Download - Cellule Staminali Del Cancro I
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Stem cells Rare cells with the ability to perpetuate themselves through self-renewal and to
generate mature cells of a particular tissue through differentiation.
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Asymmetric and symmetric stem cell divisions: stem cell strategies
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Mechanisms of asymmetric stem cell division
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Symmetric stem cell division in the adult germline
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Stem cell can facultatively use both symmetric and asymmetric division
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Stem cells occur in many different somatic tissues and are important for their physiology
Jordan et al. 2006 NEJM 355:1253-61
Neural stem cells generate cells in the central nervous system (Panel A). Hematopoietic stem cells generate mature blood cells (Panel B). Mammary stem cells generate breast tissue (Panel C).
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HSCs can be subdivided into long-term self-renewing HSCs, short-term self-renewing HSCs and multipotent progenitors (red arrows indicate self-renewal). They give rise to common lymphoid progenitors (CLPs; the precursors of all lymphoid cells) and common myeloid progenitors (CMPs; the precursors of all myeloid cells). Both CMPs/GMPs (granulocyte macrophage precursors) and CLPs can give rise to all known mouse dendritic cells. ErP, erythrocyte precursor; MEP, megakaryocyte erythrocyte precursor; MkP, megakaryocyte precursor; NK, natural killer.
Development of Hematopoietic Stem Cells
Stem Cells
Multipotent Progenitors
Oligolineage Progenitors
Mature Cells
Reya et al. 2001 Nature 414:105-111
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In vivo transfer to irradiated recipient mice
Recapitulation of the haematopoietic system in the recipient mouse
Positive and
negative s
election Peripheral Blood, Bone Marrow
or Cord Blood-derived cells
CD34+ cells
rare HSC in progenitor cell background
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Origin of the Theory of Cancer Stem Cells
Only a small subset of cancer cells is capable of extensive proliferation
Liquid Tumors In vitro colony forming assays:
- 1 in 10,000 to 1 in 100 mouse myeloma cells obtained from ascites away from normal hematopoietic cells were able to form colonies
In vivo transplantation assays: - Only 1-4% of transplanted leukaemic cells could form spleen colonies
Solid Tumors - A large number of cells are required to grow tumors in xenograft models - 1 in 1,000 to 1 in 5,000 lung cancer, neuroblastoma cells, ovarian cancer cells, or breast cancer cells can form colonies in soft agar or in vivo
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Two models for tumor heterogeneity and propagation
Normal cellular hierarchy
Clonal evolution model: all undifferentiated cell have similar
tumorigenic capacity
Cancer stem cell model model: only CSC can generate tumor, based on
its self-renewal properties and enormous proliferative potential
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Both models of tumor maintenance may underlie tumorigenesis
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Early in situ lesion low heterogeneity
Advanced lesion high heterogeneity
cancer stem cells
proliferating tumor cells
mutation
asymmetrycal division
symmetrycal division
senescent/dying cancer cells
Tumor formation according to the CSC hypothesis. A mutated stem cell can expand by symmetrical and asymmetrical division, giving rise to daughter stem cells and progenitor cells, which in turn generate other tumor cells without self-renewal capability. Proliferating tumorigenic cells are the target of additional mutations that eventually result in tumor progression. As CSC divide and mutate, the tumor can become more heterogeneous, although rapidly dividing CSC derivatives are likely to be positively selected.
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Defects in regulation of the switch between symmetric and asymmetric divisions can be deleterious
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Sublethally irradiated NOD/SCID Mice
FACS Cell Sorter
Cancer Cells (ex: Leukaemia cells)
Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell
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Prospective identification of tumorigenic breast cancer cells.
FACS Cell Sorter
Solid Tumor Single Cell Suspension CD24 Expression
CD
44 E
xpre
ssio
n
Mince (small pieces)
Surgical Implantation
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Prospective isolation of human CSC from freshly dissociated tumors
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Cancerstemcellsfeatures:exvivoandinvivoassaysforCSCs
Abilitytopropagateindenitely.
Giverisetotheirdieren>atedprogeny
Resemble,uponinjec>oninvivo,thetumoroforigin
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Reciprocalinterac>onsbetweentheCSCanditsmicroenvironmentorniche
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Tumor tissue Dissociation
Brain Tumor Stem Cell Culture
Xenografts
Differentiation
GFAP Neu-N
MRI
H&E
H&E H&E
DICT IF
Glioblastoma stem cells
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Nature. 2007; 445(7123):111-5.
Cell Death and Differentiation. 2008; 15: 504-514.
