1. ISOLATION AND ANALYSIS OF CIRCULATING TUMOR CELLS IN RENAL CELL CARCINOMA AS ENABLED BY THE VERSA.
E. Jason Abel, Jacob J. Tokar, Benjamin P. Casavant, Stephanie Thiede, Lindsay Strotman, David J. Beebe, Joshua M. Lang
University of Wisconsin Carbone Cancer Center
Acknowledgements
Process
Blood Collection Ficoll-Paque
Density Centrifugation
VERSA Isolation,
Staining, and Imaging
Clinical Data
Blood from patients with metastatic clear cell carcinoma of the
kidney were were drawn with a UW-IRB approved protocol and
their CTCs were stained and analyzed for intact nuclei (DAPI),
Carbonic Anhydrase IX and Cytokeratins. Single cells and cell
clumps were identified from patient samples.
Conclusions and Future Work
Isolation and purification of RCC cells from peripheral blood
using CAIX is possible using the VERSA platform.
Ongoing studies will be aimed at identifying CTC heterogeneity,
evaluation of DNA and RNA isolated from CTCs to provide evi-
dence of their biologic potential in the metastatic cascade.
Future studies will also investigate the potential of CTCs for
prognostic indicators beyond methods of enumeration.
Adhesion Invasion Metastases
This work was supported by a grant from the Wisconsing Partnership
Program and the DOD PCRP W81XWH-09-1-0192
Background
Metastases, not primary tumors, are the most common cause for cancer pa-
tient mortality. Circulating tumor cells are cells within the metastatic cas-
cade that are accessible by a simple blood draw, and can act as a `liquid
biopsy’for understanding tumor progression. Their enumeration has shown
prognostic relevance, however methods to integrate analyses and take
CTCs beyond enumeration have been limited.
Intravasation and Transit
Adhesion/Arrest Extravasation
CTCs
Abbreviations:
PMP - Paramagnetic ParticleCTC - Circulating Tumor Cell
PBMC - Peripheral Blood Mononuclear Cell
The VERSA:
Isolation:
bead-bound
cells of interest
Non-target cells
Magnet
2) Magnet pull
1) Add PMPs
Vertical Exclusion Based
Rare Sample Analysis
Principle:
Two aqueous solutions can be placed in adjacent
wells, and due to the relative dominance of surface
tension on the microscale, stay pinned, creating a‘vir-
tual wall’at the interface.
virtual
wall
Aqueous phases
(cell suspensions, stains, washes)
Oil phases
INTRODUCTION AND OBJECTIVES: Circulating tumor cells (CTCs) have dem-
onstrated prognostic ability in several cancers but similar studies in Renal
Cell Carcinoma (RCC) are limited because the current techniques rely on
positive selection of cells using cell surface markers rarely expressed in RCC
cells. We have designed a novel platform to permit the use of any antibody
of interest bound to paramagnetic particles (PMPs) to isolate and purify
PMP-bound cells via immiscible oil barriers. The purpose of this study was
to demonstrate and validate RCC cell isolation from blood by positive se-
lection of cells expressing carbonic anhydrase nine (CAIX).
METHODS: The Vertical Immiscible Filtration Assisted by Surface Tension
(VerIFAST) platform was designed (BC, DB, patent filed) using the relative
dominance of surface tension in the microscale to create virtual walls be-
tween oil and aqueous phases filtering contaminants in a single step, while
maintaining cell viability for further analysis. Initial experiments used
samples of whole blood spiked with known RCC cell lines (786-0). After op-
timization of technique and confirmation of capture, samples from patients
with locally advanced and metastatic RCC were evaluated.
RESULTS: Initial results demonstrate a high capture efficiency of the spiked
786-0 cells (~75% capture efficiency) and the ability to integrate intracellu-
lar staining, with successful staining of both cytokeratins and the prolifera-
tive marker, Ki-67. Patient CTCs were isolated and stained for intracellular
cytokeratins for initial patient validation.
