314 Background: Cells shed by a tumor into the bloodstream upon metastasis present an opportunity to study the properties of these cells for biomarker discovery, drug development, and personalized therapy. Enrichment and expansion of circulating tumor cells (CTCs) in culture enables molecular characterization not readily available for rare cells. Here, we demonstrate the successful identification and culture of rare circulating tumor cell clusters from peripheral blood of metastatic castration resistant prostate cancer patients (mCRPC). Methods: The approach to isolate CTC clusters relies on nucleated cell enrichment via Ficoll-based centrifugation from 15 mL of peripheral blood. A collagen-based cell-binding substrate was used to capture cell clusters with preferential affinity for collagen (i.e. epithelial-like cells). Hypoxic and pressurized culturing conditions were used to maintain and propagate adhered CTC clusters. After 7 days in culture, immunofluorescence imaging, qPCR and/or mRNAseq were performed on CTC clusters that had formed colonies. Results: Blood samples processed within 3 hours from time of draw yielded a 25% success rate (4/16) for identifying PSMA+/EpCAM+/CD45- CTC colonies. Morphologically distinct CTC colonies cultivated at 1% oxygen and increased hydrostatic pressure (2 PSI) were positive for PSMA and EpCAM staining as observed via confocal microscopy, with individual colonies ranging from approximately 50 to 1000 in cell number. Colonies consistently revealed white blood cell contaminants (T-cells and dendritic cells, 10 to 30% of colony) that did not appear to perturb colony growth. CTC colonies were collected for qPCR analysis and mRNAseq via laser-capture microdissection and/or micropipetting. Genomic analysis revealed differential gene expression patterns between CTC colonies cultured from the same patient sample, with a subset expressing neuroendocrine like signatures (AURKA), stem cell markers (SOX2, OCT4), and immuno-oncology relevant markers (PD-L1, CTLA4). Conclusions: In conclusion, we show that viable CTCs are amenable to ex vivo culture, providing both functional and molecular insights into a sub-population of CTCs with propagating potential. The presence of T-cells and dendritic cells on CTC colonies warrants further investigation, and may provide unique insights into immune-tumor interactions. The culturing platform is currently being evaluated as a research tool for biomarker discovery and as a clinical tool for disease monitoring and treatment decision-making.
