Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma.
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Powrnima Joshi1, Barbara Jacobs2, Adeeb Derakhshan3, Lee R. Moore1, Paul Elson2,4, Pierre L. Triozzi2, Ernest Borden2, Maciej Zborowski1
1 Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH
2 Taussig Cancer Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH
3 Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, 9500 Euclid Avenue, Cleveland, OH
4 Quantitative Health Sciences, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH
Maciej Zborowski, email:
Keywords: circulating melanoma cells, CTC, Melan-A, S100B, magnetic separation, negative selection
Received: December 11, 2013 Accepted: February 1, 2014 Published: February 3, 2014
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.
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