral spread
Infected basal
cells
proliferation in
viral entry
Koilocytosis
Immortalize
but not
alone
Permissive
milieu
Immortalize
Dominant
oncogene
Cross-
linking
VLP-
vaccine
Shape
and
stability
Immortalized E6E7- no tumorigenic in vitro E6E7+V-rasfos - tumorigenic
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Changes in the HPV16 life cycle
during the development of cervical
cancer
CIN1 CIN2 CIN3
VS
bull CIN1 generally resemble productive lesions
bull In CIN2 and CIN3 lesions the order of life cycle events is unchanged
but the extent of E7 (dominant oncogene) expression is increased
bull Viral genome integration into cellular DNA
bull Loss of E2 leads to increased E6E7 expression
bull In cervical ca the productive stages of the virus life cycle are no longer
supported and viral episomes are usually lost
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Revisited Concept
ASCUS cells caused by incomplete E4E5 activity
Abortive koilocyte
Queryocyte
Regressing koilocyte not enough cleared by immune sys
HR-LSIL
Need to monitor carefully but no overconcern
LR-HSIL
Occur rarely in single but often combined need to be further studied
CIN2
definitely heterogeneous in HPV life cycle to manifest combined
productive and proliferative phase
1
2
3
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Performance characteristics
Designation ProbesprimersReaction
product
Analytical
sensitivity fg
detectable
types
Hybridi
zation
HC2 HPV DNA
assay
Mixture of
RNA probes
DNARNA
hybrids25ndash75 13
PCRMY0911 Dot blot Degenerate primer 450 bp 01ndash100 39
PCR PGMY0911
reverse LBA
Mixture of
consensus primers450 bp 01 27
PCR GP5+GP6+ EIA
ELISA systemConsensus primers 150 bp 05ndash10 20
PCRGP5+6+ reverse
LBAConsensus primers 150 bp 05ndash10 37
PCRSPF-PCR reverse
LiPA
Mixture of
consensus primers65 bp 01ndash10 43
Iftner T et al J Natl Cancer Inst Monogr 2003(31)80-8
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
The concept of HPV load
with clinical behaviorSnijders P et al J Pathol 2003 201 1ndash6
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Analytical VS Clinical Sensitivity
Snijders P et al J Pathol 2003 201 1ndash6
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Take Home MessagesQ1) What is the most abundant genome in LSIL
1 E6 2 E7 3 E4 4 E5
A1) 3 E4
Q2) What reacts first when infected basal cells
A2) 4 E5
Q3) What is inducing this change
A3) 4 E5
Q4) What is persistently identified in cytoplasm
A4) 3 E4
Q5) What is dominant oncogene
A5) 2 E7
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Ovary Ca Represents Many Disease
bull Many ovarian ca are derived from non-ovarian tissue
bull Different ovarian histiotypes share few molecular similarities
bull Many EM hg should be reclassifed as serous ca bull Many mucinous ca should be reclassified as
secondary ca bull Serous ca hg share molecular profiles with basal-like
breast ca bull Clear cell ca share with renal CCC in sunitinib effect
bull Favoring pelvicperitoneal cancer over ovary ca
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Salpingectomy best choice to prevent ovary cain case risk group
Mucinous type-exclusion Dx
Appendectomy-Best choice to manage PP
Endometriosis-High risk factor of ovary ca
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Genesis and Evolution of Ovary Ca
bull Type I-low grade slow growing tumor (25) ndash Developing from borderline tumorndash Normal karyotypendash Rasmut high BRCA β-cat PTEN p53mut lack ndash PI3KCA ARID1A loss (EMCCC aw endometriosis)ndash Mucinous LG serous LG EM clear cell candash Poorly responsive to platinum-based CTx
bull Type II-rapid growing highly aggressive (75)ndash Rare Rasmut 70-80even 100 p53mut
ndash De novo Genetic instabilityndash HG serous ca HG endometrioid ca MMMT undiff sarcoma
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Tumor Microenvironment-linking to Pathology-
연세의대 병리학교실
조 남훈
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Cell cycle