CONCLUSIONS: Isolation and purification of RCC cells from peripheral
blood using CAIX is possible using this novel platform. Further, the ability
to perform intracellular analyses for therapeutic targets promotes the abil-
ity of the VerIFAST to act as a liquid biopsy, informing clinical decision-
making through analysis of cells directly within the metastatic cascade. On-
ngoing studies with the VerIFAST will be aimed at identifying CTC heterog-
eneity with the targets of cMet and mTOR, realizing the potential of CTCs for
prognostic indicators beyond methods of enumeration
Abstract
Vertical Cell Isolation
Vertical device orientation, featuring the long axis on the vertical instead of
horizontal axis, allows non-target cells to passively settle out of the opera-
tional path of the PMP-bound target cells
FG FM
Input
Magnet
Oil
Traverses
Wash Output
0
100
200
300
400
500
600
700
800
900
NumberofCells
PBMC
LNCaP
Purity
Traverse Number
1 2 3
77% 82%
86%
1 2 3
VERSA Device Side-View
Force
Vectors Cell Data
This allows us to capture a few cells from a large background:
0
20
40
60
80
100
5M 20M 100M
PercentRecovery
Background PBMCs
(M = million)
One cell in 20M PBMCs
target well
Variable PBMCs
PMP were bound to an antibody
specific for Carbonic Anhydrase IX.
The immortalized renal cancer cell
line, 786-O, were incubated with
CAIX-PMPs in the VERSA chip then
captured as above. We show that
capture efficiencies of 60-75% that
is dependent on cell surface expres-
sion of CAIX based on trypsiniza-
tion versus gentle cell dissociation.
Sieve-Assisted Staining
A microporous membrane is fabricated into one of the VerIFAST wells such that
fluid can be added and replaced from an adjacent well without touching the
sample, critical for rare or delicate cell samples and enabling sophisticated flu-
idic procedures in-device.
Membrane
Aspirate
Add Fluid (Wash, Fixative, etc.)
Incubate
Repeatasnecessary
Top View Side View
Fluid Exchanges
Rear
Well
Front
Well
Magnet
PMP Removal
0.5
0.6
0.7
0.8
0.9
1
1.1
1.2
1.3
1 2 3 4 5
Wash Number
Normalized
NumberofCells
Loss Due to Washing
786-O Cells are then fixed, permeabilized, and stained with primary
and secondary antibodies for extra and intracellular proteins
Brightfield KI-67
Cytokeratins DAPI
Merged
MergedMerged
DAPIDAPI
Cytokeratins Total C-MET
CA IX CA IX
Total Nucleic Acid Extraction
After imaging, cells are lysed with a RIPA buffer and PMPs bound to an oligo-dT
are added to capture mRNA. An external magnetic force is applied and the
PMP-mRNA conjugates are purified into the final rear well of the chip. A high
salt buffer is then added to the sieve well to lyse nuclei. Silica beads are then
added to bind DNA. An external magnetic force is applied and the PMP-DNA
conjugates are purified into the final front well of the chip. Nucleic acids are
eluted from PMPs in a 15uL volume and used for quantitative PCR for GAPDH
and compared to a commercial Qiagen kit.
786-O cells were processed by the VERSA and mRNA extracted for gene expres-
sion analysis of a housekeeping gene, P0, HIF1, HIF2, and FIH.
Input
Live Cell
Staining
Intracellular
Staining
DNA
Purification
mRNA
Purification
1
10
100
1,000
10,000
100,000
1000 100 10 1
RelativeDNASignal
Number of Cells
Qiagen AllPrep
VERSA
1
10
100
1,000
10,000
100,000
1000 100 10 1
RelativeRNASignal
Number of Cells
Qiagen AllPrep
VERSA
DAPI
CKs
CA IX
Merged
DAPI
CKs
CA IX
Merged