Combined microfluidic enrichment and staining workflow for single-cell analysis of circulating tumor cells in metastatic prostate cancer patients.

Circulating tumor cells (CTCs) are precursors of cancer in the blood and provide an attractive source for dynamic monitoring of disease progression and tumor heterogeneity. However, the scarcity of CTCs in the bloodstream has limited their use in clinical practice. In this study, we present a workflow for easy detection of CTCs by cytokeratin staining using the FDA-cleared Parsortix device for size-based microfluidic enrichment. To minimize sample handling, the isolated cells are stained inside the separation cassette and harvested for subsequent single cell isolation and whole genome copy-number analysis. We validated the workflow on a panel of four prostate cancer cell lines spiked into healthy donor blood collected in CellRescue or EDTA tubes, resulting in mean recoveries of 42% (16-69%). Furthermore, we evaluated the clinical utility in a cohort of 12 metastatic prostate cancer patients and found CTCs in 67% of patients ranging from 0 to 1172 CTCs in 10 mL blood. Additionally, we isolated single patient-derived CTCs and identified genomic aberrations associated with treatment response and clinical outcome. Thus, this workflow provides a readily scalable strategy for analysis of single CTCs, applicable for use in monitoring studies to identify genomic variations important for guiding clinical therapy decision.