3mar15-ramhold departmental seminar (edited)
TRANSCRIPT
Targeting Metastatic Triple Negative Breast Cancer Using Phage Display NanotechnologyCHRIS RAMHOLD & DR. VALERY PETRENKO11-MARCH-15DEPARTMENT OF PATHOBIOLOGY AUBURN UNIVERSITY CVM
General Outline Cancer
Significance Statistics Models
Phage Display Technology Introduction Selection Modification of pre-existing cancer nanomedicines Targeting
Summary Future Work
Highly invasive tumor embolus. UD- Bio-imaging Core Center
Why Cancer?
What Is Cancer? Product of malignant
progression Loss of proliferative
control Escape apoptotic signals Escape Hayflick limit
Loeb, Lawrence A. "Human cancers express mutator phenotypes: origin, consequences and targeting." Nature Reviews Cancer 11.6 (2011): 450-457.
Cancer (Incidence)
This year, the American Cancer Society estimates 232,670 new cases of breast cancer alone in the United States
Siegel, Rebecca, et al. "Cancer statistics, 2014." CA: a cancer journal for clinicians 64.1 (2014): 9-29
Cancer (Incidence) Incidence rates of breast cancer
are projected to stabilize Lung cancer projected to decline More management options are
needed
Siegel, Rebecca, et al. "Cancer statistics, 2014." CA: a cancer journal for clinicians 64.1 (2014): 9-29
Cancer (Deaths)
Deaths in the U.S. attributed to breast cancer are estimated at 40,000 annually Deaths worldwide are over 500,000
Breast Cancer (Metastatic) The 5-year relative survival
rates of invasive metastatic breast cancer is at 29%
High likelihood of recurrence
http://www.cancercenter.com/breast-cancer/statistics/tab/breast-cancer-survival-statistics/
Breast Cancer Survival Rates
http://www.cancer.org/acs/groups/content/@research/documents/document/acspc-042725.pdf
5-year relative survival rates are lower in younger women
Typically more aggressive Why are these types of cancer
so deadly?
“If we can put a man on the moon, why can’t we cure cancer?” Tumors are heterogeneous
Cancer genome unstable Cancer cells exhibit
plasticity
Wang, Anxin, et al. "Heterogeneity in cancer stem cells." Cancer letters 357.1 (2015): 63-68.
Two Models of Tumorigenesis
Girouard, S. D., & Murphy, G. F. (2011). Melanoma stem cells: not rare, but well done. Laboratory Investigation; a Journal of Technical Methods and Pathology, 91, 647–664. doi:10.1038/labinvest.2011.50
Cancer Stem Cell Hypothesis “Malignant tumors are initiated
and maintained by a population of tumor cells that share similar biologic properties to normal adult stem cells.” – Brenton Thomas Tan
Selective pressures induce evolution of cancer cells which may acquire mutations in the mechanism for EMT (epithelial-mesenchymal transition) allowing tumor formation.
Owens TW and Naylor MJ (2013) Breast cancer stem cells. Front. Physiol. 4:225. doi: 10.3389/fphys.2013.00225
CSCs by Any Other Name… Tumor populations are heterogeneous Not all cells are capable of forming new
tumors Some tumorigenic cells have been
characterized CSCs=Tumor cells with tumorigenic
potential
TobogganToboggan
CSCs and Tumor Initiators The metastases originate
with tumor initiating cells (TICs).
Metastases account for nearly all breast cancer related deaths
Goal: Target the TICs… Stop proliferation Stop metastases Stop recurrence
Cancer Stem Cells from http://www.currinbiotech.com/categories/20101004
Breast Cancer Molecular Subtypes
Prat, Aleix, and Charles M. Perou. "Mammary development meets cancer genomics." Nature medicine 15.8 (2009): 842-844.
Killing Cancer Chemotherapies
Prevent mitosis Induce apoptosis Effective against rapidly proliferating cells
Challenges Low weight- Cleared quickly Low accumulation in tumors Hydrophobicity- Large volume of distribution Tumors are heterogeneous Tumor vasculature is leaky Toxicity towards healthy cells
Art by JerryKongArt http://jerrykongart.deviantart.com/art/Killing-Cancer-Cells-185013819
How Do We Target Cancer?
Targeted delivery of the drug to the site of pathology by DDS
Pathology Pathology Pathology
<1% 10% 20-30%
Untargeted Drug Nanomedicine
Random distribution of the drug leading to side effects
Localization of nano-medicines in tumor due to vasculature defects
Targeted Nanomedicine
Development of Targeted Nanomedicines
Wang, Tao, et al. "On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters." Molecular pharmaceutics 8.5 (2011): 1720-1728.
