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

Sara R Bang-Christensen, Viatcheslav Katerov, Amalie M Jørgensen, Tobias Gustavsson, Swati Choudhary, Thor G Theander, Ali Salanti, Hatim T Allawi, Mette Ø Agerbæk

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.

OriginalsprogEngelsk
Artikelnummer5881
TidsskriftCancers
Vol/bind13
Udgave nummer23
ISSN2072-6694
DOI
StatusUdgivet - 23 nov. 2021

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