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Michael J Young, Natalia Vila, Vasco Bravo, Petr Y Baranov, Burke Lieppman, Alexis Palazzolo, Miguel N Burnier; Phenotypic heterogeneity of uveal melanoma cell lines. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5310.
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© ARVO (1962-2015); The Authors (2016-present)
Uveal melanoma (UM) is the most common primary intraocular tumor in adults, with 2,500 new cases diagnosed every year in United States. The “cancer stem cell” paradigm has been explored in skin melanoma and has led to better understanding of disease manifestation, progression and metastasis. The expression of stem cell markers (ABCB5, CD133, Sox2, Pax6, nestin) by small subpopulation of uveal melanoma cells in vitro and in vivo has also been previously described, although the possibility of using this “stem” subpopulation for drug discovery has not been addressed. The goal of this study was to identify new targets for drug therapy, which would be focused on suppressing proliferative potential of dedifferentiated melanoma cells.
Immunocytochemistry, flow cytometry and RT-PCRT analysis was performed for 3 adherent UM cell lines: MKT-BR, OCM1, 92.1. The expression of stemness (Oct4, Nanog, Sox2, Klf4, cMyc, NMyc, LMyc, Lin28, hTERT), proliferative (FGFR, HGFR, Ki67, EpoR), eye field and melanocyte (Lhx2, Mitf, Nestin, TRP1, TRPM1), known skin melanoma markers (ABCA5, ABCB5, ABCG2) as well as several cell surface markers, expressed through neural crest (CD73, CD38, PSA-NCAM, PTK7, A2B5, CD133, HLA-ABC).<br /> For proliferation assays we have plated cells at 10,000 cells/well in 96 well plate, cultured them for 24 hours before drug treatment and assessed the population change at 48 hours after stimulation using CyQuant. The dose-response curves (7-point dilution) were calculated for several small molecules and growth factors.
We have observed the uniform expression of early development markers (Sox2, Klf4), proliferative marker Ki67 and melanoma markers (ABCA5, ABCB5, ABCG2) within population. We have also confirmed the expression of growth factor receptors. Cultured cell lines (92.1, MKT-BR and OCM1) contain a small subpopulation of Lhx2, CD38 (2%, 3%, 8%), CD24 (2%, 11%, 5%), PSA-NCAM (2%, 3%, 4%) and CD133 (15%, 6% and 3%, respectively) - positive cells.<br /> Proliferation studies showed the cytostatic effect of Iodoacetic acid, Rapamycin, PI3K inhibitor, Akt inhibitor, DAPT, AMPK inhibitor and Valproic acid. Wnt-3a, EGF and FGF stimulated the proliferation.
We have identified several immature markers that are heterogeneously expressed in 3 uveal melanoma cell lines. This identified subpopulation may be considered as a target for drug therapy.
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