Detection of VAR2CSA-Captured Colorectal Cancer Cells from Blood Samples by Real-Time Reverse Transcription PCR

SIMPLE SUMMARY: Circulating tumor cells are cancer cells that have entered blood or lymphatic vessels wherefrom they might get access to distant body parts and form metastases. The presence of cancer cells in a blood sample can be exploited for non-invasive diagnostic purposes. However, as blood consists of a vast number of healthy red and white blood cells the task of identifying the few potential cancer cells in a sample is a technical challenge. In this study we explore strategies for detecting circulating tumor cells after a pre-enrichment through binding to VAR2CSA protein coupled to magnetic beads. We evaluate the performance of a novel workflow that recognizes and detects the cancer cells based on their gene expression and compare this with the more traditional detection strategy using antibodies for cell staining. The highly sensitive assay presented here could potentially provide a novel strategy for early cancer detection. ABSTRACT: Analysis of circulating tumor cells (CTCs) from blood samples provides a non-invasive approach for early cancer detection. However, the rarity of CTCs makes it challenging to establish assays with the required sensitivity and specificity. We combine a highly sensitive CTC capture assay exploiting the cancer cell binding recombinant malaria VAR2CSA protein (rVAR2) with the detection of colon-related mRNA transcripts (USH1C and CKMT1A). Cancer cell transcripts are detected by RT-qPCR using proprietary Target Enrichment Long-probe Quantitative Amplified Signal (TELQAS) technology. We validate each step of the workflow using colorectal cancer (CRC) cell lines spiked into blood and compare this with antibody-based cell detection. USH1C and CKMT1A are expressed in healthy colon tissue and CRC cell lines, while only low-level expression can be detected in healthy white blood cells (WBCs). The qPCR reaction shows a near-perfect amplification efficiency for all primer targets with minimal interference of WBC cDNA. Spike-in of 10 cancer cells in 3 mL blood can be detected and statistically separated from control blood using the RT-qPCR assay after rVAR2 capture (p < 0.01 for both primer targets, Mann-Whitney test). Our results provide a validated workflow for highly sensitive detection of magnetically enriched cancer cells.