2014 CFTCC Annual Symposium Keynote Speaker Brian Kirby, PhD

1. http://www.kirbyresearch.com
Circulating Tumor Cells:
linking analysis to surgical and medical care
100 nM PTX
Brian J. Kirby, PhD
Sibley School of Mechanical and
Aerospace Engineering, Cornell
University, Ithaca
Department of Medicine, Division of
Hematology and Medical Oncology,
Weill Cornell Medical College, NYC

2. Acknowledgements
Kirbylab Group Members:
• Nitya Arasanipalai
• Alex Barbati
• Mike Bono
• Jason Gleghorn PhD
• Charlie Huang
• Tim Lannin
• Erica Pratt
• Steven Santana
• Jim Smith
• Yusef Syed
• Fredrik Thege
Collaborator Group Members:
• He Liu PhD
• Mona Jodari
• Ganjun Gakhar PhD
• Mirjam Blattner
• Asya Stepansky PhD
Collaborators (WCMC):
• David Nanus MD
• Evi Giannakakou PhD
• Neil Bander MD
• Scott Tagawa MD
• Mark Rubin MD
• Linda Vahdat MD
• Maureen Lane PhD
Other Collaborators
• Jim Hicks PhD, Cold Spring
Harbor
• Peter Kuhn PhD, Scripps
• Mitch Gross MD, USC
• Kasia Rezniak PhD, Moffitt
• Andy Rhim MD, UPenn
• Ben Stanger MD PhD, Upenn
• Eric Shaqfeh, Stanford

3. Our group works at the interface of microscale fluid physics,
microdevice technology, and cellular bioanalysis
flow in
microdevices
interface science,
adhesion
forces on
particles, cells
micropatterned
devices
GEDI GEDI-MASTR
DEP-GEDI
DES: dielectric
spectrometer
captured, sorted,
classified cells
CTC-Quant
Jason Gleghorn
Erica Pratt
Jim Smith
Nitya Arasanipalai
Mike Bono
Jim Smith
Nitya ArasanipalaiTim LanninSteven Santana
Charlie Huang
Alex Barbati
Jason Gleghorn
Jim Smith
Alex Barbati
Steven Santana
Fredrik Thege
Alex Jim
Charlie Tim

4. Our group works at the interface of microscale fluid physics,
microdevice technology, and cellular bioanalysis
algae monitoring for
biodiesel
(Mike)
CPCs for early
detection of
pancreatic cancer
(Fredrik)
engineered ex-vivo
metastatic lesions for
personalized chemo
(Steven)
membrane
capacitance as tool
to inform antibiotic
discovery (Nitya)
personalized
detection of taxane
response in prostate
cancer (Yusef)
unwrapping the
genomic evolution
of tumors and
metastases (Erica)
captured, sorted,
classified cells

5. Cancer is the 2nd leading cause of death in the US, yet aspects of
R&D are hindered by tissue accessibility and inability to track
patient status in real-time
• Cancer causes 1 in 4
deaths in the US1
– Over 500k deaths in 2011
• Therapeutic monitoring is
challenging
• Tissue is largely
inaccessible!
Siegel et al. CA 2011
[1] Cancer Facts & Figures 2011
[2] Kling et al., Nature Biotech. 2012 Erica Pratt

6. Late-stage prostate cancer illustrates the inherent limitations in
patient care and the need for real-time
Simplified
patient staging
for PC:
modified from Scher+ JCO 2011
Clinically localized
disease
(radiation/surgery)
Rising PSA
(indication of
metastasis and
disease progression)
initial treatment
hormone therapy
(chemical
castration)
castrate-resistant,
hormone-sensitive:
abiraterone
castrate-resistant,
hormone-independent,
clinical metastases:
docetaxel
docetaxel fail:
cabazitaxel,
abiraterone
(1)
(2) (3)
(4)

7. Capture of circulating tumor cells enables assessment of
patient metastatic state and chemotherapeutic response
• Circulating Tumor Cells
(CTCs)
– Found in the blood of metastatic
cancer patients1,2
• Approximately 1 CTC per
billion blood cells
• CTCs have great clinical
value
– Liquid biopsy
– Genetic information3
– Prognostic4,5
[1] Allard+, Clin Canc Res 2004
[2] Danila+,Clin Canc Res 2007
[3] Scher+, Lancet Onc 2009
[4] Paris+, Cancer Lett 2009
[5] Okegawa+, J of Urology 2009
[6] Hayes+ CCR 2006
National Cancer Institute
CTC
Erica Pratt
Time (Ref[6])
Fractionalsurvival

