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

PLoS One. 2020 Oct 23;15(10):e0241123. doi: 10.1371/journal.pone.0241123. eCollection 2020.

Abstract

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Antigens, Neoplasm / metabolism*
  • Biomarkers, Tumor / isolation & purification
  • Biomarkers, Tumor / metabolism*
  • Cell Count
  • Cell Line, Tumor
  • Cell Separation / instrumentation
  • Cell Separation / methods*
  • Gene Expression Profiling / methods
  • Healthy Volunteers
  • Humans
  • Microfluidics / instrumentation
  • Microfluidics / methods
  • Neoplasms / blood
  • Neoplasms / diagnosis*
  • Neoplasms / immunology
  • Neoplastic Cells, Circulating / immunology*
  • Neoplastic Cells, Circulating / metabolism
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction
  • Sensitivity and Specificity

Substances

  • Antigens, Neoplasm
  • Biomarkers, Tumor
  • RNA, Messenger

Grants and funding

We would like to disclose that the Angle Europe Limited provided the Parsortix® PR1 instrumentation and disposable cassettes for evaluation purposes and JMR serves on the scientific review board. Furthermore, one of the molecular assays, the QuantiGene Plex, was provided by Thermo Fisher Scientific eBioscience for evaluation purposes. No financial support was provided by HTG Molecular for the EdgeSeq gene expression assay, but MD Anderson has subsequently established a collaborative research agreement with HTG to develop a smaller version of the gene expression panel presented here.