1. Targeting Metastatic Triple
Negative Breast Cancer
Using Phage Display
Nanotechnology
CHRIS RAMHOLD & DR. VALERY PETRENKO 11-MARCH-15
DEPARTMENT OF PATHOBIOLOGY AUBURN UNIVERSITY CVM

2. 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

3. Why Cancer?

4. 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.

5. 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

6. 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

7. Cancer (Deaths)
 Deaths in the U.S. attributed to breast cancer are estimated at 40,000 annually
 Deaths worldwide are over 500,000

8. 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/

9. 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?

10. “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.

11. 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

12. 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

13. 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
Toboggan
Toboggan

14. 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

15. Breast Cancer Molecular Subtypes
Prat, Aleix, and Charles M. Perou. "Mammary development meets cancer genomics." Nature medicine 15.8 (2009): 842-844.

16. 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

17. 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

18. 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.

19. 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

20. 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

21. 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.

22. 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.

23. Phage Display
Selection Overview

24. 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.

25. 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

26. 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.

27. 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.

28. 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.

30. 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
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
1.00E+07
1.00E+08
1.00E+09
CFU(TiterxVolume)
Phage Fractions
Phage Titers Throughout Round 1 of Selection

32. 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

33. 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
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
Eluate yield (%) Lysate yield (%)
%PhageRecovery(Output/Input)x100
Phage Fraction
Phage % Recovery from Round 1 of Selection
against MDA-MB-231 cells

35. 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
1.00E+00
1.00E+01
1.00E+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

36. 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
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Eluate Lysate
%PhageRecovery(Output/Input)x100
Phage Fraction
Phage % Recovery Comparison
Round 1
Round 2
Round 3

38. 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

39. 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

40. Sequencing Results (Peptide Sequences)
Unique Clones from Eluate (Repeats) Unique Clones from Lysate (Repeats)
AGLNYNVDQ DYDSLHINS (15) GMVSGQADD (4) VDYSEVGSL
DARQGVMME EFGGPDYDT GPSYSENPD (2) VEPGGWTGD
DGSGSLDGD EPYSGSISN GSLDEALNQ (12) VGENGGSAD (2)
DGYRSSEDS (4) ERFQEGGTD (4) GSQTVMTDD (5) VGSDYGTGD (16)
DLDLPGVND EVDAHVNLD GSSMSFQDT VLRDFLDTD
DLQAQWAGD (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)

41. Sequencing Results (Families)
(A)GS(Y) (E/N)GG(N) GGH NVD (W)TGD YSE
AGSNNEGMT 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 EWNRSELGD
DGY 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

42. 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

43. 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

44. Binding Assay (Comprehensive)
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
7b1
VADSAVGHD
DLQAQWAGD
VGGHGDDFD
VDADRFSGD
VSEPTSNES
DHGGGGHDS
EPYSGSISN
DYDSLHINS
DARQGVMME
DGSGSLDGD
GSYDEVSSA
VNHETLTAD
VTASGMSDD
VDYSEVGSL
EYSQGQEGS
VDTAEISSL
VGSMSDGYN
DFAVGPGSD
EWNRSELGD
DGYRSSEDS
ETSRYSDID
DSSMSWSGD
DTGMLEGGN
DLDLPGVND
GSEQSWTGD
GLNGGNWDD
GFNTEFGDT
ERFQEGGTD
GSSMSFQDT
VAVSEPGMD
VPPDFLDTD
AGSNNEGMT
VGSDYGTGD
GSLLSSQED
AGSYGDMDT
GSQTVMTDD
VDIAEQSTA
GAEYVGDTT
VSTSSDFDP
DFNLHDAMD
GETGGADND
DGIWEHGDS
DMTNGSVPE
EFGGPDYDT
AGLNYNVDQ
VGENGGSAD
GDYTEAVGA
DVPRETGLD
VLRDFLDTD
ESALWGGDS
GYDPVNDYN
%Yield
Phage Clones
Binding Assay Complete
% Yield Target % Yield Serum

45. Binding Assay (Top 15 Yields)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
%Yield
Phage Clones
Binding Assay (Top 15 % Yields)
% Yield Target % Yield Serum

46. Binding Assay (Top Target Specificity *16)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
%Yield
Phage Clones in order of increasing target specificity (Target % yield / Serum % yield)x100
Binding Assay Clones With Target Specificity > 10
% Yield Target % Yield Serum

47. Potential Champion Clones (10)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
EFGGPDYDT AGLNYNVDQ VGENGGSAD GDYTEAVGA DVPRETGLD VLRDFLDTD ESALWGGDS GYDPVNDYN DSVYDEENS
%Yield
Phage Clones
Potential Champion Clones
% Yield Target % Yield Serum

48. Specificity Assay
0.00E+00
5.00E-02
1.00E-01
1.50E-01
2.00E-01
2.50E-01
%Yield
Phage Clones
Specificity Assay
% Yield 231 % Yield MCF7 % Yield MCF10A % Yield Serum

49. 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

50. 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

51. References
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.
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
http://www.virology.wisc.edu/virusworld/ICTV8/1fd-enterobacteria-phage-fd.jpg
http://www.cancer.org/acs/groups/content/@research/documents/document/acspc-042725.pdf
http://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. * Modified
Owens TW and Naylor MJ (2013) Breast cancer stem cells. Front. Physiol. 4:225. doi: 10.3389/fphys.2013.00225
Prat, 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): 9-29
Wang, 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.