8. A variety of methods have been used for the isolation
of CTCs and model CTCs
• Several methods of CTC isolation
exist
– Filtration1,2,3
– Nano-features4,5
– Streamline focusing6
– Electrokinetic7-12
• Immunocapture techniques have
shown the greatest potential13
– Antibodies specific to cancer cells
Nagrath et al,
Nature 2007
Zheng et al, J.
Chromatogr. A 2007
Hughes et al,
Langmuir 2010
[1] Zheng et al., J. Chromatogr. A 2007
[2] Hosokawa et al., Anal Chem 2010
[3] Chen, Du et al., J Micromech Microeng 2006
[4] Hughes et al., Langmuir 2010
[5] Wang et al., Angew. Chem. Int. Ed. 2011
[6] Zheng et al., J Micromech Systems 2008
[7] Tai et al., Biomed Microdev 2007
[8] Becker et al., PNAS 1995
[9] Yang et al., Anal Chem 1999
[10] Gascoyne et al., Electrophoresis 2009
[11] Kim et al., PNAS 2007
[12] Shafiee et al., LOC 2010
[13] Pratt*, Huang* et al., Chem Eng Sci 2010
Erica Pratt

9. The GEDI technique leverages fluid mechanical design to
maximize immunocapture interactions of target cells
[1] Zheng et al., J Chromatogr A, 2007
• Geometrically Enhanced
Differential Immunocapture
(GEDI) technique creates
size-dependent cell-
antibody interactions
• High-avidity antibodies
• Fluid transport drives cell
trajectories based on size
• CTC 12-25 mm1
• Blood cell 5-15 mm1
• Gleghorn+ Lab Chip 2010, Smith+
BMMD 2013, Smith+
Electrophoresis 2013
J591-terminated
50 mm
Flow
Jason Gleghorn, Steven Santana, Jim Smith, Erica Pratt

10. We aim to facilitate personalized treatment via capture
and analysis of rare cells
Patient
Sample
CTC Capture
• our unique contribution: viable CTCs on chip
• our current clinical domains: castrate-resistant prostate
cancer; precancerous pancreas disease
Functional
Response
Genomic
Characterization
/CD45+
CK+/CD45+
CK-/CD45+
CK+/CD45-
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK+/CD45-
CK-/CD45+
Enumeration

11. I have divided this talk into three sections
CTC capture
100 nM PTX
Functional
assays in CRPC
captured
cells
Circulating cell capture in
pancreas
CTCspermL

12. Our GEDI microdevice is a micropatterned silicon chip
designed to capture rare cells from whole blood
• One-step operation
for multi-institutional
trials and unskilled
operators
• 1 mL of whole blood
is injected into the
device over 1 hour
• Gleghorn+ Lab Chip
2010, Kirby+ PLoS
ONE 2012
Si
microdevice
blood input
blood output
silicone
gasket
captured
cells
Jason Gleghorn, Erica Pratt, Steven Santana

13. Jason Gleghorn, Jim Smith
Our approach (GEDI) combines immunocapture with
size-dependent particle trajectories and collision rates
• GEDI technique
combines size and
surface specificity to
exploit differences in
size between CTCs and
leukocytes
• Computational models
allow us to tune device
geometry to maximize
CTC collisions while
eliminating leukocyte
collisions
• Obstacle shape and
array geometry (not
surface area) determine
collision frequency—
distortion of streamlines
is critical
A: 25 um
B: 12 um
C: 8 um
D: <1 um
AB
C
D
collisionrate
cell size

14. Collisions change the distribution of particle
locations—the GEDI device uses this to advantage
FLOW
Jim Smith, Jason
collisionrate
offset

15. The collisions particles experience in an obstacle array
are a strong function of the row offsets
• red: non-reversing array; blue: reversing array
• collisions: not
reported to date
• displacement: cf.
Frechette and
Drazer PRL 2009

16. Collision rates as a function of radius illustrate the size
dependence of the capture device
• red: non-reversing array; blue: reversing array
• collision
dependence on
radius is the
central principle
of GEDI
• displacement: cf.
Huang+ Science
2004, Frechette+
PRL 2009, JFM
2009

