Abstract
BackgroundPersonalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis; however, the technical difficulties in isolating and analyzing rare CTCs have limited progress to date. In this preclinical study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology.MethodsWe developed a cell capture platform using On-chip Sort (On-Chip Biotechnologies), a novel bench-top cell sorter equipped with a disposable microfluidic chip. Spike-in experiments comprising a series of lung cancer cell lines with varying epithelial cell adhesion molecule (EpCAM) expression levels were conducted to assess the capture and purification efficiency of the platform. Samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, followed by sample fixation and labeling. The enriched and labeled samples were then sorted by On-chip Sort based on cytokeratin, vimentin, and CD45 expression. Captured cells were immediately subjected to whole genome amplification followed by mutation analysis using deep targeted sequencing, and copy number analysis using quantitative polymerase chain reaction (qPCR).ResultsSpike-in experiments revealed an excellent overall mean capture rate of 70.9%. A 100% success rate in the detection of EGFR, KRAS and BRAF mutations from captured cells was achieved using pyrosequencing and deep sequencing. The mutant variant detection rates were markedly higher than those obtained with the CellSearch profile kit. qPCR analysis of amplified DNA demonstrated reproducible detection of copy number changes of the EGFR in captured tumor cells.ConclusionsUsing a novel cell sorter, we established an efficient and convenient platform for the capture of CTCs. Results of a proof-of-principle preclinical study indicated that this platform has potential for the molecular characterization of captured CTCs from patients.
Highlights
Personalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells
The CK+ and/or vimentin+ events were subjected to CD45 negative gating to distinguish tumor cells from white blood cells (WBCs) and/or debris
Tumor cell events that appeared in the CD45-Alexa Fluor 700 vs. FL5 density plot (FL5 is a detection channel adjacent to that for Alexa Fluor 700) were distinguished from the WBCs and debris population (Figure 1C, middle)
Summary
Personalized cancer treatment relies on the accurate detection of actionable genomic aberrations in tumor cells. Circulating tumor cells (CTCs) could provide an alternative genetic resource for diagnosis; the technical difficulties in isolating and analyzing rare CTCs have limited progress to date. In this preclinical study, we aimed to develop an improved capture system for molecular characterization of CTCs based on a novel cell sorting technology. The presence or absence of various actionable genomic aberrations has been shown to predict response to molecularly targeted treatments [1]. Rebiopsy remains challenging, mainly because of the invasiveness of the procedure
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