Circulating tumor cells (CTCs) are shed from solid tumors and found at extremely low frequencies in the blood of patients in most cancers. A subset of these cells can seed and give rise to metastases, which is the primary cause of cancer-related mortality. Isolation and characterization of these cells from blood as a ‘liquid biopsy’ can be a sensitive, non-invasive method for early detection, disease monitoring and therapy selection. CTCs can be found even at early disease stages in preclinical models and patients. There is increasing evidence that clusters of CTCs in blood are associated with higher metastatic potential; however, efficient isolation and interrogation of these rare clusters is challenging. In this study, we utilize an in-line rare cell enrichment platform developed by Becton Dickinson (BD), coupled with the BD FACSTM Influx cell sorter to rapidly isolate both single cells and clusters from blood. This platform utilizes magnetic particle-based depletion of unwanted leukocytes and combines acoustic focusing to remove red blood cells and debris while enriching for rare cells of interest. We achieved 30-fold enrichment of tumor cells spiked into blood along with >6-fold improvement in sort efficiency with the enrichment process as compared to without. Importantly, the large 200μm nozzle and low sheath pressure (3.5 psi) on the Influx minimizes shear forces and maintains cell viability and integrity of clusters during sorting, thereby enabling discrimination of single cells and clusters based on side scatter. Using this strategy, we could sort enriched populations of viable cell clusters (range 2-10 cells). Finally, this workflow can be seamlessly integrated with downstream molecular analysis such as next-generation sequencing. We successfully performed whole-transcriptome single-cell sequencing (sc-RNAseq) on cells isolated with this streamlined platform where pooled cells and bulk RNA were used as comparisons. As proof of principle, we used this optimized workflow to isolate and characterize CTCs from a pancreatic cancer mouse model (KPCY) in which all tumor cells are labeled with Yellow Fluorescent Protein (YFP). We sorted YFP+ CTCs and CD45+ white blood cells (WBCs) from the blood of 7 KPCY mice along with YFP+ cells from the matched tumor. We recovered an average of 88 CTCs/ml of blood (range 5–258). RNA sequencing was completed on pooled and single cells from all samples. Epithelial genes including Krt18 and Krt19 as well as lineage specific genes such as Sox9 were significantly overexpressed in CTCs and tumors compared to WBCs, suggesting the utility of this approach. This integrated CTC workflow provides a sensitive approach to identify pancreatic cancer-specific markers, which will be evaluated for their ability to improve sensitivity of positive-selection of CTCs in patient blood and for development of a clinically relevant diagnostic assay.

Citation Format: Neha Bhagwat, Charles H. Pletcher, Ling Wang, William DeMuth, Keely Dulmage, David Balli, Stephanie S. Yee, Liping Yu, Jonni S. Moore, Ben Z. Stanger, Eric P. Dixon, Erica L. Carpenter. A novel sensitive flow-cytometry based platform for isolation and molecular characterization of circulating tumor single cells and clusters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3801. doi:10.1158/1538-7445.AM2017-3801