Abstract
Purpose: :
Due to the lack of lymphatics in the eye, hematogenous dissemination is the predominant means by which uveal melanoma (UM) cells escape the primary site. Our laboratory has recently demonstrated the presence of circulating malignant cells (CMC’s) in the blood using both animal models and clinical trails involving UM patients. Current data suggests that all UM patients are positive for CMC’s, at some point in time after diagnosis. Phenotypic changes in those cells that are necessary to establish metastatic disease may occur while in circulation. The objective of this study was to develop a method to isolate CMCs from blood using immunomagnetic beads.
Methods: :
Uveal melanoma cells were isolated using the CELLection Pan Mouse IgG Kit consisting of 4.5um diameter superparamagnetic polystyrene beads coated with monoclonal human anti–mouse IgG. Cells were isolated via the direct technique as described by the manufacturer. Uveal melanoma cell lines 92.1, MKT–BR, OCM–1 SP6.5, UW–1 and 92.1 transfected with GFP were adjusted to a concentration of 1x 106 cells/ml five tenfold dilutions were performed in order to yield six cell concentrations ranging from 1x106 to 10 cells/ml PBS/0.1%BSA. Cell suspension were added to CELLection Pan Mouse IgG Dynabeads pre–labeled with anti–melanoma antibodies NKI/C3, NKI/Beteb and 9.2.27. Negative controls consisted of cells incubated with unlabeled beads. Cells were then eluted and plated in six well plates containing 1ml RPMI–1640 medium supplemented with 5% heat inactivated FBS, 1% fungizone, and 1% penicillin–streptomycin. Cultures were monitored daily for growth via microscopic evaluation.
Results: :
Cells were isolated from all five human uveal melanoma cell lines using a mixture of monoclonal antibodies against surface molecules specific to melanoma cells (NKI/C3, NKI/Beteb, 9.2.27). When used in isolation none of these antibodies were capable of capturing all the uveal melanoma cell lines at levels lower than 10,000 cells/ml. However, the mixture of all three antibodies allowed for the isolation of cells from blood spiked at a concentration of 10 cells/ml. No cells were isolated when using the negative controls. The viability of the cells isolated was verified by staining with trypan blue.
Conclusions: :
To the best of our knowledge this is the first time that an immunomagnetic isolation method to diagnose CMCs from uveal melanoma cell lines has been described using a panel of antibodies. This method may be useful to test uveal melanoma patients for CMCs and is significantly faster and cheaper to run than traditional nested–PCR methods.
Keywords: melanoma • oncology