Malignant melanoma of the choroid is the most common, primary intraocular malignancy. Histopathologic analysis of enucleated tumor bearing eyes has revealed the existence of distinct cell types comprising subgroups of choroidal melanoma. Little is known about the biology of these tumor cells. The first major objective of this research is to establish choroidal melanoma cell lines in vitro. In addition to conventional tissue culture techniques, melanoma growth stimulatory activity (MGSA) and other growth factors will be utilized to induce growth and proliferation of tumor cells in vitro. Cloning techniques and differential centrifugation will be utilized to isolate tumor stem cells. Alternatively, tumors will be passaged as xenografts in the anterior chamber of immunosuppressed mice prior to tissue culture. Choroidal melanoma cells will be analyzed by an in vitro differentiation model by manipulation of growth factors and/or terminal differentiating agents (e.g., phorbol esters). Experimental evidence suggests that choroidal melanoma cells express both unique and shared melanoma-associated antigens (MAA). The second objective of this research is to identify and characterize choroidal MAA by the generation of specific monoclonal antibodies. This will entail the production of different heteroantisera against spindle and/or epithelioid tumor cells or purified membrane components. The major membrane antigens will be isolated by affinity and other forms of chromatography. Monoclonal antibodies will be generated against purified membrane antigens and screened using radioimmunoassays and ELISA. MAA will be identified by polyacrylamide gel electrophoresis and their expression delineated by immunoperoxidase/immunofluorescence staining of frozen tissue sections. The ultimate goal of this research is to provide experimental tools which can be utilized for study and analysis of this important ocular tumor.