Phage Display Technology Genetically engineered to express random 9 amino acid insertion at the N-terminus of the pVIII protein. Billions of unique sequences constitute a phage display library
Phage Display using filamentous bacteriophage fd
Løset, Geir Åge, et al. "Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage." PLoS One 6.2 (2011): e17433. * Modified
9-mer insert
Phage Display Library The library contains roughly one billion randomized clones. These clones are subjected to a process of affinity selection.
Negative selection- Depletion of non-specific binders such as plastic, serum, and a “normal” cell line.
Positive selection- Depletion of phage that doesn’t or weakly binds target cells and enrichment of phage that binds and penetrates target cells.
Following several rounds of selection, the phage enriched for binding target cells are characterized.
http://www.virology.wisc.edu/virusworld/ICTV8/1fd-enterobacteria-phage-fd.jpg
Phage Fusion Peptides Intrinsic membrane proteins Capable of self-integration N-terminus bearing targeting
peptide remains exterior to liposome
PEGylated liposomes
Jayanna, Prashanth K., et al. "Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency." Nanomedicine: Nanotechnology, Biology and Medicine6.4 (2010): 538-546.
Phage Fusion Protein Modified Nanomedicines
Leaky vasculature allows accumulation of liposomes at site of tumor Passive- Takes advantage of enhanced permeability and retention effect Active- Phage fusion protein targets cancer cells
Lammers, Twan, et al. "Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress." Journal of controlled release 161.2 (2012): 175-187.
Phage DisplaySelection Overview
Selection- Round 1 (Depletion) Round 1 includes depletion against non-specific
binders (plastic, serum, “normal” cells) and incubates phage with cells at room temperature.
“Normal” cell line used was MCF-10A cell line (ATCC). Classified as non-tumorigenic. Derived from fibrocystic breast tissue (previously
termed “fibrocystic breast disease,” now replaced with fibrocystic breast condition as it is not a disease).
Morphology comparable to normal cells Immortalized due to a loss of p16 at both loci-
otherwise diploid and genetically stable
Botlagunta, Mahendran, Paul T. Winnard, and Venu Raman. "Neoplastic transformation of breast epithelial cells by genotoxic stress." BMC cancer 10.1 (2010): 343.
Selection- Round 1 (Target Cells)
MDA-MB-231 cell line was used as target cells. Tumorigenic- derived from metastatic site (primary
adenocarcinoma) Triple negative (no expression of estrogen and
progesterone receptors as well as HER2 oncogene). Falls into the claudin-low molecular subtype Enrichment for EMT markers Shares features with stem-like cells
Breast Cancer Molecular Subtypes Cell lines are heterogeneous CD44+/CD24- used as standard
detection of stem-like cells Not always tumorigenic CD44+/CD24-/ESA+ cells more
consistent Showed tumorigenicity across
33 cell lines Genomic profile associated
with EMT signaling, loss of proliferative control
Prat, Aleix, and Charles M. Perou. "Mammary development meets cancer genomics." Nature medicine 15.8 (2009): 842-844.
Why MDA-MB-231? Nearly 100% of MDA-MB-231
cells are CD44+/CD24- MDA-MB-231 cell line has one of
the highest % for CD44+/CD24-/ESA+
Fillmore, Christine M., and Charlotte Kuperwasser. "Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy." Breast cancer res 10.2 (2008): R25.
Hypothesis Targeting breast cancer
stem-like cells will decrease the population of tumorigenic cells, increasing cytotoxic effects and reducing recurrence and metastasis.
Reya, Tannishtha, et al. "Stem cells, cancer, and cancer stem cells." nature414.6859 (2001): 105-111.
Selection- Round 1 (Phage Titers)
After incubation of phage with target cells (1 hr), unbound or weakly bound phage are removed by a series of sequential washes
Input- Starting phage concentration
Unbound and washes- non-specific and weakly binding phage
Dilute eluate through lysate- phage specific for target cells, both surface binding and membrane penetrating
Input
Unbou
nd
1st wash
2nd w
ash
3rd wash
4th wash
5th wash
6th wash
7th wash
8th wash
9th wash
10th
wash
Dilute E
luate
1st PE
wash
2nd P
E wash
PE su
pernata
ntLys
ate1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
Phage Titers Throughout Round 1 of Selection
Phage Fractions
CFU
(Tite
r x V
olum
e)
Selection- Quantification of Phage Phage recovered from both
the eluate and lysate fractions were amplified for use as inputs for subsequent rounds of selection.
