Abstract 3779: Incorporating in-line sample pre-enrichment with FACS and single cell mRNA-seq to facilitate isolation and characterization of prostate cancer circulating tumor cells

For decades, circulating tumor cells (CTCs) have been used as biomarkers to indicate disease progression and survival in patients with metastatic tumors. Detection of CTCs is an attractive, non-invasive blood test for early diagnosis of many types of cancer. However, CTCs are present at extremely low frequency in blood, and it is challenging to recover them in typical multi-step sample processing workflows. EpCAM, the widely accepted marker for CTC, has been found being down regulated in protein expression and RNA expression analysis in many cases thus EpCAM-dependent CTC detection might not be sensitive enough. We have developed a new sample pre-enrichment technology incorporated in line with fluorescence activated cell sorting (BD FACS™) platforms to demonstrate a one-step sample processing workflow to minimize cell loss and maximize CTC recovery. The pre-enrichment technology combines magnetic depletion of white blood cells and acoustic removal of lysed red blood cells, sending enriched tumor cells directly to a cell sorter for single CTC isolation. The in-line enrichment and sorting technology has shown 90% removal of CD45+ cells and over 90% recovery of target cells going through the system. Furthermore, we have incorporated molecular indexing technology for next generation sequencing (NGS) into the single CTC isolation to streamline gene expression analysis. We conducted a pilot study in which prostate cancer cell lines PC3, DU145, and LNCaP were spiked in healthy donors’ peripheral blood, quantitatively recovered, and individually sorted into 96-well BD™ Precise barcoded plates using a BD Influx™ sorter equipped with the in-line pre-enrichment module. The sorted cells were then sequenced for a prostate cancer targeted gene panels with approximate 100 genes, including EpCAM. Single cell sorting and sequencing were also performed on cell line cells directly and confirmed the workflow did not affect the gene expression. In LNCaP and PC3 cells, EpCAM was not the high expression marker in the genes analyzed, and its protein expression was also heterogeneous. The highly expressed genes discovered by single cell mRNA sequencing such as GSTP1 and CAV1 were investigated to provide additional cell surface markers for more sensitive CTC detection. Finally, CTCs from blood samples collected from prostate cancer patients were isolated based on marker panels beyond EpCAM and characterized at the single cell level. The differences between gene expression profiles