Capture and analysis of prostate cancer circulating tumor cells (CTCS) using geometrically enhanced differential immunocapture (GEDI).

Abstract

53 Background: EpCAM-based immunocapture of prostate cancer (PC) CTCs yields relatively low purity and specificity. We developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that incorporates flow dynamics and utilizes a mAb to prostate-specific membrane antigen (PSMA) to optimize isolation and analysis of CTCs from PC patients. Methods: GEDI microfluidic silicon chips, fabricated using standard photolithography techniques, were functionalized by chemical cross-linking ending with a neutravidin terminated surface to which anti-PSMA biotinylated-mAb J591 was bound. C4-2 (PSMA+) and PC-3 (PSMA-) cells were used for chip optimization. 1 mL of peripheral blood from PC patients was flowed (1mL/hour) over functionalized chips. Captured cells were washed with PBS × 30 min, fixed with 3.7% formaldehyde, immunofluorescently stained for DAPI, androgen receptor (AR), tubulin and EpCAM and analyzed by high resolution point-scanning confocal microscopy. PSMA+, DAPI+, and CD45- cells were manually scored. RNA was extracted from unfixed captured CTCs using lysis buffer flowed thru the chip. Results: ∼80% capture efficiency was achieved from 26 PSMA positive C4-2 cells spiked into 1 mL blood flowed through the GEDI chip. RNA extracted from 50 C4-2 cells in 1 mL blood flowed thru the chip detected a known AR point mutation by RT-PCR and Sanger sequencing. Immunofluorescence staining of PSMA+ cells captured on the chip detected changes in AR subcellular localization and microtubule structure following treatment with DHT or paclitaxel, respectively. 10 patients with metastatic PC were analyzed by CellSearch (range 0-201 cells/7.5 mL) and GEDI chip (range 35->1200 cells/mL) yielding a 7->350 fold enrichment using GEDI. Captured PC cells isolated from PC patient incubated in 50 nm paclitaxel (ex vivo) overnight demonstrated microtubule bundling, indicative of drug-target engagement. Conclusions: PSMA based GEDI microfluidic CTC capture is highly specific and sensitive in capturing PSMA positive PC CTCs; and allows detailed CTC analysis including protein expression and subcellular localization, mutational analysis and drug sensitivity assessment. [Table: see text]