Antigen-agnostic microfluidics-based circulating tumor cell enrichment and downstream molecular characterization.

Circulating tumor cells (CTC) isolated from the peripheral blood of cancer patients by a minimally invasive procedure provide surrogate markers of the tumor that can be repeatedly sampled. However, the selection and enumeration of CTCs by traditional methods based on surface proteins like EPCAM may not detect CTCs with a mesenchymal phenotype. Here, we employed an antibody-agnostic platform, the Parsortix® PR1 system, which enriches CTCs based on cell size and membrane deformability. We evaluated the linearity, sensitivity, and specificity of the Parsortix PR1 system in tandem with 3 downstream molecular characterization techniques using healthy donor blood spiked with cultured cell lines. Signal amplification of mRNA using a QuantiGene 25-gene assay was able to quantitate multiple epithelial genes, including CDH1, EGFR, ERBB2, KRT18, and MUC1, from high numbers of spiked cells and was able to detect KRT18 when only 50 MCF-7 or SUM190 cells were spiked into healthy donor blood. However, target amplification of mRNA by quantitative polymerase chain reaction (qPCR) showed better sensitivity; qPCR without pre-amplification was able to detect CTC-related genes in Parsortix PR1-enriched cells when as few as 5 SKBR3 cells were spiked into blood. Finally, the HTG EdgeSeq nuclease protection assay was able to profile mRNA expression of over 2,560 cancer-related genes from Parsortix PR1 enriched cells, showing enrichment in cancer signaling pathways and ERBB2, KRT19, and KRT7. Overall, the Parsortix PR1 platform may be amenable to transition into routine clinical workflows.