Isolation of circulating tumor cells by immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS) for molecular profiling

Methods. 2013 Dec 1;64(2):114-8. doi: 10.1016/j.ymeth.2013.07.029. Epub 2013 Jul 26.

Abstract

Circulating tumor cells (CTCs) are cells shed by the primary tumor into the blood stream capable of initiating distant metastasis. In the past decade, numerous assays have been developed to reliably detect these extremely rare cells. However, methods for purification of CTCs with little or no contamination of normal blood cells for molecular profiling are limited. We have developed a novel protocol to isolate CTCs by combining immunomagnetic enrichment and fluorescence-activated cell sorting (IE/FACS). The two-part assay includes (1) immunomagnetic capture using magnetic beads conjugated to monoclonal antibody against an epithelial cell adhesion marker (EpCAM) to enrich for tumor cells; and (2) FACS analysis using EpCAM to purify tumor cells away from mononuclear cells of hematopoietic lineage. Downstream molecular analyses of single and pooled cells confirmed the isolation of highly pure CTCs with characteristics typical that of malignant cells.

Keywords: Array comparative genomic hybridization; Circulating tumor cells; Fluorescence-activated cell sorting; Immunomagnetic enrichment; Single cell analysis.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies, Monoclonal
  • Antigens, Neoplasm / immunology
  • Breast Neoplasms / pathology
  • Cell Adhesion Molecules / immunology
  • Cell Count
  • Cell Line, Tumor
  • DNA Copy Number Variations
  • Epithelial Cell Adhesion Molecule
  • Female
  • Flow Cytometry / methods*
  • Humans
  • Immunomagnetic Separation / methods*
  • Neoplastic Cells, Circulating / pathology*
  • Single-Cell Analysis

Substances

  • Antibodies, Monoclonal
  • Antigens, Neoplasm
  • Cell Adhesion Molecules
  • Epithelial Cell Adhesion Molecule