Functional characterization of circulating tumor cells with a prostate-cancer-specific microfluidic device.

Brian J Kirby,Vicente N Navarro,Gunjan Gakhar,James P Smith,Neil H Bander,Paraskevi Giannakakou,Chantal Chanel-Vos,He Liu,Jason P Gleghorn,Steven M Santana,David M Nanus,Scott T Tagawa,Mona Jodari,Matthew S Loftus,Erica D Pratt,Natasha Kyprianou

doi:10.1371/journal.pone.0035976

Abstract

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

Highlights

The development of metastases in patients with solid tumor malignancies is believed to result from tumor cells entering the circulatory system and migrating to distant organs, where they extravasate and multiply [1,2,3]
This is attributable to the lack of readily available tumor tissue that can be obtained from patients over the course of their disease and analyzed for molecular signatures and functional response
The identification of circulating tumor cells in the peripheral blood of metastatic patients and the development of specialized CTCcapture technologies has provided, for the first time, investigators, with tumor tissue in the form of a ‘‘liquid biopsy’’ that can be used to study the process of metastasis and aid in the development of effective therapies [26]

Summary

Introduction

The development of metastases in patients with solid tumor malignancies is believed to result from tumor cells entering the circulatory system and migrating to distant organs, where they extravasate and multiply [1,2,3]. A variety of technologies has been developed to improve the detection and capture of CTCs from the peripheral blood. These include density gradient centrifugation, immunomagnetic bead separation using monoclonal antibodies targeting epithelial cell-surface antigens, cell sorting using flow cytometry, filtration based size separation [6] and microfluidic devices. The epithelial celladhesion molecule (EpCAM), represents the antigen of choice for the majority of microfluidic devices that have been developed to capture circulating tumor cells [7,8,9,10]

Methods

Results

Conclusion