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Maria Santiago-Varela, Diego Perez-Sotelo, Nerea Lago-Baameiro, Manuel F Bande, Susana Bravo, Francisco Ruiz-Oliva, María José Blanco, Maria Pardo, Antonio Piñeiro; DECIPHERING THE ROLE OF EXTRACELULAR VESICLES IN HUMAN UVEAL MELANOMA. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3972.
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© ARVO (1962-2015); The Authors (2016-present)
Tumor-released extracellular vesicles (EVs) and their role on cancer pathogenesis has created great expectation as a very sophisticated way of intercellular signaling favoring tumor development and progression. Moreover, EVs may become an important source of cancer biomarkers. Since there is barely any knowledge about uveal melanoma (UM) secreted EVs, neither about their role in this ocular tumor, we aim to isolate and characterize uveal melanoma shed EVs as potential non-invasive uveal melanoma prognostic trackers.
UM-A human melanoma cell line was labeled during 72 hours by CILAIR: Comparison of Isotope-Labeled Amino acid Incorporation Rates with Arg and Lys (L-[13C6]-Lys/L-[13C6,15N4]-Arg). Labeled serum deprived secretomes were collected and ultracentrifugated for EVs isolation and characterization by immunoblotting, light scattering and electronic microscopy. Labeled secretomes and isolated labeled vesicles were processed for LC-MALDI protein identification followed by functional analysis.
Uveal melanoma cells in culture shed exosomal extracellular vesicles of 100 nm positive for CD9 and CD63. Accordingly, cellular component classification after mass spectrometry analysis of isolated vesicles shows that 80% of identified proteins were exosomal; others were classified as from extracellular matrix or extracellular space. From the former, 50% were isotopically labeled showing that quantitative proteomics analysis by CILAIR proves to be an effective method for EVs characterization avoiding external contaminants. UM exosome proteome functional analysis reveals proteins implicated in cell growth and maintenance, protein metabolism, and angiogenesis and cell surface interactions integrins.
We demonstrate for the first time the secretion of vesicles of exosomal nature from UM cells. The proteomics characterization of UM exosomes suggest their implication in tumor-metastatic niche preparation and therefore reveals new potential therapeutic targets in uveal melanoma.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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