Single‐cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT‐related genes in metastatic prostate cancer

Abstract

BACKGROUND Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial-mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer. METHODS A microfiltration system was used to trap CTCs from peripheral blood based on size selection of large epithelial-like cells without CD45 leukocyte marker. These cells individually retrieved with a micromanipulator device were assessed for cell membrane physical properties using atomic force microscopy. Additionally, 38 CTCs from eight prostate cancer patients were used to determine expression profiles of 84 EMT-related and reference genes using a microfluidics-based PCR system. RESULTS Increased cell elasticity and membrane smoothness were found in CTCs compared to noncancerous cells, highlighting their potential invasiveness and mobility in the peripheral circulation. Despite heterogeneous expression patterns of individual CTCs, genes that promote mesenchymal transitioning into a more malignant state, including IGF1, IGF2, EGFR, FOXP3, and TGFB3, were commonly observed in these cells. An additional subset of EMT-related genes (e.g., PTPRN2, ALDH1, ESR2, and WNT5A) were expressed in CTCs of castration-resistant cancer, but less frequently in castration-sensitive cancer. CONCLUSIONS The study suggests that an incremental expression of EMT-related genes in CTCs is associated with metastatic castration-resistant cancer. Although CTCs represent a group of highly heterogeneous cells, their unique EMT-related gene signatures provide a new opportunity for personalized treatments with targeted inhibitors in advanced prostate cancer patients.

Gene Expression Profiling of Circulating Tumor Cells in Breast Cancer

“…Whereas some of the studies (23 ) used an EpCAM-based enrichment approach, other studies, mostly dealing with single cells in prostate cancer, relied on complex techniques that are not suitable for translation to the clinic (38 ).…”

Circulating Tumor Cells and Circulating Tumor DNA: Challenges and Opportunities on the Path to Clinical Utility

“…Importantly, CTCs also showed markedly different gene expression profiles compared with those in single cells from breast cancer cell lines, suggesting that CTC analysis might complement cell-line analysis in drug development. Similarly, prostate cancer also appears to be characterized by CTC heterogeneity, with distinct differences in single-cell expression of EMT-related genes between CTCs from castration-sensitive and castrate-resistant cancers (48).…”

Challenges and unanswered questions for the next decade of circulating tumour cell research in lung cancer

“…In contrast, most CTC enrichment technologies require an additional step for CTC visualisation and isolation. This has been achieved in a variety of ways including fluorescenceactivated cell sorting (FACS) (31), CTC visualisation followed by isolation via micromanipulation (21,(32)(33)(34), and the DEPArray™ automated system which incorporates cell manipulation via electrostatic charge (35,36) ( Table 1).…”