Novel method for rapid enrichment of high purity circulating tumor cells (CTCs) for prostate cancer (PCa) gene expression profiling.

Abstract

271 Background: CTC RNA analysis currently involves single cell recovery that is laborious and expensive, or alternatively lysis of preserved whole blood which yields RNA predominantly from leukocytes which vastly outnumber CTCs. To effectively characterize gene expression in large patient cohorts, new enrichment methodologies are needed that yield high purity CTC populations while preserving RNA integrity. Here we describe a simple yet robust method for enrichment of prostate CTCs for gene expression analysis. Methods: Blood was drawn with informed consent under an IRB-approved protocol. For initial optimization, CFSE-stained PCa cells were spiked into healthy blood and recovered using various combinations of 2 methods: microfluidic enrichment (Parsortix™ system) and CD45 depletion. For assay qualification, a prostate-specific multiplexed qRT-PCR gene expression panel was developed. Enrichment and gene expression were tested initially using PCa cell lines spiked into healthy blood, then metastatic castrate resistant prostate cancer (mCRPC) blood samples in parallel with CellSearch enumeration. Results: Optimal enrichment of live cells was achieved with CD45 depletion followed by microfluidic enrichment, resulting in an average spiked cell recovery of 30% and approximately 100 contaminating background leukocytes. Using this enrichment method, prostate specific genes were detectable by multiplexed qRT-PCR down to 25 cells spiked into 7.5 ml whole blood, and transcripts were not measurable in matched healthy blood controls. When applied to mCRPC patient blood containing CTCs by CellSearch, multiplexed qRT-PCR successfully detected prostate specific genes in all samples. Conclusions: We developed a novel enrichment method capable of rapidly and efficiently recovering live CTCs with high purity, free of magnetic beads and with very few background leukocytes. Captured cells yielded high-quality RNA with high sensitivity and specificity for prostate-specific transcripts. This approach is applicable to high throughput gene expression profiling assays and offers an alternative to laborious single cell recovery or non-cancer-specific whole blood fixation.