Colon and lung cancer stem cells have been identified for the first time at ISS
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Cancer Stem Cells
Cancer stem cells have been isolated from solid tumors by surface marker sorting (i.e. CD133) or by specific culture conditions that allow CSC-containing tumor spheres to growth.
Tumor Sphere
Xenograft
Sorting
Surgical biopsy (colon cancer)
Culture in serum free medium, containing EGF and
FGF2
CD
133
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Conventional chemotherapy is unable to kill colon cancer stem cells
Stem cells Differentiated cells
Untreated 5-FU Irinotecan Oxaliplatin
CD
133+
cel
ls (%
) C
ell v
iabi
lity
(%)
0 20 40 60 80
100
5-FU Irinitecan Oxaliplatin
0 2 4 6 8
10
In vitro
In vivo
Control
Oxaliplatin
drug
0
0.4
0.8
1.2
1.6
2
2.4
4 6 8 10 12
Weeks
Tum
our s
ize
(cm
3 )
drug
0
CSC-based xenografts
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Our approach to isolate lung cancer stem cells from primary tumors
In our laboratory, human CSC were isolated based on their ability to survive in serum-free conditions and to proliferate as cellular aggregates, so called tumor spheres.
This experimental strategy represents the best approach so far to obtain unlimited expansion of the tumorigenic cancer cell population from primary tumors, providing a powerful tool to allow extensive studies on these cells.
Tumor samples are obtained from consainting patients.Tissue dissociation is carried out by mechanical disgregation and enzymatic digestion with collagenase II 1.5 mg/ml and DNaseI 20g/ml. Recovered cells were cultured at clonal density in serum-free medium and supplemented with 20 g/ml EGF and 10 g/ml bFGF. Non-treated flasks for tissue culture were used to reduce cell adherence and support growth as tumor spheres.
Tumor dissociation Tumor sphere
Experimental Design
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Lung cancer stem cells are resistant to conventional chemotherapyThe establishment of exponentially growing lung CSC cultures may allow the direct evaluation of the cytotoxicity of antineoplastic agents on the cells responsible for tumor growth and spreading, which represented the optimal cellular target for successful therapies.
Lung CSC were more resistant to chemotherapeutic drugs than differentiated cells in line with the poor therapeutic effect of conventional chemotherapy on lung cancer patients.
Cisplatin 5g/ml Etoposide 10g/mlGemcitabine 250 m Paclitaxel 30 ng/ml
LCNEC
AC SCC
Cel
l via
bilit
y (%
)
Cel
l via
bilit
y (%
) C
ell v
iabi
lity
(%)
Cel
l via
bilit
y (%
)
0
20
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60
80
100
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0
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0 20 40 60 80
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Control Cisplatin Etoposide Gemcitabine Paclitaxel
SCLC
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5
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15
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NT CIS ETO PACLI GEMC
spheres differentiated
Cell d
eath
(%)
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Models of tumor drug resistence
Conventional model: rare cells with genetic alterations that confer multidrug resistance (MDR) form a drug resistant clone (yellow). Following chemotherapy, these cells survive and proliferate, forming a recurrent tumour
Cancer-stemcell model: tumours contain a small population of tumour stem cells (red) and their differentiated offspring, which are committed to a particular lineage (blue). Following chemotherapy, the committed cells are killed, but the stem cells, which express drug transporters, survive. These cells repopulate the tumour
Acquired resistance stem-cell model: the tumour stem cells (red), which express drug transporters, survive the therapy, whereas the committed but variably differentiated cells are killed. Mutation(s) in the surviving tumour stem cells (yellow) and their descendants (purple) can arise conferring a drug-resistant phenotype
Intrinsic resistance model: both the stem cells (yellow) and the variably differentiated cells (purple) are inherently drug resistant, so therapies have little or no effect, resulting in tumour growth.