Concentration measured by absorbance @ 269nm
Selection- Round 1 (Phage Recovery % Yield)
Low % recovery is typical as the large pool of ~ 1 billion clones is not enriched for the target cells
Phage recovered from the eluate vs lysate fractions is more pronounced in Round 1 as incubation at room temperature creates unfavorable conditions for phage to penetrate into target cell membranes
Successive rounds of selection should… Indicate enrichment of target-specific clones Have an increase in % recovery for both the
eluate and lysate fractions
Eluate yield (%) Lysate yield (%)0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
Phage % Recovery from Round 1 of Se-lection against MDA-MB-231 cells
Phage Fraction
% P
hage
Rec
over
y (O
utpu
t/In
put)
x100
Selection- All Rounds(Eluate Phage Titers)
Round 2 & 3 incubation steps are carried out at 37oC providing an environment conducive to membrane penetrating phage
Results comparable to Round 1
Enrichment of targeted phage
Input
Unbou
nd
1st wash
2nd w
ash
3rd wash
4th wash
5th wash
6th wash
7th wash
8th wash
9th wash
10th
wash
Dilute E
luate
1st PE
wash
2nd PE
wash
PE su
pernata
ntLys
ate1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
Phage Titers Throughout Rounds 1-3 of Selection
Round 1 Round 2 Round 3
Selection- Comparison of Phage % Recovery
Was there enrichment of target specific clones? Yes, an increase in % recovery is seen
each round Caveat* some of the phage may simply
be “fast growers” Did the % of phage recovered in the
lysate fraction increase with an increase in temperature? Higher temperatures allowed more
phage to penetrate target cells as shown by an increase in the amount of phage recovered through cell lysis
Eluate Lysate0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Phage % Recovery Comparison
Round 1Round 2Round 3
Phage Fraction
% P
hage
Rec
over
y (O
utpu
t/In
put)
x100
Isolation of Phage Clones Well isolated, individual colonies of infected K91 Blue
Kan E. coli cells from the Eluate, Lysate, and Post elution wash titer plates were chosen at random and inoculated on a 100 grid plate. (100 colonies from eluate, lysate, and 50 each from the post elution wash plates).
Following an overnight culture, 95 colonies were then used in PCR to provide… Confirmation that the colony contains phage with major
coat protein Product to be sequenced for peptide identification
Electrophoresis of PCR Products
Band indicating presence of major coat protein Primer Dimers Clear Negative control
Eluate Clones 1-24 LtR TtB Eluate Clones 25-48 LtR TtB Eluate Clones 49-72 LtR TtB Eluate Clones 73-95 LtR TtB
Solid Contaminant
Sequencing Results (Peptide Sequences)
Unique Clones from Eluate (Repeats) Unique Clones from Lysate (Repeats)
AGLNYNVDQ DYDSLHINS (15) GMVSGQADD (4) VDYSEVGSLDARQGVMME EFGGPDYDT GPSYSENPD (2) VEPGGWTGDDGSGSLDGD EPYSGSISN GSLDEALNQ (12) VGENGGSAD (2)DGYRSSEDS (4) ERFQEGGTD (4) GSQTVMTDD (5) VGSDYGTGD (16)DLDLPGVND EVDAHVNLD GSSMSFQDT VLRDFLDTDDLQAQWAGD (15) EWNRSELGD (2) GSYDEVSSA (2) VNHETLTAD (3)DMQWSSGDT (3) EYSQGQEGS VADSAVGHD VPPDFLDTD (6)DSGWERNVD (2) GDYTEAVGA VAVSEPGMD (3) VSEPTSNES (2)DSSMSWSGD (63) GETGGADND (3) VDADRFSGD VSYMESETD (2)DTGMLEGGN GLNGGNWDD (8) VDTAEISSL (4) VTASGMSDD (2)
