title: A novel flow cytometry-based cell capture platform for the detection, capture and molecular characterization of rare tumor cells in blood.
abstract: BACKGROUND
  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; 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.
  
  
  METHODS
  We 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).
  
  
  RESULTS
  Spike-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.
  
  
  CONCLUSIONS
  Using 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.
authors.forename: Masaru
authors.initials: M
authors.lastname: Watanabe
authors.forename: Masakuni
authors.initials: M
authors.lastname: Serizawa
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sawada
authors.forename: Kazuo
authors.initials: K
authors.lastname: Takeda
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takahashi
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Koizumi
authors.affiliation: Drug Discovery and Development Division, Shizuoka Cancer Center Research Institute, Shizuoka, Japan. ykoh@wakayama-med.ac.jp.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Koh
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism; D045744:Cell Line, Tumor; D000071858:Epithelial Cell Adhesion Molecule; D066246:ErbB Receptors / Q000235:genetics; D005434:Flow Cytometry; D011905:Genes, ras; D006801:Humans; D018189:Immunomagnetic Separation; D008175:Lung Neoplasms / Q000097:blood* / Q000235:genetics; D009154:Mutation; D009360:Neoplastic Cells, Circulating; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics
chemical_list: D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf