Proteomic Profiling of Human Extracellular Vesicles Reveals Diagnostic Biomarkers for Colon Adenocarcinoma

Early detection of colon adenocarcinoma (COAD) remains suboptimal. Fecal tests fail to diagnose 30% of stage I cancer, and serum CEA lacks sensitivity (< 40%). Extracellular vesicles (EVs) circulate systemically and package tumor-related cargo, making them attractive non-invasive biomarkers for cancer diagnosis. We profiled the EV proteome from 233 human patients using LC-MS/MS, including stage I‑IV tumors with matched non‑tumor colon tissues (n = 50 each; n = 100), paired pre‑/post‑operative plasma (n = 90) and healthy plasma (n = 43). Circulating EVs contained both tumor-specific and stromal/immune cell-derived proteins, reflecting the systemic nature of EV biology in the cancer setting. Proteomic analysis identified 745 proteins enriched in tumor-derived EVs (e.g., SRPK1, THBS2) and 127 proteins enriched in adjacent tissues. Plasma EVs revealed 166 proteins enriched in COAD (e.g., UBA1, FCN1) and 233 enriched in healthy controls. Pathway analysis linked tumor EV cargo to angiogenesis, mRNA splicing, TGF‑β signalling and RNA translation. Notably, a cross-cancer comparison (pancreatic = 10, lung = 14 cases) revealed that 76% of tumor EV proteins were COAD-specific, highlighting tissue of origin specificity. We further developed a 10-protein EV panel comprising seven tumor-associated and three healthy-enriched EV proteins, which effectively distinguished COAD patients from healthy controls in the two validation cohorts (n = 104 and n = 215), achieving > 90% sensitivity for differentiating COAD from healthy and non-COAD colorectal conditions. Six weeks after curative resection, tumor-associated EV proteins decreased by > 70%, whereas healthy-associated proteins rebounded to baseline, indicating surgical responsiveness. Collectively, EV protein signatures provide a sensitive and tissue-specific window into tumor-host communication, further supporting blood-based early detection of COAD.