17. Predicted and measured collision dynamics in GEDI
devices identify sharp cutoffs to create size specificity
• we maximize CTC-
obstacle collisions
and minimize
nonspecific
interactions
• design space is also
informed by system
requirements
(footprint, clogging,
robustness of design)
• experiments have
validated the
simulated collision
rates
Column
Spacing
Row
Spacing
Offset
Cell
Diameter
Obstacle
Diameter
Flow
GEDIdesignspace
Jim Smith

18. Captured CTCs in castrate-resistant prostate cancer
have been used to functionally evaluate drug efficacy
CTC capture
100 nM PTX
Functional
assays in CRPC
captured
cells
Circulating cell capture in
pancreas
CTCspermL

19. Stages (3) and (4) hinge on drug-target engagement,
i.e., does the drug do what it is supposed to
modified from Scher+ JCO 2011
Clinically
localized disease
(radiation/surgery
)
Rising PSA
(indication of
metastasis and
disease progression)
initial treatment
hormone therapy
(chemical
castration)
castrate-resistant,
hormone-sensitive:
abiraterone
castrate-resistant,
hormone-independent,
clinical metastases:
docetaxel
docetaxel fail:
cabazitaxel,
abiraterone
(1)
(2) (3)
(4)
• We need access to
tissue to ask
questions about
drug efficacy!
• characterize GEDI
capture
performance and
functional assay

20. Capture efficiency was characterized by spiking a
prostate tumor cell line into PBS or patient blood
• 150-220 LNCaP cells/mL
spiked in healthy whole
blood or PBS+1% BSA
Labeled
Cells
Cell type #/mL # captured
LNCaP 200 170
Blood 5 x 109 91
dotted lines: [Nagrath et al, Nature, 2007]
Jason Gleghorn
Gleghorn+ LOC 2010

21. PCTCs were isolated from healthy volunteer and CRPC
patient samples using the GEDI mdevice
p<0.001
(Wilcoxon)
med=3 med=54
Erica Pratt, Mona Jodari, Matt Loftus
intact morphology+
CD45-DAPI+PSMA+
n=45
Kirby+ PLoS ONE 2012
Kirby et al., PLoS ONE 2012

22. Functional assay: taxanes should induce tubulin
bundling and impair AR nuclear accumulation
testosterone testosterone
plus taxane
• immunostain for
tubulin and androgen
receptor
nuclear AR
accumulation
cytoplasmic
sequestration
perinuclear
bundling10 μm10 μm
Matt Loftus, Medha Darshan, Evi Giannakakou
Darshan+ Canc Res 2011
Kirby+ PLoS ONE 2012
100 nM PTX

23. castrate-resistant,
hormone-independent,
clinical metastases:
docetaxel
docetaxel fail:
cabazitaxel,
abiraterone
The TAXYNERGY trial will determine the efficacy of switchover
between drugs based on clinical and microfluidic indicators
modified from Scher+ JCO 2011
Clinically localized
disease
(radiation/surgery)
Rising PSA
(indication of
metastasis and
disease progression)
initial treatment
hormone therapy
(chemical
castration)
castrate-resistant,
hormone-sensitive:
abiraterone
(1)
(2) (3)
(4)

24. TAXYNERGY: Phase II Trial to Evaluate Benefit of Early Switch from first-
Line Docetaxel/Prednisone to Cabazitaxel/Prednisone and the opposite
sequence, exploring molecular markers and mechanisms of taxane
resistance in men with metastatic CRPC who have not received prior
chemotherapy
R
A
N
D
O
M
I
Z
E
2:1
100 men with
Chemotherapy
naïve metastatic
CRPC
Docetaxel
75 mg/m2
Cabazitaxel
25 mg/m2
4 cycles
>30% PSA
reduction
<30% PSA
reduction
>30% PSA
reduction
<30% PSA
reduction
Docetaxel
Continued
Cabazitaxel
Continued
Switch to
Cabazitaxel
Switch to
Docetaxel
Primary Clinical Endpoint: Explore benefit of an early switch from DTX to CBZ
Evaluate the association of biomarkers with clinical response/resistance to Rx
- Molecular Drug Target Engagement (DTE) in CTCs
- RNA sequencing on CTCs to assess for tubulin mutations and AR isoforms