AGSNNEGMT (2) ETSRYSDID (2)AGSYGDMDT (11) GAEYVGDTT (2)DFAVGPGSD (2) GFNTEFGDT (2)DFNLHDAMD (2) GSEQSWTGD (2)DGIWEHGDS (2) GSLLSSQED (2)DHGGGGHDS (2) GYDPVNDYN (2)DMTNGSVPE (2) VDIAEQSTA (2)DSVYDEENS (2) VGGHGDDFD (2)DVPRETGLD (2) VGSMSDGYN (4)ESALWGGDS (2) VSTSSDFDP (2)
Sequencing Results (Families)(A)GS(Y) (E/N)GG(N) GGH NVD (W)TGD YSEAGSNNEGMT DTGMLEGGN DHGGGGHDS AGLNYNVDQ GSEQSWTGD GPSYSENPD
AGSYGDMDT ERFQEGGTD VGGHGDDFD DSGWERNVD VEPGGWTGD VDYSEVGSL
GSYDEVSSA VGENGGSAD GLN QEG VGSDYGTGD V**DFLDTD
AVG GLNGGNWDD AGLNYNVDQ ERFQEGGTD VDA VLRDFLDTD
DFAVGPGSD EPG GLNGGNWDD EYSQGQEGS EVDAHVNLD VPPDFLDTD
GDYTEAVGA VAVSEPGMD GSD SGD VDADRFSGD Orphans
VADSAVGHD VEPGGWTGD DFAVGPGSD DMQWSSGDT VGS DARQGVMME
DFD / DYD EQS VGSDYGTGD DSSMSWSGD VDYSEVGSL DFNLHDAMD
VGGHGDDFD GSEQSWTGD GSL(D) VDADRFSGD VGSDYGTGD DLQAQWAGD
VSTSSDFDP VDIAEQSTA GSLLSSQED SGS VGSMSDGYN DMTNGSVPE
DYDSLHINS ETG VDYSEVGSL DGSGSLDGD VND ETSRYSDID
EFGGPDYDT DVPRETGLD DGSGSLDGD EPYSGSISN DLDLPGVND EWNRSELGDDGY GETGGADND GSLDEALNQ (S)SMS GYDPVNDYN GMVSGQADD
DGYRSSEDS GD(S/T) HGD DSSMSWSGD VSEP GSQTVMTDD
VGSMSDGYN DGIWEHGDS DGIWEHGDS GSSMSFQDT VAVSEPGMD VDTAEISSL
EFG DMQWSSGDT VGGHGDDFD VGSMSDGYN VSEPTSNES VNHETLTAD
EFGGPDYDT ESALWGGDS MSD YDE VSYMESETD
GFNTEFGDT GAEYVGDTT VGSMSDGYN DSVYDEENS
GFNTEFGDT VTASGMSDD GSYDEVSSA
Sequencing Results (Potential Targets)
Utilizing BLAST MimoDB, peptides that are similar or identical to the sequenced phage peptides can be matched, giving clues to potential targets.
Our phage from selection against MDA-MB-231 cells may bind to… Hepatocellular carcinoma line Mahlavu Vβ1, Vβ3, and Vβ6 integrins Phosphorylated/Unphosphorylated Erβ EGFR PC3 prostate carcinoma cell line Human lewis lung carcinoma cells TNF-α SK-OV-3 (Human ovarian tumor cell
line)
Breast cancer tumor (Human- in vivo) OS-732 (osteosarcoma cell line) LNCaP (Prostate carcinoma cells) NCI-H1299 non-small cell lung cancer
cell line 9L Glioma cell line HT29 colon cancer cell line
Binding Assays 2 mL propagation of phage clones. 7b1 phage used as negative control. MDA-MB-231 used as target cell
line. Media containing serum (DMEM/F12
+ 10% FBS + 1% Ab/Am) was used for baseline comparison.
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Binding Assay (Comprehensive)
7b1
DLQAQWAGD
VDADRFSGD
DHGGGGHDS
DYDSLH
INS
DGSGSLD
GD
VNHETLTA
D
VDYSEV
GSL
VDTAEIS
SL
DFAVGPG
SD
DGYRSS
EDS
DSSMSW
SGD
DLDLPG
VND
GLNGGNWDD
ERFQ
EGGTD
VAVSEPG
MD
AGSNNEG
MT
GSLLSS
QED
GSQTV
MTDD
GAEYVGDTT
DFNLH
DAMD
DGIWEH
GDS
EFGGPD
YDT
VGENGGSA
D
DVPRET
GLD
ESALW
GGDS
DSVYD
EENS
0.00E+00
2.00E-02
4.00E-02
6.00E-02
8.00E-02
1.00E-01
1.20E-01
1.40E-01
1.60E-01
Binding Assay Complete
% Yield Target % Yield Serum
Phage Clones
% Y
ield
Binding Assay (Top 15 Yields)
GAEYVGDTT
VSTSS
DFDP
DFNLH
DAMD
GETGGADND
DGIWEH
GDS
DMTNGSV
PE
EFGGPD
YDT
AGLNYN
VDQ
VGENGGSA
D
GDYTEA
VGA
DVPRET
GLD
VLRDFLD
TD
ESALW
GGDS
GYDPV
NDYN
DSVYD
EENS
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Binding Assay (Top 15 % Yields)
% Yield Target % Yield Serum
Phage Clones
% Y
ield
Binding Assay (Top Target Specificity *16)
GSQTV
MTDD
DVPRET
GLD
GSYDEV
SSA
GYDPV
NDYN
AGLNYN
VDQ
DYDSLH
INS
VGSDYG
TGD
VPPDFLD
TD
GSSMSFQ
DT
VLRDFLD
TD
GDYTEA
VGA
EFGGPD
YDT
DGIWEH
GDS
VGENGGSA
D
ESALW
GGDS
DSVYD
EENS
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Binding Assay Clones With Target Specificity > 10
