Extracellular vesicles (EVs) carry molecules derived from donor cells and are able to alter the properties of recipient cells. They are important players during the genesis and progression of tumors. Uveal melanoma (UM) is the most common primary intraocular tumor in adults and is associated with a high rate of metastasis, primarily to the liver. However, the mechanisms underlying this process are poorly understood. In the present study, we analyzed the oncogenic potential of UM-derived EVs and their protein signature. We isolated and characterized EVs from five UM cell lines and from normal choroidal melanocytes (NCMs). BRCA1-deficient fibroblasts (Fibro-BKO) were exposed to the EVs and analyzed for their growth in vitro and their reprograming potential in vivo following inoculation into NOD-SCID mice. Mass spectrometry of proteins from UM-EVs and NCM-EVs was performed to determine a protein signature that could elucidate potential key players in UM progression. In-depth analyses showed the presence of exosomal markers, and proteins involved in cell-cell and focal adhesion, endocytosis, and PI3K-Akt signaling pathway. Notably, we observed high expression levels of HSP90, HSP70 and integrin V in UM-EVs. Our data bring new evidence on the involvement of UM-EVs in cancer progression and metastasis.

Extracellular vesicles (EVs) play a key role in cellular communication both in physiological conditions and in pathologies such as cancer. Emerging evidence has shown that EVs are active carriers of molecular cargo (e.g. protein and nucleic acids) and a powerful source of biomarkers and targets. While recent studies on EV‐associated DNA (EV‐DNA) in human biofluids have generated a large amount of data, there is currently no database that catalogues information on EV‐DNA. To fill this gap, we have manually curated a database of EV‐DNA data derived from human biofluids (liquid biopsy) and in‐vitro studies, called the Extracellular Vesicle‐Associated DNA Database (EV‐ADD). This database contains validated experimental details and data extracted from peer‐reviewed published literature. It can be easily queried to search for EV isolation methods and characterization, EV‐DNA isolation techniques, quality validation, DNA fragment size, volume of starting material, gene names and disease context. Currently, our database contains samples representing 23 diseases, with 13 different types of EV isolation techniques applied on eight different human biofluids (e.g. blood, saliva). In addition, EV‐ADD encompasses EV‐DNA data both representing the whole genome and specifically including oncogenes, such as KRAS, EGFR, BRAF, MYC, and mitochondrial DNA (mtDNA). An EV‐ADD data metric system was also integrated to assign a compliancy score to the MISEV guidelines based on experimental parameters reported in each study. While currently available databases document the presence of proteins, lipids, RNA and metabolites in EVs (e.g. Vesiclepedia, ExoCarta, ExoBCD, EVpedia, and EV‐TRACK), to the best of our knowledge, EV‐ADD is the first of its kind to compile all available EV‐DNA datasets derived from human biofluid samples. We believe that this database provides an important reference resource on EV‐DNA‐based liquid biopsy research, serving as a learning tool and to showcase the latest developments in the EV‐DNA field. EV‐ADD will be updated yearly as newly published EV‐DNA data becomes available and it is freely available at http://www.evdnadatabase.com.

Background Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. Methods The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. Results MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

Purpose: Extracellular vesicles (EVs) are small (50nm-800nm) membrane-enclosed structures with highly heterogeneous content. Recently, the importance of EVs in tumor progression as well as biomarkers has become apparent in many malignancies. However, there has been very limited research done to investigate EVs derived from uveal melanoma (UM). 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 was (i) to characterize the protein signatures of UM cell line-derived EVs to determine potential biomarkers, (ii) to determine EV uptake by recipient cell lines, (iii) to analyze the activation of the MAPK pathway in the recipient cells, and to examine the potential of UM EV-educated BRCA1 deficient fibroblasts (Fibro-BKO) to develop tumors in NOD-SCID mice. Methods: EVs were isolated from the conditioned media of UM cell lines (MP41, MP46, 92.1, MEL285, MEL270, OMM2.5) and normal choroidal melanocytes cells using an ultracentrifugation method. Western blots and immunogold-labelling Transmission Electron Microscopy were employed to validate various EV markers (CD63, CD81 and TSG101). Proteomic analysis by mass spectrometry was performed to determine the UM-derived EV proteome signature that could elucidate potential key players in UM metastasis. After exposure to EVs, recipient cells were analysed for the activation of downstream signaling pathways, and were inoculated in NOD-SCID mice to analyze their behaviour. Results: We observed 2069 proteins in EVs using Scaffold Viewer. FUNRich analysis revealed that 95% of these EV proteins were found in the Vesiclepedia database. Gene Ontology analysis confirmed the presence of exosomal markers (CD81, TSG101and Syntenin) as well as cell-cell adhesion-related proteins. The DAVID Functional Annotation chart revealed proteins related to endocytosis, PI3K-Akt signaling pathway and focal adhesion. In addition, we observed high hits in HSP90 and HSP70, well-known biomarkers in UM. Integrin alpha V, which are known for preparing premetastatic niches in the liver environment, were also present. Western blot results confirmed the increase in MAPK pathway in the recipient cells. Furthermore, UM EVs-educated Fibro-BKO transformed and gave rise to tumors in vivo. Conclusion: In conclusion, our study reports an essential step towards understanding protein emission through EVs from UM cells. Our analyses revealed UM EVs cargo candidates that may play a role in pre-metastatic niche formation and metastasis in specific organs, such as the liver. The in vivo study model confirmed that EVs derived from UM are capable of inducing tumorigenesis. We believe that our data pave the way to the application of liquid biopsy to the monitoring of UM-affected patients. Citation Format: Thupten Tsering, Alexander Laskaris, Mohamed Abdouh, Prisca Bustamante Alvarez, Goffredo Arena, Julia Valdemarin Burnier. Characterization of extracellular vesicles derived from uveal melanoma cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6455.