title: 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.
authors.affiliation: Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH.
authors.forename: Powrnima
authors.initials: P
authors.lastname: Joshi
authors.forename: Barbara
authors.initials: B
authors.lastname: Jacobs
authors.forename: Adeeb
authors.initials: A
authors.lastname: Derakhshan
authors.forename: Lee R
authors.initials: LR
authors.lastname: Moore
authors.forename: Paul
authors.initials: P
authors.lastname: Elson
authors.forename: Pierre L
authors.initials: PL
authors.lastname: Triozzi
authors.forename: Ernest
authors.initials: E
authors.lastname: Borden
authors.forename: Maciej
authors.initials: M
authors.lastname: Zborowski
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D016022:Case-Control Studies; D002469:Cell Separation; D006801:Humans; D007124:Immunoenzyme Techniques; D018189:Immunomagnetic Separation / Q000379:methods*; D017493:Leukocyte Common Antigens / Q000097:blood; D058965:MART-1 Antigen / Q000097:blood; D008545:Melanoma / Q000097:blood*; D035683:MicroRNAs / Q000235:genetics; D009360:Neoplastic Cells, Circulating / Q000737:chemistry / Q000473:pathology*; D011379:Prognosis; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D064568:S100 Calcium Binding Protein beta Subunit / Q000097:blood; D015996:Survival Rate; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D058965:MART-1 Antigen; D035683:MicroRNAs; D012333:RNA, Messenger; D064568:S100 Calcium Binding Protein beta Subunit; C579160:S100B protein, human; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human