25. Correlative studies in TAXYNERGY focus on CTC
biomarkers
R
A
N
D
O
M
I
Z
E
2:1
100 men with
Chemotherapy
naïve metastatic
CRPC
Docetaxel
75 mg/m2
Cabazitaxel
25 mg/m2
4 cycles
>30% PSA
reduction
<30% PSA
reduction
>30% PSA
reduction
<30% PSA
reduction
Docetaxel
Continued
Cabazitaxel
Continued
Switch to
Cabazitaxel
Switch to
Docetaxel
GEDI CTCs
Screening
Baseline
C1D1
Initial Rx
C1D8
C5D1
C5D8
Progression
TAXYNERGY at http://clinicaltrials.gov/

26. Correlative studies in TAXYNERGY focus on CTC
biomarkers
R
A
N
D
O
M
I
Z
E
2:1
100 men with
Chemotherapy
naïve metastatic
CRPC
Docetaxel
75 mg/m2
Cabazitaxel
25 mg/m2
4 cycles
>30% PSA
reduction
<30% PSA
reduction
>30% PSA
reduction
<30% PSA
reduction
Docetaxel
Continued
Cabazitaxel
Continued
Switch to
Cabazitaxel
Switch to
Docetaxel
GEDI CTCs
TAXYNERGY at http://clinicaltrials.gov/
1700 analyses total; 700
completed

27. We are currently combining the GEDI technique with
SNS to determine genetic provenance of CTCs
CTC cell membrane lysis
Elution of CTC nuclei
Patient sample GEDI CTC capture
SNS of CTC nucleiPhylogenetic Tree
Erica Pratt

28. Capture and release of cancer cells allows
sophisticated genetic analysis
• Single-nucleus
sequencing of
captured and
released nuclei
(Navin+ Nature 2011)
allows
characterization of
amplifications and
deletions in nuclei
of captured cells
Erica Pratt, Asya Stepansky, Jim Hic
amplifications
deletions
Pristine genome (male)
Bin number
Copynumber

29. Capture and release of cancer cells allows
sophisticated genetic analysis
• Single-nucleus
sequencing of
captured and
released nuclei
(Navin+ Nature 2011)
allows
characterization of
amplifications and
deletions in nuclei
of captured cells
Erica Pratt, Asya Stepansky, Jim Hic
amplifications
deletions
Healthy genome (male)
Bin number
Copynumber

30. Capture and release of cancer cells allows
sophisticated genetic analysis
• Single-nucleus
sequencing of
captured and
released nuclei
(Navin+ Nature 2011)
allows
characterization of
amplifications and
deletions in nuclei
of captured cells
• Cancer cells show
dramatic genetic
instability
Erica Pratt, Asya Stepansky, Jim Hic
amplifications
deletions
Single cancer cell nucleus
Bin number
Copynumber

31. Capture and release of single cancer cells allows
sophisticated genetic analysis
• Single-nucleus sequencing of captured and released nuclei
(Navin+ Nature 2011) allows characterization of amplifications
and deletions in nuclei of captured cells
Erica Pratt, Asya Stepansky, Jim HicksPratt+ Lab Chip, submitted, 2014

32. Circulating cells are important in early-stage disease
as well as late-stage disease
CTC capture
100 nM PTX
Functional
assays in CRPC
captured
cells
Circulating cell capture in
pancreas
CTCspermL

33. Pancreatic cancer has no early clinical detection
strategy
• 5-year survival rate is 6%
• Detected disease is almost
always metastatic
• The majority of pancreatic
cancer is detected
presymptomatically but
clinically too late
Hopkins-Turner+ 2004

34. Circulating cells can be detected in precancerous
pancreatic cyst patients
• Circulating cells can
be captured in
precancerous
pancreas patients
• Cell counts are
highest in pancreatic
adenocarcinoma,
zero in normals
• Points to the
potential in pancreas
to detect circulating
cells BEFORE cancer
tumors form
Fredrik Thege, Steven Santana, Andrew RhimRhim+ Gastroenterology 2014

35. We have shown that CTC Capture in GEDI microdevices allows for
genetic and functional characterization
Patient
Sample
CTC Capture
Functional
Response
Genomic
Characterization
Enumeration 100 nM PTX
CTCspermL
Functional
Response
/CD45+
CK+/CD45+
CK-/CD45+
CK+/CD45-
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK+/CD45-
CK-/CD45+
Enumeration