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Cancer stem cells display enhanced resistance to radiation
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drug resistance
conventional therapy
bulk tumor surviving CSCs tumor relapse
bulk tumor non-self renewing
tumor cells tumor
eradication
CSC-oriented therapy degeneration
proliferating tumoral cell
self-renewing cancer stem cell
Therapeutic implications of Cancer Stem Cells
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Cancer stem cells Cancer stem cells are responsible for tumor cell development, maintenance and spreading
Tumor relapse results from residual cancer stem cells surviving the therapeutic treatment
Cancer stem cells should represent the primary target for new therapeutic strategies aimed at tumor eradication
Cancer stem cell analysis may provide considerable information for prognostic study and patient stratification
Options Characterization: immunoistochemistry screening, mRNA and protein analysis
Functional assays: in vitro drug screening, in vivo drug validation,
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Results of analyses with the Cox Proportional-Hazard Models Overall Survival Progression-Free Survival
Variable Hazard Ratio (95% CI)
P Value
Hazard Ratio (95% CI)
P Value
CSC generation (vs. no CSC generation) Symptom duration 3 months (vs > 3 months) Partial surgery (vs. complete surgery) Age < 50 years (vs. 50) MGMT status negative (vs. positive) p53 status positive (vs. negative)
4.03 (1.82-8.93) 2.98 (1.25-7.09) 3.09 (1.43-6.69)
3.80 (1.44-10.04) 1.08 (0.52-2.22) 1.24 (0.54-2.84)
0.0006 0.0133 0.0042 0.0071 0.8369 0.6161
4.45 (2.03-9.78) 1.89 (0.89-4.01) 3.40 (1.58-7.31) 2.42 (1.04-5.63) 1.03 (0.52-2.05) 0.86 (0.39-1.88)
0.0002 0.0956 0.0018 0.0403 0.9231 0.7045
CD133/Ki67 positive (vs. negative) Symptom duration 3 months (vs > 3 months) Partial surgery (vs. complete surgery) Age < 50 years (vs. 50) MGMT status negative (vs. positive) p53 status positive (vs. negative)
4.87 (1.94-12.22) 4.95 (2.05-11.96) 3.21 (1.45-7.10) 1.95 (0.76-5.03) 1.06 (0.51-2.18) 1.06 (0.47-2.39)
0.0007 0.0004 0.0041 0.1674 0.8808 0.8925
6.64 (2.65-16.64) 3.46 (1.56-7.67) 3.80 (1.73-8.30) 1.35 (0.58-3.13) 0.92 (0.45-1.85) 0.86 (0.40-1.86)
3 months) Partial surgery (vs. complete surgery) Age < 50 years (vs. 50) MGMT status negative (vs. positive) p53 status positive (vs. negative)
1.81 (0.91-3.59) 1.04 (1.0-1.09)
3.44 (1.43-8.30) 3.23 (1.49-6.99) 2.53 (0.98-6.53) 1.13 (0.54-2.37) 0.99 (0.44-2.22)
0.0883 0.0674 0.0060 0.0029 0.0545 0.7467 0.9796
1.79 (0.9-3.56) 1.06 (1.02-1.10) 2.57 (1.19-5.52) 3.19 (1.48-6.93) 1.59 (0.66-3.81) 1.35 (0.65-2.82) 0.96 (0.42-2.16)
0.097 0.0061 0.0158 0.0033 0.2965 0.4185 0.957
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P=0.0002
0 6 12 18 24 30 0
10 20 30 40 50 60 70 80 90
100
CSC generation
No CSC generation
Months
Prob
abili
ty o
f Ove
rall
Surv
ival
(%)
Cancer stem cell generation correlates with worse prognosis
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Colon cancer stem cell characterization
Mutational profiling RasG12V
BRAFV600E PTEN PI3KCA TP53 Smad4 MSH6 MLH1
ctsc11 +/+ +/+ +/+ E542K +/+nfR361C +/+ +/+
ctsc12 +/+ +/+ +/+ E542K +/+nfR361C +/+ +/+
ctsc18 G12V +/+ +/+ +/+ +/fs +/+ +/+ down
ctsc85 +/+ +/+ down +/+ +/+nfR361C +/+ +/+
ctsc26 +/+ +/+ +/+ +/+ +/+nf+/+ +/+ down
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Cancer stem cell banking
Tumor dissociation
Stem cell culture and expansion
Tumor database
H&E
Diagnosis and tissue banking
Available Resources
High throughput microRNA expression
Transcriptional profiling
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Entero-endocrine FABP2 CHGA CHGB
REG1A SPON1 Paneth DEFA6 DEFA5 NELL2
SERPINI1 Enterocyte
TFF3 MUC3B MUC20 MUC1 MUC6
MUC12
CTS
C11
MSI2 SFRP5 (11,12)
MSI1 LGR4
LGR5 (85) NANOG
KLF4 POU5F1
LIN28 KLF5
PROM1 CD44
CTS
C18
CTS
C85
CTS
C26
CTS
C12
HC
T116
HT2
9
NM
uc
CTS
C22
0
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Stem cell Culture
Differentiation
Tumor sphere
DIC
Absence of Growth Factors
Protein Lysate
Data Analysis
Arrayer
Nitrocellulose coated slides Antibody-based
phosphoprotein detection
Experimental design for reverse phase-based high throughput proteomic analysis
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Compounds withunknown mechanismknown mechanism
Experimental protocol
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In vitro drug testing
Resistant Sensitive
In vivo drug testing 1 2 3 4 5 6 7 8 9
80% 45-55%
Path
way
inhi
bito
rs
Patients
RPPA