Hallmarks of Cancer
apoptosis
TME TME
TSG
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Tumor microenvironment- Plethora of interaction
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Chemical factorsMechanical Factors
Cellular factors
Tumor microenvironment
Interaction Arena Terra Incognita
Extracellular matrix (ECM) Stiffness
Focal adhesion complex (FAC) Actin polymerization
Cell adhesion molecule (CAM) Integrin
Tumor resident cell Ca-associated fibroblast (CAF) Ca-associated adippocyte (CAA)
Tumor infiltrating cell Tumor-ass macropahge (TAM)
Vascular factors
Cytokine Chemokine Soluble factors Signal transduction
Interstitial fluids Endothelial cell angiogenesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
TME ldquoseed-soilrdquoampldquoPing-pongrdquo
bull Stephen Paget ldquoseed-soilrdquondash 1994 76 articles
ndash 2004 331 (3 folds1decade)
ndash 2010 4861 (15 folds half decade)
bull Post-Paget era
ndash bdquoPing-pong‟ experiments (IP Witz Cancer Res 1989)
bull BALBc 3T3 in vitro- in vivo injection (ping)
bull Recultured from in vivo tumor (pong)
bull In vivo passage cells was considerably augmented as compared to that of in vitro maintained clonal ancestors
bull In vivo ldquoTME induces of pro-tumor effectrdquo
ndash Class II cancer gene (R Sager PNAS 1997)
bull Many of class II genes are regulated by TME
bull Shifting focus from DNA to RNA
Non-tumor cells in TME are different from those in normal ME
ndash Structural and functional alteration in TME
ndash Paracrine signal from tumor cell or normal cell
ndash Promalignant factors in TME
ndash Hypoxia (necrosis) in TME
ndash Low glucose concentration in TME
Question
ndashNormalization of abnormal non-tumor
constituents may reduce the malignancy
phenotype
ndashTumor reversion is indeed possible
ndashNot sufficiently cured by tumor cell
eradication alone
ndashIs there a hierarchy of interaction
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Tumor microenvironment- Double-edged sword
bull Contemporary issuebull Cellular soluble factors
ndash Balance score of pro-(MCP-1 IL-6 MMP) and anti-malignant (IP-10) factors 136~ 244 (Witz IJCa 2003)
ndash No MECE
bull P-P interaction
bull Tumor heterogeneity vs signal heterogeneity
bull Cross-talk (agonistic vs antagonistic)ndash TNF family
raquo Proinflammatory cytokine as promalignant effect (since R Virchow 1863)
ndash TGF-βraquo Inhibit normal mammary epithelail cells
raquo Enhance tumor cell invasionmets
- loss of anti-proliferative role
- mutation in TGFBR
- inhibition of SMAD pathway
- suppressing anti-tumor immune response
- augmenting angiogenesis
macrophage
MCP-1TNF-α
MMP
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Extracellular Matrix (ECM)
Compression buffer with structural frameCell-cell interaction regulation
Blackbox (footpath)One thing arises from all things and all things arise from
one thing (Aristotle)
Tumor migrationStiffness and destruction
No de novo synthesis
Proteoglycan
Heparan sulfate
Chondroitin
sulfate
Keratan sulfate
Non-proteoglycan
Fiber
Fibronectin
Collagen-14 types
Elastin
Laminin
Hyaluronic acid
Soil researcher as in Scientific Botanist
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Matrix metalloproteinase FM27 types
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Integrin as heterodimer in biphasic roles
α1
α2
α3
α4
α5
α6
α7
α8
α9
α10
α11
β7
αE
β1
β4 - lamininβ5 β6 β8
αV
β3 - vitronectin
β2
αL
αM
αX
αD
18 α+ 8 b = 24 heterodimers
fibronectincollagen
Outs
ide-
in s
ignal
Insid
e-out
sig
nal
HYD-1 (RGD mimicker)decapeptide
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Focal Adhesion bull Subcellular macromolecule that mediates anchorage of
ECM (lt15nm close to membrane)bull Outside-in Signal hub
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Integrin-targeted therapy
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
TZ
1 2 3 4 5
NZIZ
DCIS
Laminin-γ2
GAPDH
Integrin β4
TZ
1 2 3 4 5
NZIZ
IDCA
