A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood

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.

Isolation and molecular analysis of circulating tumor cells from lung cancer patients using a microfluidic chip type cell sorter

“…Amplified DNA samples were analyzed for EGFR L858R and T790M using pyrosequencing, and exon 19 deletion using fragment analysis for detection. We have previously shown the detection of rare alleles down to 10% frequency, even in a few target cells …”

“…In our previous study, varying numbers of various tumor cell lines were spiked into blood, and tumor cell isolation was evaluated using our On‐chip Sort system . The calculated detection efficiency was constant and >70% when 5–25 tumor cells were present per 4 mL blood, and there was a 100% success rate in the detection of EGFR , KRAS , and BRAF mutations from captured tumor cells . The limit of blank of the On‐chip Sort system was determined as a finite number of false‐positive events of CTCs detected per assay.…”

“…Immunomagnetic enrichment was carried out as described previously . Briefly, 5 mL of each blood sample was lysed by lysing solution (BD Biosciences, San Jose, CA, USA), and then was negatively enriched using Dynabeads coated with anti‐CD45 mAb (Thermo Fisher Scientific Inc., Waltham, MA, USA) to remove white blood cells, followed by fixation and labeling with the FITC‐conjugated anti‐CK mAb CK3‐6H5 (1:25 dilution; Miltenyi Biotec, Bergisch‐Gladbach, Germany), the phycoerythrin‐conjugated anti‐vimentin mAb D21H3 (1:50 dilution; Cell Signaling Technology, Danvers, MA, USA), and the Alexa Fluor 700‐conjugated anti‐CD45 mAb F10‐89‐4 (1:20 dilution; AbD Serotec, Oxford, UK).…”

“…Enumeration and sorting of cells were carried out by On‐chip Sort according to the manufacturer's instructions and as described previously . Briefly, the flow path was prewashed with 1× Through Path Plus (On‐chip Biotechnologies) and the On‐chip sample buffer (1× Through Path Plus with 1.5% polyvinylpyrrolidone; On‐chip Biotechnologies).…”

Materials and microfluidics: enabling the efficient isolation and analysis of circulating tumour cells

“…34 Some researchers have reduced this variability by counting spiked cells on the cap of a blood sample prior to mixing 63 or by using a micromanipulator to physically pick individual cells for spiking. 64 …”