Efficient leukocyte depletion by a novel microfluidic platform enables the molecular detection and characterization of circulating tumor cells

Abstract

RT-qPCR is a highly sensitive approach to detect rare transcripts, as derived from circulating tumor cells (CTCs) in the blood of cancer patients. However, the presence of unwanted leukocytes often leads to false positive results. Here, we evaluated whether the micro-fluidic Parsortix™ technology is appropriate to remove these leukocytes and thereby finally to improve the overall approach.In this study, we established a workflow including the micro-fluidic Parsortix™ technology for the molecular detection of CTC related transcripts. Background levels of EpCAM, PPIC, TUSC3, and MAL2 were efficiently removed due to an up to 106-fold depletion of leukocytes. The presence of these gene markers was observed in Parsortix™-enriched blood samples from patients with primary and recurrent gynecological cancer (32% and 14%), as well as in 86% of the metastatic breast cancer samples, at a very high specificity. In the ovarian cancer samples, PPIC was the most prominent gene marker, contributing to all positive cases and at least to 70% of the positive cases after pre-amplification of the respective target genes. Expanding the analytical panel up to 29 gene markers further increased the positivity rate (primary gynecological cancer: 95%, recurrent gynecological cancer: 100%, metastatic breast cancer: 92%).The established workflow strongly improved the overall molecular analysis of the target cells by the efficient removal of contaminating cells, and, thereby offers great promise for the molecular characterization of CTCs.

The Parsortix™ Cell Separation System—A versatile liquid biopsy platform

“…Cell viability ensures integrity of nucleic acids, allowing molecular interrogation of the harvests with both increased sensitivity of detection (concentration effect) and decreased background (enrichment effect). The Parsortix System has been used to successfully capture and/or harvest CTCs from a number of different cancer types, with the harvested CTCs successfully evaluated by a variety of subsequent analysis techniques, including: cytological/histological staining and/or immunostaining for identification, enumeration and detection of protein biomarkers on captured and/or harvested CTCs (Figure B,C) ; individual gene expression (mRNA) analysis (e.g., ddPCR and qPCR) ; multiplex nucleic acid or protein evaluation (e.g., via Ziplex® System Flow‐Thru Chip® technology [ANGLE Biosciences, Toronto, CA]); evaluation of DNA aberrations (e.g., aCGH and HER2 FISH) ; whole transcriptome or genome sequencing (e.g., RNA‐seq and DNA‐seq) ; mouse xenograph models . …”

“…As mentioned earlier, cells captured by the Parsortix System can be interrogated by cytological/histological staining techniques such as hematoxylin and eosin (H&E) or Wright‐Giemsa staining, as well as by immunochemical techniques, including immunofluorescence . Importantly, the captured rare cells do not have to be harvested from the separation cassette in order to be evaluated by such techniques.…”

“…The typical rate of separation for whole blood on the system is approximately 5 ml/h. The Parsortix System allows for the harvest of the captured cells from the separation cassette into a small volume (100–210 μl) of buffer, such as PBS, enabling a high level of versatility in how the cells could be evaluated and/or clinically interrogated .…”

Liquid biopsies

“…The same system when used in combination with single CTC analysis, revealed the heterogeneity of PIK3CA mutational status within patient‐matched EpCAM high and EpCAM low/negative CTCs . This isolation system offers great promise for the molecular characterization of CTCs as it is very efficient in removing contaminating cells . The fluorescent EPISPOT assay is based on the identification of viable cells through the detection of proteins secreted/released/shed by functional single epithelial cancer cells.…”

“…26 This isolation system offers great promise for the molecular characterization of CTCs as it is very efficient in removing contaminating cells. 27 The fluorescent EPISPOT assay is based on the identification of viable cells through the detection of proteins secreted/released/shed by functional single epithelial cancer cells. When combined with an enrichment step, as the RosetteSep and Parsortix, it enables functional studies in CTCs.…”