% Yield Target % Yield Serum
Phage Clones in order of increasing target specificity (Target % yield / Serum % yield)x100
% Y
ield
Potential Champion Clones (10)
EFGGPDYDT AGLNYNVDQ VGENGGSAD GDYTEAVGA DVPRETGLD VLRDFLDTD ESALWGGDS GYDPVNDYN DSVYDEENS0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
Potential Champion Clones
% Yield Target % Yield Serum
Phage Clones
% Y
ield
Specificity Assay
DFPPSS
AE
VGENGGSA
D
VLRDFLD
TD
GSSMSFQ
DT
VPPDFLD
TD
VGSDYG
TGD
DYDSLH
INS
AGLNYN
VDQ
GYDPV
NDYN
GSYDEV
SSA
DVPRET
GLD
GSQTV
MTDD
GDYTEA
VGA
EFGGPD
YDT
DGIWEH
GDS
VGENGGSA
D
ESALW
GGDS
DSVYD
EENS
0.00E+00
5.00E-02
1.00E-01
1.50E-01
2.00E-01
2.50E-01
Specificity Assay
% Yield 231 % Yield MCF7 % Yield MCF10A % Yield Serum
Phage Clones
% Y
ield
Future Work Designate champion clones from best binding clones Isolate protein of champion clones and modify Lipodox®
Characterize modified nanomedicine (size, zeta potential, cytotoxicity, uptake)
Isolate breast cancer stem-like cells (potentially select CD44+high/CD24-low /ESA+ cells)
Select phage specific for breast cancer stem-like cells Modify nanomedicine to target tumor initiating cells Cytotoxicity assay on CSC population in cell lines representing
all molecular subtypes Potential for in vivo applications
Acknowledgements
Special thanks to lab members Dr. Valery Petrenko Dr. Anatoliy Puzyrev James Gillespie Amanda Gross Logan Stallings
Funding from AURIC Graduate Research
Fellowship NIH Grant
ReferencesFillmore, Christine M., and Charlotte Kuperwasser. "Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy." Breast cancer res 10.2 (2008): R25.Girouard, S. D., & Murphy, G. F. (2011). Melanoma stem cells: not rare, but well done. Laboratory Investigation; a Journal of Technical Methods and Pathology, 91, 647–664. doi:10.1038/labinvest.2011.50http://www.virology.wisc.edu/virusworld/ICTV8/1fd-enterobacteria-phage-fd.jpghttp://www.cancer.org/acs/groups/content/@research/documents/document/acspc-042725.pdfhttp://www.cancercenter.com/breast-cancer/statistics/tab/breast-cancer-survival-statistics/Jayanna, Prashanth K., et al. "Landscape phage fusion protein-mediated targeting of nanomedicines enhances their prostate tumor cell association and cytotoxic efficiency." Nanomedicine: Nanotechnology, Biology and Medicine6.4 (2010): 538-546.Lammers, Twan, et al. "Drug targeting to tumors: principles, pitfalls and (pre-) clinical progress." Journal of controlled release 161.2 (2012): 175-187.Loeb, Lawrence A. "Human cancers express mutator phenotypes: origin, consequences and targeting." Nature Reviews Cancer 11.6 (2011): 450-457.Løset, Geir Åge, et al. "Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage." PLoS One 6.2 (2011): e17433. * ModifiedOwens TW and Naylor MJ (2013) Breast cancer stem cells. Front. Physiol. 4:225. doi: 10.3389/fphys.2013.00225Prat, Aleix, and Charles M. Perou. "Mammary development meets cancer genomics." Nature medicine 15.8 (2009): 842-844.Siegel, Rebecca, et al. "Cancer statistics, 2014." CA: a cancer journal for clinicians 64.1 (2014): 9-29Wang, Anxin, et al. "Heterogeneity in cancer stem cells." Cancer letters 357.1 (2015): 63-68.Wang, Tao, et al. "On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters." Molecular pharmaceutics 8.5 (2011): 1720-1728.