Functional Characterization of Circulating Tumor Cells with a Prostate-Cancer-Specific Microfluidic Device

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.

Circulating tumor cell technologies

“…Many affinity-based enrichment technologies use epithelial markers, which are down-regulated during EMT, to capture CTCs. In response, researchers have expanded the antigen repertoire used in affinity-based capture to include stem cell markers (e.g., CD133), mesenchymal markers (e.g., cellsurface vimentin, CSV) (Satelli et al, 2014), and cancerspecific antigens (e.g., HER2, PSMA) (Galletti et al, 2014;Kirby et al, 2012;Winer-Jones et al, 2014 Another commercially available platform for CTC detection is AdnaTest (Adnagen AG). AdnaTest enriches CTCs through the use of magnetic, antibody-coated beads.…”

Automated electrorotation shows electrokinetic separation of pancreatic cancer cells is robust to acquired chemotherapy resistance, serum starvation, and EMT

“…1 These cells originate from tumors and a subpopulation has been shown to be pre-cursors to metastasis. 2 In addition to this putative role, it is well known that these circulating tumor cells correlate with disease progression, 3 that they can be captured even in early disease, 4 and that they can be used to gauge functional cell responses to pharmaceuticals 5 or the genetic character of the cancer. 6 This information from captured CTCs is valuable to both clinicians and researchers particularly in the context of pancreatic cancer, in which the presence of circulating pancreatic cells precedes a) Author to whom correspondence should be addressed.…”

Enhancing sensitivity and specificity in rare cell capture microdevices with dielectrophoresis

“…2 There is a need for devices that can capture CTCs and other rare cells at high purity (i.e., high specificity; capturing the target rare cells while minimizing the capture of contaminating cells) to facilitate subsequent downstream analyses. This is particularly true for single-cell genetic analyses, such as gene expression [3][4][5][6] and copy number analysis studies; 7-9 a higher sample purity reduces the time and cost required by reducing the number of single-cell analyses performed on contaminating cells.…”

“…Microfluidic devices have demonstrated impressive advances in the high efficiency isolation of CTCs and other rare cells using immunocapture (i.e., operating with high sensitivity; capturing most of the target rare cell population), 6,10-13 but researchers often report low purity. We have previously shown that the immunocapture can be combined with dielectrophoresis (DEP) to achieve a synergistic effect in a Hele-Shaw characterization microdevice, increase the capture of pancreatic cancer cells while decreasing the capture of contaminating cells.…”

“…18 Separately, these obstacle arrays have been used to generate size-dependent collision dynamics, bringing target cells into contact with the antibodies on the obstacle surface, while displacing contaminating cells onto trajectories with infrequent cell-obstacle collisions; this technique has been termed geometrically enhanced differential immunocapture (GEDI). 6,11,27 This work focuses on combining differential DEP response in obstacle arrays with immunocapture, so as to capture target cells on the obstacle surfaces using antibody-antigen binding. Although the ideal DEP-obstacle array system would generate a uniformly high electric field around the obstacles, attracting cells experiencing pDEP to the obstacle capture surface, such a device would require electrodes on the side of each obstacle and would be difficult to fabricate using conventional techniques.…”