Cadherin-11 (Cad11) for detection of epithelial-mesenchymal transition (EMT) in circulating prostate cancer (PCa) cells.

Abstract

e22047 Background: CTC are believed to require EMT to metastasize. Cells undergoing EMT may lack or express low levels of epithelial markers such as EpCAM and cytokeratins (CK) and experience a cadherin switch from epithelial to mesenchymal. However, most current detection methods only rely on epithelial markers for enrichment, resulting in failure to recognize other CTC phenotypes. We previously showed that PCa cells expressing the mesenchymal (osteoblast-lineage) marker Cad11 are more likely to home to and colonize bone (Cancer Res 2010). Whether Cad11+ PCa CTC lose CK expression is uncertain. Since Cad11 is an EMT marker relevant to the bone tropism of PCa, we characterized its expression in CTC from patients (pts) with bone metastasis. Methods: Blood from PCa pts (n=86) with different extents of bone involvement was analyzed employing Biocept’s microfluidic CTC technology. For cell capture, we used a PCa CTC-optimized cocktail of antibodies that included epithelial, mesenchymal and putative stem cell markers. Captured cells were scored based on standard stain criteria (CK+/CD45−/DAPI+) and Cad11 expression. To ascertain malignant identity of CK−/CD45−/DAPI+cells, we used FISH to characterize androgen receptor [AR], Myc and PTEN status. Results: Among 69 pts with detectable CK+ CTC, 39% had at least 1 cell co-expressing Cad11. Very rare CK−Cad11− CTC displaying different degrees of aneuploidy were also observed, but only in 4 pts. No single CK−Cad11+ FISH “abnormal” cell was seen. FISH profiling of individual CK+ CTC revealed an association between Myc gain and PTEN loss (P=0.00004). We also found positive correlations between AR and PTEN amplification (r=0.55) as well as AR amplification and Myc loss (r=0.56; both P<10-6). Interestingly, CK−Cad11+cells (range, 1-910) were observed in 45 pts, all with a “normal” pattern of AR, Myc and PTEN expression. Conclusions: Complete loss of epithelial marker expression in CTC (identified by concurrent gene alterations) is a very infrequent event in PCa pts with established bone metastasis. Simultaneous individual CTC profiling of gene gains/losses may provide insights into PCa biology. Evaluation of the identity of CK−Cad11+ cells is ongoing.