Frozen melanoma tissues yield extracellular vesicles with preserved diagnostic and immunogenic properties

BACKGROUND: Extracellular vesicles (EVs) isolated from tumor tissues carry disease-associated proteins and surface antigens, making them promising candidates for cancer diagnostics and immunotherapy. For EV-based approaches to reach clinical application, it is essential that functional EVs can be obtained from clinically accessible materials, including cryopreserved tumor tissues stored in biobanks. Whether cryopreservation alters EV integrity and function remains unclear. This study evaluates whether EVs derived from cryopreserved tumor tissues retain key molecular and biological properties required for diagnostic and therapeutic use. METHODS: EVs were isolated from human metastatic melanoma tissues processed immediately after surgical resection (fresh) or after storage at - 80 °C (frozen), using a protocol consistent with standard biobank procedures. EV isolation was performed through ultracentrifugation followed by an iodixanol density cushion. The resulting EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and mass spectrometry to assess EV morphology, purity, and molecular composition. The diagnostic potential was evaluated by examining the presence of previously identified cancer-associated membrane proteins. Furthermore, therapeutic potential was assessed in vivo by co-administering EVs from fresh or frozen melanoma tissues with synthetic bacterial vesicles and evaluating their effects on tumor growth in melanoma-bearing mice. RESULTS: EVs from fresh and frozen tissues showed similar morphology, size distribution, yield, and purity. Moreover, the protein composition, including cancer-associated markers such as MT-CO2, COX6c, SLC24A22, HLA-DR, and Erlin2, was highly consistent between EVs derived from fresh and frozen tissues, with no relevant enrichment of intracellular or mitochondrial contaminants in frozen-derived EVs. Functionally, EVs from cryopreserved tissues combined with synthetic bacterial vesicles significantly inhibited tumor progression in vivo, demonstrating antitumor effects comparable to those of EVs from fresh tissues. CONCLUSIONS: Our results validate cryopreserved tissues as a reliable source of functional EVs, comparable to fresh tissues. This supports the potential use of existing biobanks for retrospective EV-based biomarker discovery and functional research.