36. 0 2000 4000
-1000
0
1000
2000
3000
CD45
CK
Fluorescent imaging
Image gallery from chip
Patient sample GEDI CTC capture
Automated image
interpretation
Cell counts,
charateristics,
categories
Lannin+ Cytometry A 2014
Ongoing work is focused both on new technologies
and pressing clinical/translational questions
-4 -2 0 2 4 6 8
-4
-2
0
2
4
6
8
Theoretical Normalized Quantiles
NormalizedQuantiles
CK-/CD45+
CK-/CD45+
CK+/CD45-
CK-/CD45+
CK-/CD45+
CK+/CD45+
CK-/CD45+
CK+/CD45-
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK-/CD45+
CK+/CD45-
CK-/CD45+

37. Ongoing work is focused both on new technologies
and pressing clinical/translational questions
Flow
FDEP Electric
field
Side view
Electrodes
Flow
V+
V-
Top view
250
mm
Flow
50 mm
Huang+ BMMD 2013, Huang+ Electrophoresis 2013, Huang+ Biomicrofluidics 2014

38. Ongoing work is focused both on new technologies
and pressing clinical/translational questions
Santana Phys. Biol 2014
• hydrogel GEDI devices (the GEDI-
MASTR) can capture cells in a
tissue-engineering framework
• We hope to engineer “personalized
metastases” to allow direct inquiry
into the interaction between
genomics, functional response,
and metastatic niche
10 mm
100 µm
Alginate CaCl2

39. http://www.kirbyresearch.com
Circulating Tumor Cells:
linking analysis to surgical and medical care
100 nM PTX
Brian J. Kirby, PhD
Sibley School of Mechanical and
Aerospace Engineering, Cornell
University, Ithaca
Department of Medicine, Division of
Hematology and Medical Oncology,
Weill Cornell Medical College, NYC

40. The conclusion is that geometric choices have
enormous impact on capture efficiency and purity
• GEDI technique
combines size and
surface specificity to
exploit differences in
size between CTCs
and leukocytes
A: 25 um
B: 12 um
C: 8 um
D: <1 um
collisionrate
offset
captureefficiency
LNCaP capture
Jason Gleghorn, Erica Pratt, Jim Smith

41. PSMA-captured cells can show low EpCAM levels,
indicating epithelial-to-mesenchymal transition
• 210 PSMA+ CTCs
extracted from 1 mL of
peripheral blood of a
CRPC patient by use of a
GEDI microdevice
• immunostain CTCs for
PSMA & DAPI (CD45- not
shown)
• immunostain CTCs for
vimentin or EpCAM;
compare with cell line
controls, below
Vimentin-H
PC3:
Vimentin-
H
EpCAMVimentin-L
C4-2:
Vimentin -
L
C4-2: EpCAM
PSMA PSMA PSMA
120 /210
(57%)
90/210
(43%)
0/210
(0%)
• arrows: PSMA+ cells with high (vimentin-H) or low
(vimentin-L) scores; EpCAM not detected in cells from this
patient
Matt Loftus

42. GEDI mdevice capture allows for molecular and
functional assays of rare CTCs
Mona Jodari, Ganjun Gakhar, Matt Loftus, Erica Pratt
• Results show (1)
functional assay of drug-
target engagement (2)
detection of SNPs in
spiked cells and (3)
fusion in CTCs
healthy
blood
C4-2
culture
50C4-2cells
C A T C A G T T C A C T T T T G A C C T
C A T C A G T T C G C T T T T G A C C T
C A T C A G T T C G C T T T T G A C C T
ERG MergeDAPI CD45
ERG MergeDAPI CD45
DAPI CD45 CK18 Tubulin Merge
DAPI CD45 CK18 Tubulin Merge
DTX100nMPTX100nM

43. Ongoing work includes design of release mechanisms
and downstream assays
• We are characterizing the
shear dependence of cell
adhesion with different linkers
and demonstrating that rare
cells can be captured and then
released
Incubation/release experiment
Flow/release experiment
Jason Gleghorn
Erica PrattErica Pratt, Mija Blattner
Novel mutations in captured/released cells