Cancer marker-free enrichment and direct mutation detection in rare cancer cells by combining multi-property isolation and microfluidic concentration

Abstract

Genetic analysis, rather than simply counting the number of circulating tumor cells (CTCs), which are rare cancer cells in peripheral blood, has great potential for non-invasive biopsy or “liquid biopsy.” However, a practical problem in conventional enrichment of CTCs is that the isolated target cells are mixed with numerous residual leukocytes, and are suspended in a large volume. Hence, further isolation (i.e., cytokeratin (CK)-positive cell picking) or DNA purification is required for downstream genetic analysis after isolation. Here, we propose a novel cancer marker-free method of CTC enrichment by size-based Filtration and Immunomagnetic Negative selection followed by Dielectrophoretic concentration (CTC-FIND) for direct detection of genetic mutations in rare cancer cells suspended in whole blood. A combination of two independent isolation methods based on physical (filtration) and biochemical properties (immunomagnetic negative selection) in CTC-FIND allowed highly efficient cancer marker-free purification (5.1-log depletion of leukocytes). The isolated cells were trapped and concentrated using a microfluidic step-channel device using dielectrophoresis for discrimination and downstream genetic analysis. The feasibility of cancer marker-free enrichment by CTC-FIND was successfully demonstrated by directly detecting mutations in various cancer cells with a very high sensitivity of 1 cell per mL, including EpCAM and CK-negative cells, which were used to spike 8 mL of whole blood. Thus, CTC-FIND can be used with liquid biopsy to detect genetic mutations in wide-ranging CTC subsets, independent of cancer cell-specific marker expression.