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Martina Angi, Rosalind Jenkins, Bertil E. Damato, Sarah E. Coupland, Helen Kalirai; Optimization of Culture Conditions for the Analysis of the Secretome of Primary Uveal Melanoma Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3392.
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© ARVO (1962-2015); The Authors (2016-present)
Analysis and characterisation of the cancer secretome using proteomic techniques is a key step in the search for prognostic biomarkers and potential therapeutic targets. In uveal melanoma (UM), analysis of biological fluids proximal to the tumor, e.g. subretinal fluid (SRF), is limited by their availability. It has been shown in other malignancies that proteins secreted by tumor cells in vitro ("secretome") are similar to those released by the same tumors in vivo. In this study, we investigated the influence of culture conditions on the composition of the UM secretome.
Primary UM cells from two enucleation specimens as well as two UM cell lines (Mel270 and Omm2.5) were grown under adherent and non-adherent culture conditions and in different media. Cell proliferation was evaluated using 0.4% sulforhodamine-B. Following progressive lowering of serum levels and incubation in 100% serum-free medium for 48 hours, the UM secretome was collected, concentrated and further processed for mass spectrometry analysis. DNA and proteins were extracted from the remaining cells and investigated using Multiplex Ligation-dependent Probe Amplification (MLPA) and Western blot, respectively.
In all cultures, the number of proliferating viable cells was significantly higher in Minimum Essential Medium Alpha (αMEM)-based medium. MLPA results for the primary UM cells at the end of the culture period were consistent with the data obtained from the initial tumor biopsy. The proteins identified in the secretome of UM cells included putative secreted proteins, such as osteopontin and secreted melanoma-associated ME20 antigen, as well as some associated with cell leakage. Six-fold more proteins were detected in the secretome of adherent cultured cells compared with the suspension cultured cells.
Culture of UM cells in an αMEM-based medium under adherent conditions is optimal for in vitro UM secretome analysis. Future work will compare the secretome of primary UM specimens with adjacent ocular fluids, such as SRF, from the same patient. In this way, we hypothesize that prognostic biomarkers of clinical relevance in UM will be identified.
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