Multi-omics characterization of extracellular vesicles derived from virus-positive Merkel cell carcinoma cells

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer, with approximately 80% of cases driven by Merkel cell polyomavirus (MCPyV). Although extracellular vesicles (EVs) are increasingly recognized as mediators of intercellular communication within the tumor microenvironment, their molecular cargo in MCPyV-positive MCC has not been comprehensively characterized. Here, we performed a multi-omics characterization of EVs released by two MCPyV-positive MCC cell lines. EVs were isolated by differential ultracentrifugation and characterized by nanoparticle tracking analysis, imaging flow cytometry, cryo-electron microscopy, and immunoblotting, demonstrating a heterogeneous population of small and large EVs. Proteomic and transcriptomic analyses revealed that MCC-derived EVs possess distinct protein, mRNA, and miRNA cargo compared with their parental cells, with enrichment of molecules associated with gene expression, RNA processing, intracellular signaling, and vesicle-mediated transport. Despite differences in the molecular composition of EVs derived from WaGa and MKL-1 cells, functional enrichment analyses revealed highly similar biological pathways. To investigate whether the viral oncoprotein small T antigen (sT) contributes to EV cargo composition, EVs from inducible sT knockdown cells were analyzed. Loss of sT was associated with modest changes in the EV proteome and mRNA cargo, whereas the overall EV-associated miRNA profile remained largely unchanged. Collectively, these findings provide the first comprehensive molecular characterization of EVs released by MCPyV-positive MCC cells and establish a foundation for investigating the contribution of EV-mediated communication to MCC biology and tumor–microenvironment interactions.