Abstract
Purpose :
Extracellular Vesicles (EVs) are oval membrane-enclosed structures with highly heterogeneous content. EVs are known to play important roles in intercellular communication by transporting their contents between cells whereby they alter the properties of both donor and recipient cells. Recently, the importance of EVs in tumor progression as well as biomarkers has become apparent in many malignancies. Uveal melanoma (UM) is the most common primary intraocular tumor in adults and it displays a high frequency of metastases to the liver. The aim of this study is to characterize the protein signature and determine the biomarker potential of circulating EVs derived from UM cell lines.
Methods :
EVs were isolated from the conditioned media of UM cell lines (MP41, MP46, MEL270, 92.1) using an ultracentrifugation method. Western blots and immunogold-labelling Transmission Electron Microscopy (TEM) were performed to validate various EV markers (CD63, CD81, TSG101). Proteomic analysis by mass spectrometry was performed to identify a UM-derived EV proteome signature that could elucidate potential key players in UM metastasis.
Results :
We observed 1330 proteins in EVs using Scaffold viewer. FUNRich analysis revealed that 92% of these EV proteins were found in the Vesiclepedia database, and we report an additional 104 novel proteins. The biological processes with the highest hits were protein metabolism, cell growth/maintenance and transportation. Gene Ontology analysis confirmed the presence of exosomal markers (CD81, TSG101, Flotillin and Syntenin) as well as cell-cell adhesion related proteins. DAVID Functional annotation chart revealed proteins related to endocytosis, PI3K-Akt signaling pathway and focal adhesion. In addition, we observed a high hit in heat shock protein 90 (HSP90) and HSP 70, well known biomarkers in UM. Moreover, integrin a V/ integrin b V, which are known for preparing premetastatic niches in liver environment, were also present.
Conclusions :
In conclusion, our study reports an essential step towards understanding protein emission through EVs from UM cells. Emitted proteins are a significant part of liquid biopsy biomarker approaches involving the detection of oncoproteins in the blood of cancer patients. This EV-related proteomic approach may also highlight unique biological activities worthy of exploration from the therapeutic perspective.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.