title: Immunomagnetic isolation and in vitro expansion of human uveal melanoma cell lines.
abstract: OBJECTIVE
  Uveal melanoma (UM) is the most common intra-ocular tumor in adults. Despite advances in diagnosis and treatment, the survival rate of UM has not increased in the last several decades. Approximately 50% of patients will die as a consequence of metastatic disease with the majority of metastases localized to the liver. Due to the lack of lymphatics in the eye, hematogenous dissemination is the predominant means by which UM cells escape the primary site. Our laboratory has recently demonstrated the presence of circulating malignant cells (CMCs) in the blood using both animal models and clinical trails involving UM patients. Current data suggests that all UM patients will be positive for CMCs after diagnosis. Furthermore, some of the phenotypic changes that are necessary for metastatic growth may occur while the cells are circulating in the blood. In this study, we evaluated the efficiency of a panel of antibodies to immunomagnetically isolate CMCs for the purpose of in vitro expansion and genetic, immunological, and phenotypic characterization.
  
  
  METHODS
  In this study, five human uveal melanoma cell lines (92.1, MKT-BR, OCM-1, SP6.5, and UW-1) were immunostained with a panel of antibodies against known melanoma cell surface markers. Staining with monoclonal antibodies PAL M2, NKI C3, NKI/Beteb, and 9.2.27 permitted the generation of a cell surface expression profile in these cell lines. The five human UM cell lines and 92.1 transfected with GFP were subsequently spiked into human blood at concentrations ranging from 1x10(6) cells/ml to 10 cells/ml. Cells were immuno-magnetically isolated at concentrations as low as 10 cells/ml.
  
  
  RESULTS
  Immunomagnetic isolation of all five human UM cell lines tested at concentrations down to 10 cells/ml human blood was achieved only when antibodies were used in combination. Individually, the antibodies did not permit isolation of cells at physiologically relevant concentrations.
  
  
  CONCLUSIONS
  The immunomagnetic isolation method presented in this study can be used to isolate CMCs at physiologically relevant concentrations and at sensitivities comparable to those seen in polymerase chain reactions (PCR). In addition, our data suggests that our method is more efficient and reliable for the isolation of CMCs in UM than the methods currently used.
authors.affiliation: The Henry C. Witelson Ophthalmic Pathology Laboratory and Registry, McGill University Health Center, Montreal, PQ, Canada. jonathan.cools-lartigue@mail.mcgill.ca
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Cools-Lartigue
authors.forename: Cristin S
authors.initials: CS
authors.lastname: McCauley
authors.forename: Jean-Claude A
authors.initials: JC
authors.lastname: Marshall
authors.forename: Sebastian
authors.initials: S
authors.lastname: Di Cesare
authors.forename: Francois
authors.initials: F
authors.lastname: Gregoire
authors.forename: Emilia
authors.initials: E
authors.lastname: Antecka
authors.forename: Patrick
authors.initials: P
authors.lastname: Logan
authors.forename: Miguel N
authors.initials: MN
authors.lastname: Burnier
mesh_terms: D001769:Blood; D002455:Cell Division; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods / Q000592:standards; D008545:Melanoma / Q000473:pathology*; D009360:Neoplastic Cells, Circulating / Q000473:pathology; D014162:Transfection; D014604:Uveal Neoplasms / Q000473:pathology*
chemical_list: D049452:Green Fluorescent Proteins