Dataset Information

Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma.

ABSTRACT: Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate. These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture. However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma. We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malignant cell characteristics. CMCs were enriched by centrifugation of blood samples from healthy (N = 10) and patient (N = 11) donors, followed by RBC lysis and immunomagnetic depletion of CD45-positive leukocytes in a specialized magnetic separator. CMCs were identified by immunocytochemistry using Melan-A or S100B as melanoma markers and enumerated using automated microscopy image analyses. Separation was optimized for maximum sensitivity and recovery of CMCs. Our results indicate large number of CMCs in Stage IV melanoma patients. Analysis of survival suggested a trend toward decreased survival with increased number of CMCs. Moreover, melanoma-associated miRs were found to be higher in CMC-enriched fractions in two patients when compared with the unseparated samples, validating this method as applicable for molecular analyses. Negative selection is a promising approach for isolation of CMCs and other EpCAM -negative CTCs, and is amenable to molecular analysis of CMCs. Further studies are required to validate its efficacy at capturing specific circulating cells for genomic analysis, and xenograft studies.

SUBMITTER: Joshi P

PROVIDER: S-EPMC4058018 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma.

Joshi Powrnima P Jacobs Barbara B Derakhshan Adeeb A Moore Lee R LR Elson Paul P Triozzi Pierre L PL Borden Ernest E Zborowski Maciej M

Oncotarget 20140501 9

Circulating tumor cells have emerged as prognostic biomarkers in the treatment of metastatic cancers of epithelial origins viz., breast, colorectal and prostate. These tumors express Epithelial Cell Adhesion Molecule (EpCAM) on their cell surface which is used as an antigen for immunoaffinity capture. However, EpCAM capture technologies are of limited utility for non-epithelial cancers such as melanoma. We report a method to enrich Circulating Melanoma Cells (CMCs) that does not presuppose malig ...[more]

PMID: 24811334

Similar Datasets

Project description:Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.

Dataset Information

Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma.

Publications

Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets