Abstract 931: Enrichment and characterization of propagating circulating tumor cells from late stage prostate and pancreatic cancer patients
Authors: Lim James I. , Ryan Charles J. , Krahn Thomas , Stoecklein Nikolas H. , Fischer Johannes , Adams Bruce 
Published: August 2015

Capturing cells shed by a tumor into the bloodstream represents an opportunity to study the properties of these cells for biomarker discovery in support of drug development and personalized therapy. Enrichment and expansion of circulating tumor cells (CTCs) in culture enables molecular characterization not readily available for rare cells. We report on a primary cell culture platform to enrich and expand rare circulating cells from the blood of patients with metastatic castration resistant prostate cancer and pancreatic ductal adenocarcinoma. The approach relies on tumor cell enrichment via cell-binding substrates comprised of a collagen-based hydrogel for CTC capture, and hypoxic culturing conditions to maintain and propagate subset of these cells with tumor-initiating potential.

Here, we demonstrate the successful identification and expansion of rare circulating tumor cells from peripheral blood and leukapheresed blood samples from prostate and pancreatic cancer patients, respectively. Upon successful culture of samples for a period of 5 to10 days after plating, immunofluorescence imaging was performed. Morphologically distinct CTC colonies positive for PSMA, EpCAM and cytokeratin staining were observed, with individual colonies ranging from approximately 50 to 1000 in cell number. Interestingly, colonies consistently revealed white blood cell contaminants (T-cells and dendritic cells) that did not appear to perturb colony growth.

Targeted DNA sequencing was performed on pancreatic CTC colonies from six patients using a genetic panel covering 237 genes. We achieved 100-200X coverage for each patient sample and present a preliminary genetic characterization of their CTCs in culture. Our results show unique genetic signatures indicative of aggressive forms of pancreatic ductal adenocarcinoma based on SNP/INDEL detection, CNV analysis and gene fusion predictions. In summary, cultured CTCs had 20-30X more variants than corresponding white blood cell controls, with mutations observed in MLL2, ARID1A, BRAF, KRAS and BRCA2. CNV analysis and gene fusion predictions revealed previously unreported biomarker candidates.

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.

Citation Format: James I. Lim, Charles J. Ryan, Thomas Krahn, Nikolas H. Stoecklein, Johannes Fischer, Bruce Adams. Enrichment and characterization of propagating circulating tumor cells from late stage prostate and pancreatic cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 931. doi:10.1158/1538-7445.AM2015-931