Purpose: Circulating tumor cells (CTCs) hold promise to be a non-invasive measurable biomarker in all cancer stages. Because the analysis of CTCs is still a technical challenge, we compared different types of microfluidic enrichment protocols to isolate these rare cells from the blood.
Methods: Blood samples from patients with early and metastatic breast cancer (BC) were processed using the microfluidic Parsortix® technology employing (i) a single-step cell separation using the standard GEN3D6.5 microfluidic cassette, (ii) a two-step separation with an upfront pre-enrichment, and (iii) a two-step separation with a different type of cassette. In the enriched cells, the gene expression levels of CTC-related transcripts were assessed using quantitative real-time PCR (qPCR) by Taqman® and Lightcycler (LC) technology.
Results: 23/60 (38.3%) BC samples were assigned as positive due to the presence of at least one gene marker beyond the threshold level. The prevalence of epithelial markers was significantly higher in metastatic compared to early BC (EpCAM: 31.3% vs. 7.3%; CK19: 21.1% vs. 2.4%). A high level of concordance was observed between CK19 assessed by Taqman® and LC technology, and for detection of the BC-specific gene SCGB2A2. An upfront pre-enrichment resulted in lower leukocyte contamination, at the cost of fewer tumor cells captured.
Conclusion: The Parsortix® system offers both reasonable recovery of tumor cells and depletion of contaminating leukocytes when the single-step separation using the GEN3D6.5 cassette is employed. Careful selection of suitable markers and cut-off thresholds is an essential point for the subsequent molecular analysis of the enriched cells.
Keywords: Density gradient centrifugation; Early breast cancer; Gene expression analysis; Liquid biopsy; Microfluidic enrichment.
© 2022. The Author(s).