Extracellular vesicles isolated from frozen and fresh human melanoma tissue are similar in purity and protein composition

Extracellular vesicles (EVs) isolated from tumor tissues represent a precious source of EVs because they are enriched in disease-associated biomarkers and are highly concentrated. However, a fundamental question is whether freezing of tissues influences the EVs’ integrity and function and whether non-EVs are co-isolated with the EVs. In this work, we isolated EVs from metastatic melanoma tissue both immediately after tissue resection and after being frozen for at least two weeks. Specifically, the samples were divided into two parts: one was immediately processed for EV isolation (hereafter called fresh), and the other was frozen on dry ice and stored for two weeks before being processed for EV isolation (hereafter called frozen). Large and small EVs were isolated through ultracentrifugation, pooled, and further purified on an iodixanol density cushion. The EVs were analyzed by transmission electron microscopy, nanoparticle tracking analysis, and protein quantification as well as by quantitative mass spectrometry. The results did not show any significant difference between EVs isolated from fresh and frozen tissue. Importantly, there was no enrichment of either intracellular proteins or mitochondrial proteins in EVs isolated from frozen tissues vs. fresh tissues. Moreover, there were no significant differences in the quantity of proteins like MT-CO2, Cox6, SLC24A22, HLA-DR, and Erlin2, which were previously identified as potential markers of melanoma, ovarian cancer, and breast cancer. Overall, this study supports the use of frozen tissues as a source of EVs for research purposes because frozen tissue-derived EVs do not differ in purity or protein composition compared to their fresh counterparts. ### Competing Interest Statement RC, CL and JL have developed multiple EV-associated patents for putative clinical utilization and they own equity in Exocure Sweden AB. JL owns equity in Exocure Sweden AB and Nexocure Therapeutics AB and consults in the field of EVs through Vesiclebio AB. ROB has received institutional research grants from Bristol-Myers Squibb (BMS), Endomagnetics Ltd (Endomag), and SkyLineDx, speaker honoraria from Roche, Pfizer, and Pierre-Fabre, and has served on advisory boards for Amgen, BD/BARD, Bristol-Myers Squibb (BMS), Merck Sharp, & Dohme (MSD), Novartis, Roche, and Sanofi Genzyme and is a shareholder in SATMEG Ventures AB.