Comparison of microfluidic platforms for the enrichment of circulating tumor cells in breast cancer patients

Abstract

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.

Is a real-time quantifiable liquid biopsy achievable using a microfluidic lab-on-chip ?

“…Regardless of whether CTCs are detected and further characterized by their intracellular proteins, gene expression pattern or genetic make-up, the background of benign cells may interfere with the analysis and thus needs to be substantially reduced. Such benign cells that are particularly relevant are the residual white blood cells (WBC), because a number of genes may also be expressed in leukocytes and hence will interfere with the analysis of the CTCs, particularly at the transcriptional level [5].…”

“…Our approach has recently been highlighted [12]. In existing chip-formats, such a porous membrane is made from an elastomeric materia that can be stretched in a cyclic way to model surface strains that cells would be exposed to in vivo, for example, in the lung-alveola barrier [13] or the 8) formed in a nanostructured permeable membrane (5) to cover a perforation in the support (4,11). The nanostructured permeable membrane is tightly connected to the material of the LoC (2).…”

“…The black circle indicates the position of one of the 3D micro-niches. (B) (B) Schematic cross-sectional view of a 3D micro-niche, which comprises a 3D protuberance (8) formed in a nanostructured permeable membrane(5) to cover a perforation in the support(4,11). The nanostructured permeable membrane is tightly connected to the material of the LoC (2).…”