Tum
or
Interface
5 mm
Normal 1 2 3 4 5
Rela
tive inte
nsity
Laminin-γ2
1 2
TZ IZ NZTZ IZ NZ
Laminin-α3
4 5
TZ IZ NZTZ IZ NZ
Integrin β4
1
TZ IZ NZ
3
TZ IZ NZ
2
TZ IZ NZ
3
TZ IZ NZ
DCIS IDC
Integrin α6
GAPDH
C
B
DCIS IDC
Laminin-γ2 Integrin-β4
TZ IZ NZTZ IZ NZ
Laminin 5 and Igβ4 specific to IZ as invasive signature
Distinct ECM gene expression in the interface zone of invasive ductal carcinoma through
Screening by ECM cDNA array and validated by real-time RT-PCR and WBKim amp Cho Am J Pathol 2011
Integrin β4Laminin-γ2
Rela
tive inte
nsity
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Expression of integrin β4 in fibroblasts only when cocultured directly with invasive cancer in vitro
ACAF
Integrin
β1
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
BCAF
Integrin
β4
Events
InF NBF
SF
MM
CF
7
CM
MD
A-
MB
-231
CM
CCAF InF NBF
Fibroblast(FITC+)
Inte
grin
β1
MC
F
-7
MD
A-M
B-
231
DCAF InF NBF
Fibroblast(FITC+)
Positiv
e
(MD
A0M
B-2
31
)
Negative
(Fib
robla
st)
MC
F-7
MD
A-M
B-
231
Inte
grin
β4
Control
Integrin β1
Integrin β4
GAPDH
EMCF7 MDA-MB-231
Co
culture
Igβ4 switching in invasion
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Integrin β4Integrin α6
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
In Situ Proximity Ligation Assay (PLA)Red dots denote regions of signal amplification
consistent with a6β4 integrin interactions
MDA-MB-231
MDA-MB-231 Integrin a6β4
Integrin a6β4
Secondary antibodies (PLA probe MINUS and PLA probe PLUS)
Ligase is added and theoligonucleotides will hybridize to the two PLA probes and join to a closed circle if they are in close proximity
Amplification solution and fluorescently labeled oligontis added together with polymeraseAs a primer for a rolling-circle amplification (RCA) reaction
Ig a6 (rabbit)Ig b4 (rat or mouse)
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Red dots amplification-a6β4 integrin interactions
In Situ Proximity Ligation Assay (PLA)- Ig a6β4
On Submission Park and Cho 2012
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Polarized morphology in Cell Migration
bullIntegrin-cytoskeleton linkage 4-fold difference between the front and rear Thatrsquos Rapid and intermittent (Schmidt 1993 JBC)
bull Migration rate depends on1 stimulus gradient2 locomotion speed
(integrin-cytoskeleton)
3 directional persistence time (inverse relation to speed)
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Desmoplasia
CAFaSMA
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Correlation of Cell Migration amp Tissue Invasion
Cell biology Asymmetric polarized morphology Kinetic fluctuation in receptor-ligand binding Concentration gradient- independent
Cell migration Cell 1996 84359-69 (Bible for migration)
Tissue biology Early onset of microinvasion step-wise model Diagnostic useful universal kit BM disruption or formation with
different composition
Clinical biology TNM stage reassessment Aggressive non-invasive cancer Biomarker to predict invasion or metastasis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Plasticity of tumor invasion-more than cell level
Tumor-cell invasion and migration Friedl amp Wolf Nat Rev Cancer
2003 3362-74
Integrin recruitement
Heterogeneous TMEHomogeneous TME
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
The stressful ME of intraductal niche promote genetic instability
5-25 layers thick around comedo necrosis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
A simplified overview of signalling networks regulating epithelialndashmesenchymal transitions (EMT)
EMT InducerLoss of cell junction
ECM Remodelling
Polyak amp Weinberg Nat Rev Cancer 2009 9265-73
TF
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Cell migration in Pathology조직에 항상 적용되지 않는다
bull Early transient phenomenon
ndash Actin polymerization SMA+ more eosinophilic
ndash Collective invasion에서도 asymmetric polarity Be careful with TMAbull 대부분 EMT process는 아님
bull EMT를 조직에서 확인하기는 어려움
ndash Keep aware of aggressive non-invasive ca
