Molecular Characterization of Plasma Extracellular Vesicles in Clear Cell Renal Cell Carcinoma A Comparative Analysis in Treated Patients

Background: Clear cell renal cell carcinoma (ccRCC) represents the most common and aggressive subtype of renal cell carcinoma and is often diagnosed at advanced stages due to its asymptomatic nature and the lack of reliable early biomarkers. In this context, extracellular vesicles (EVs) have emerged as promising candidates for liquid biopsy applications, as they carry molecular cargo reflective of their cells of origin and remain stable in biological fluids. Objectives: The present study aimed to perform a comprehensive molecular characterization of plasma-derived EVs obtained from healthy donors, patients with papillary RCC (pRCC), patients with ccRCC, and ccRCC patients undergoing systemic treatment. Particular attention was given to the evaluation of Mucin-1 (MUC1), a glycoprotein implicated in ccRCC progression and metabolic reprogramming, and to the identification of EV-associated transcriptomic signatures. Methods: EVs were isolated by differential ultracentrifugation and characterized using Transmission Electron Microscopy (TEM) and Nanoparticle Tracking Analysis (NTA), confirming their typical morphology and size distribution. Phenotypic profiling through Multiplex bead-based flow cytometry (MACSPlex) demonstrated the presence of canonical EV markers, while high-sensitivity flow cytometry enabled the detection and quantification of MUC1 on EV surfaces. Additionally, RNA sequencing was performed to investigate the transcriptomic cargo of EVs. Results: Our results showed a significant increase in circulating EV concentration in ccRCC patients compared to healthy controls, with a reduction observed in treated patients, suggesting a potential association with tumor burden and therapeutic response. MUC1 analysis revealed a complex modulation of EV subpopulations across disease conditions. Transcriptomic profiling identified a largely conserved EV RNA core, with a limited set of differentially expressed genes capable of distinguishing tumor from healthy samples. Notably, treatment exerted a strong impact on EV RNA content, modulating pathways associated with tumor biology. Conclusion: Overall, these findings support the role of plasma-derived EVs as a multifaceted source of biomarkers in ccRCC, providing insights into disease biology and treatment response.