Abstract LB-327: Isolation and mRNA-seq analysis of single CTCs from blood samples using an integrated fluidic circuit for functional single cell studies
Authors: Szpankowski Lukasz , Premasekharan Gayatri , Ramalingam Naveen , Sanada Chad , Leyrat Anne , Fowler Brian , Edwards Matthew , Greene Cassandra , Holcomb Ilona , Ryan Charles J. , Paris Pamela L. , West Jay A.A. 
Published: July 2016

Abstract
As the precursor to metastatic cancer, circulating tumor cells (CTCs) hold tremendous potential for increasing our understanding of tumor metastasis, advancing personalized therapeutics, and have emerged as a significant source of genetic material for clinical guidance. Recent technical advances have enabled the enrichment and genomic characterization of CTCs at the single cell level and have revealed significant and clinically relevant heterogeneity in these rare cells. However, data is lacking for existing methods to link the functional differences of single CTCs to their underlying genomic structure and activity. We have developed an approach which integrates key aspects of biologically based experimentation at the single cell level onto an integrated fluidic circuit. This device allows for the selection and isolation of single CTCs in a size-independent fashion from a pre-enriched population based on fluorescent markers where they can be individually cultured and exposed to a variety of drugs/reagents under environmentally controlled conditions or processed directly for mRNA-seq. If desired, an extracellular matrix can be deposited into each of the single cell culture chambers. Cell activity and phenotype can be monitored in-situ in response to treatment conditions, followed by preparation of the mRNA transcriptome for RNA-seq analysis. As the entire workflow following pre-enrichment is integrated onto a single microfluidic chip, very little manipulation of the single cell is required, serving to preserve the mRNA signature of each cell. To optimize and validate the workflow, PC3 cell lines were spiked-in to blood from healthy donors followed by pre-enrichment for metastatic cancer cells using commercially available CTC isolation platforms. Our microfluidic device further purified and isolated the desired single PC3 cells, and prepared the cells for expression analysis via RNA-seq. The sequencing results further validated the ability of our workflow to isolate rare metastatic cancer cells from human blood and moreover confirmed highly correlated gene expression profiles as compared to cultured PC3 population controls (r = 0.89 across 23,562 genes) and non spike-in single cell samples (r = 0.90 across 23,562 genes). We applied this workflow to the blood of metastatic castration resistant prostate cancer (mCRPC) patients and obtained single cell cDNA from the isolated CTCs from which we were able to generate sequencing libraries. Future work using this system will help establish a robust methodology to enable more complex perturbation and functional studies of single cells from clinical samples.
Citation Format: Lukasz Szpankowski, Gayatri Premasekharan, Naveen Ramalingam, Chad Sanada, Anne Leyrat, Brian Fowler, Matthew Edwards, Cassandra Greene, Ilona Holcomb, Charles J. Ryan, Pamela L. Paris, Jay A.A. West. Isolation and mRNA-seq analysis of single CTCs from blood samples using an integrated fluidic circuit for functional single cell studies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-327.