ndash What is EMT in Pathologybull TsF more sensitive
bull cell junction molecule more specific
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
G-protein (GTPase)
bull Outside-in signaling
bull G-protein coupled receptor (GPCR)-950개
ndash Molecular switch (GTP-on GDP-off)
ndash Heterotrimeric (large) G-protein αβγ ndash GPCR-
dependent
bull Gα cAMP (1994 Alfred Gilman amp M Rodbell Nobel pr)
ndash Heterodimeric homologous Ras G-protein (small) α( Gαs Gαio
Gαq11 Gα1213 ) 10 types ( Ras Rho Rab Rap Arf Ran Rheb Rad Rit Miro)
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Motility factors triad in cellular level
Cdc42 controls the cell polarity and the
formation of filopodia and nascent focal
complexes (shown as yellow dots)
Rho influences cell adhesion
assembly and maturation in
addition to controlling stress fiber
formation and contractile activity
Rac1 primarily controls actin
assembly and adhesion in the
lamellipodium
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Chemokinebull 48 types (8~10 kD)
bull Four cystein residue forming 3-D shape
bull G-protein receptor
(γ-2) (β-28) (α-17)
(δ-1)
CXCR-1 CXCL-6 CXCL-8 (IL-8) - neutrophilCXCR-2 CXCL-1~ 7 - neutrophilCXCR-3 CXCL-9 ~11 - T-cellCXCR-4 CXCL-12 (SDF-1) - hematopoietic cells
endotheial cells neuronCXCR-5 CXCL-13 -B-cellCXCR-6 CXCL-16CXCR-7 CXCL-12 (SDF-1) -memory B-cell
CCR-1~11 CCR-11 (scavenger receptor)-no signal
XCR-1 XCL-1~2CX3CR1 CX3CL-1
TAM
AMD3100
Pro-inflammatory Pro-angiogenic
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Newly Identified zone-Interface Zone resorted from Normal Zone
Matrix stiffness
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Min
Avg
NZ-2
TZ-3
NZ-1
NZ-3NZ-4
IZ-2IZ-3
TZ-1
IZ-1
Matrisomics using ECM array in Tumor and Normal ME
Matrix stiffness inducing Igβ4
Kim and Cho BCR 2012
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
IZ as EMT playground
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
IZ as migratory zone
Scratching assay ORIS invasion assayChoi and Cho BBRC 2012Gao and Cho Oncogene 2011
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Tumor senescenceHypoxic signature EMT
CSC-nicheInvasion signature
Why Interface zone
Quiscent zone
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
EMTInvasion signatureAnoikis-resistance
Quiscent Normal
PAST
PRESENT
NEAR FUTURERECUR ZONE
FUTURE
Rho-GTP
ROCK
WNT
ECM stiffness
Ig β4
Focal Adhesion Tumor-dependent
microenvironment
IZ is permissive for tumor invasion
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
IF zone implication in practice
bull Potential candidate for molecular margin
ndash How far in tumor extent is safebull Near Normal (overlap with TZ) vs Far normal
bull Normal is no more
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed for Recurrence-zero
bull Interaction mechanismndash Lymphangiogenesis
bull Drug-delivery huddle ndash ECM target moelcule to lessen stiffness
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
IZ-signature using MALDI-Imaing MSMS
IgHA2
Kang amp Cho JPR 2012
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Sampling in TME studyNormal is no more Tumor is too more
bull Tumor periphery vs Far normal to tumor
bull Highlight the Interface zone between TM amp NL ndash Cell invasionmigration zone
ndash Cancer stem cellEMT enriched zone
bull Molecular marginndash How far in tumor extent is safe
bull Near Normal vs Far normal
bull Interface zone (IZ) within 5mm circumferential belt
bull IZ ME is much more similar to TZ ME than far normal IZ in ECM array
bull Molecular margin within 5 mm from the tumor margin even in case of no tumor should be removed (Am J Pathol 2011 178373)
ndash for Recurrence-zero
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
Take Home Message
Do not always force to match data between in vitroin vivohuman samples-blood tissue
Human samples are too heterogeneous to simplify and apply in vitro data or hypothesis
top related