Serum extracellular vesicle-derived N6-methyladenosine RNA as a diagnostic and prognostic biomarker in colorectal cancer

Background Extracellular vesicle (EV)-based liquid biopsy is increasingly recognized as a promising strategy for cancer diagnosis and prognosis, as EVs carry abundant, stable biomolecular cargo. N6-methyladenosine (m6A), the most prevalent modification in eukaryotic intracellular RNA, plays a critical role in regulating diverse cellular processes and has been implicated in tumor initiation and progression. However, the potential of EV-associated m6A-modified RNA (EV-m6A RNA) as a clinically useful biomarker for cancer detection remains unclear. Methods EV-RNA was isolated from tumor tissues and matched serum samples collected from 76 colorectal cancer (CRC) patients. Serum samples from 19 healthy donors were included as controls. EV-m6A RNA levels were quantified using an enzyme-linked immunosorbent assay (ELISA). Results Serum - EVs isolated from CRC patients and healthy donors were identified as CD9(+)/CD63(+)/ALIX(+) small extracellular vesicles (sEVs), with a mean particle diameter of 60–70 nm. The mean serum sEV concentration and sEV-RNA yield were significantly higher in CRC patients than in healthy controls (sEV concentration: 11 ± 8 × 10 ¹² /mL vs. 5 ± 2 × 10 ¹² /mL, p  = 0.006; sEV-RNA yield: 57 ± 52 ng/µL vs. 18 ± 7 ng/µL, p  = 0.002). Through m6A modification-specific ELISA quantification, receiver operating characteristic (ROC) curve analysis demonstrated good discriminatory performance of normalized serum sEV-m6A RNA levels for CRC detection (AUC, 0.8241; 95% CI, 0.7437–0.9045; p  < 0.0001). Serum sEV-m6A RNA levels were noted to be higher in late-stage (III/IV) CRC patients than in those with early-stage (I/II) disease (0.015 ± 0.001% vs. 0.009 ± 0.005%, p  < 0.001) with no significant correlation with intratumoral METTL3 expression, a m6A writer. Increased serum sEV-m6A RNA abundance further predicted a worse overall survival (OS) in CRC patients ( p  = 0.0107; HR, 3.894), while m6A RNA levels in tumor tissues showed no significant prognostic value ( p  = 0.3243; HR, 1.152). Conclusion These findings support serum sEV-m6A RNA levels as a feasible and noninvasive biomarker for colorectal cancer diagnosis, with additional potential for liquid biopsy-based prognostication and longitudinal disease monitoring