MOLECULAR ANALYSIS OF ANTIGEN PRESENTATION MACHINERY IN CIRCULATING TUMOR CELLS FROM RENAL CELL CARCINOMA AND PROSTATE CANCER.

Stephanie Thiede, Jacob Tokar, Benjamin Casavant, Lindsay Strotman, Jamie Sperger, David J. Beebe, Joshua M. Lang University of Wisconsin Carbone Cancer Center, University of Wisconsin Dept. of Biomedical Engineering

Clinical DataBlood from patients with metastatic prostate cancer or RCC were were drawn under a UW-IRB approved protocol and their CTCs were stained and analyzed for intact nuclei (hoechst), cytokeratins, CD45 and HLA expression. Below is the total number of CTCs identi�ed and those expressing HLA.

Conclusions and Future Work

The VERSA:

Isolation:

bead-boundcells of interest

Non-target cells

Magnet

2) Magnet pull1) Add PMPs

Vertical Exclusion BasedRare 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 ‘virtual wall’ at the interface.

virtualwall

Aqueous phases(cell suspensions, stains, washes)

Oil phases

INTRODUCTION AND OBJECTIVES: Tumor cells develop many mechanisms by which to avoid detection and destruction by the immune system, namely down-regulation or silencing of genes critical for antigen expression, processing and presentation. These mechanisms of evasion have been identi�ed in nearly all tumor types, including prostate cancer and renal cell carcinoma (RCC). However, there is a lack of available biomarkers to identify patients with advanced cancer. A relatively new area of interest is the use of circulating tumor cells (CTCs) as an accessible source of tumor cells for molecular analysis. These cells could provide us with more information on HLA expression across disease types and even across patients. However, interrogation of these cells with current techologies is limited. 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. After isolation, using this same device, we are able to stain and image proteins of interest and extract nucleic acids for gene ex-pression analysis. METHODS: The VERSA platform was designed using the relative dominance of surface tension in the microscale to create virtual walls between oil and aqueous phases �ltering contaminants in a single step, while maintaining cell viability for further analysis. In previous experiments, the isolation of CTCs in patients with prostate cancer was optimized using a known prostate cancer cell line (LnCAP) spiked into whole blood and captured with EpCAM. To optimize the isolation of CTCs in patients with RCC, samples of whole blood were spiked with known RCC cell lines (786-0 and 769-P) and captured with Carbonic Anhydrase IX (CAIX). Pa-tient CTCs were then isolated to interrogate for HLA Expression and tumor asso-ciated antigen expression.

RESULTS: Initial results investigating isolation of CTCs in patients with RCC show a capture e�ciency of about 50-70% when spiking known cell lines into blood. RCC and prostate CTCs were successfully isolated from 7.5mL of blood and ca-pable of being stained both extracellularly with HLA-ABC (W6/32 antibody) and intracellularly with cytokeratin. Nucleic acids extracted from these patient samples were su�cient enough to detect tumor speci�c antigens of interest in-cluding PAP, AR and SSX2.

Abstract

Sieve-Assisted StainingA microporous membrane is fabricated into one of the VERSA wells such that �uid can be added and replaced from an adjacent well without touching the sample, critical for rare or delicate cell samples and enabling sophisticated �u-idic procedures in-device.

MembraneAspirate

Add Fluid (Wash, Fixative, etc.)

Incubate

Repe

at a

s ne

cess

ary

Top View Side View

Fluid Exchanges

RearWell

FrontWell

Magnet

PMP Removal

0.50.60.70.80.9

11.11.21.3

1 2 3 4 5Wash Number

Nor

mal

ized

Num

ber o

f Cel

ls

Loss Due to Washing

Vertical Cell IsolationVertical 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

MagnetOil

Traverses

Wash Output

0

100

200

300

400

500

600

700

800

900

Num

ber o

f Cel

ls

PBMCLNCaP

Purity

Traverse Number1 2 3

77% 82%86%

1 2 3

VERS

A

Device Side-ViewForce

Vectors Cell Data

This allows us to capture a few cells from a large background:

0

20

40

60

80

100

5M 20M 100M

Perc

ent R

ecov

ery

Background PBMCs (M = million)

One cell in 20M PBMCs

target well

Variable PBMCs

PMPs were bound to an antibody speci�c for CAIX or EpCAM. The immortalized RCC lines, 786-O and 769-P, were incubated with CAIX-PMPs with and without EpCAM-PMPs in the VERSA chip, then captured as above. The best capture e�-ciency in both cell lines occured when using CAIX-PMPs alone.

Total Nucleic Acid Extraction

Patient blood samples were processed using the VERSA and mRNA extracted for gene expres-sion analysis of a housekeeping gene, P0, and tumor speci�c antigens, AR, PAP and SSX2.

AcknowledgementsThis work was supported by a Movember-Prostate Cancer Foundation Challenge award and a PCF Young Ivestigator Award to Dr. Lang, grants from the DOD PCRP Physician Research Training Award W81XWH-09-1-0192, Wisconsin Partnership Program, UWCCC Investigator Initiated Pilot.

Tumor Associated Antigen Expression in Prostatic CTCs

Patient SampleR

elat

ive

Exp

ress

ion

0.00

0.01

0.02

0.03

0.040.10.20.30.40.50.60.70.8

AR 1/2

PAP

SSX2

43 71 98 99 100 159 165124

After imaging, cells are lysed and PMPs bound to an oligo-dT are added to capture mRNA. The PMP-mRNA conjugates is magnetically are puri�ed into the �nal rear well of the chip. A high salt bu�er is then added to lyse nuclei. Silica beads are then added to bind DNA and the PMP-DNA conjugates are puri�ed into the �nal front well. Nucleic acids are eluted from PMPs in a 20uL volume for qPCR or sequencing.

Input

Live CellStaining

IntracellularStaining

DNAPurification

mRNAPurification

1

10

100

1,000

10,000

100,000

1000 100 10 1

Rel

ativ

e D

NA

Sign

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Number of Cells

Qiagen AllPrep VERSA

1

10

100

1,000

10,000

100,000

1000 100 10 1

Rel

ativ

e R

NA

Sig

nal

Number of Cells

Qiagen AllPrep VERSA

Direct DNA Sequencing of 10 LNCaP cells Direct RNA Sequencing of 10 LNCaP cellsC) D)

A) B)qPCR for DNA on Low Cell Numbers qRT PCR for mRNA on Low Cell Numbers

Circulating tumor cells can be assayed for expression of molecular machinery critical to immune recognition and antigen expression.

Signi�cant heterogeneity exists in CTC populations with regards to MHC expression that may have potential as a predictive and pharmacodynamic biomarker for immunotherapies.

Tumor associated antigen expression can be identi�ed in CTCs as a potential predictive marker for vaccine based immunotherapies.

Prospective clinical trials will be needed to validate the utility of these assays.

Prostate Patient #

Age Pathology Sites of Disease Treatment History Total CTC Number (EpCAM+/CK+/CD45-)

HLA+ CTCs

103 74 Gleason 4+5

Bone Mets Docetaxel, Enzalutamide 30 4

79 64 Gleason 4+4

Lymph Node and Bone Mets

PAP Vaccine, Provenge, Docetaxel, Abiraterone

10 7

84 58 Gleason 4+4

Bone Mets Docetaxel, TAK700, Enzalutamide

58 24

71 64 Gleason 3+3

Lymph Node and Bone Mets

Docetaxel, TAK700, Axitinib, Enzalutamide

469 171

Renal Patient #

Age Pathology Sites of Disease Treatment History Total CTC Number (CAIX+/CK+/CD45-)

HLA+ CTCs

143 73 Clear Cell Carcinoma

Pancreatic Mass Tivozanib 4 4

111 69 Clear Cell Carcinoma

Nephrectomy Bed Sunitinib, Bevacizumab 14 14

142 74 Clear Cell Carcinoma

Bone Mets Tivozanib, Everolimus 7 7

145 76 Clear Cell Carcinoma

Lymph Node Mets

Axitinib, Everolimus, Pazopanib

46 40

Hoescht Cytokeratin

CD45

Hoescht Cytokeratin

CD45HLA-ABC

Merged

HLA-ABC

Merged

Pt 84 CTCs

Hoescht Cytokeratin

CD45HLA-ABC

Merged

Pt 145 CTCs Hoescht

HLA-ABC

Merged

CD45

Cytokeratin

Merged

Hoescht

Hoescht Cytokeratin

HLA-ABC CD45

Merged