Phosphorylated filamin-A at serine 1459 from plasma-derived small extracellular vesicles as a promising biomarker for high-risk adenoma and colorectal cancer

Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer-related death worldwide. Early detection can reduce CRC mortality by more than 90%. Circulating small extracellular vesicles (sEVs) are emerging as promising biomarkers for CRC, but their role in detecting precancerous lesions remains unclear. Herein, parallel proteomic and phosphoproteomic analyses of plasma-derived sEVs were performed in healthy subjects with negative colonoscopy, patients with high-risk adenoma (HRA) and patients with CRC. A total of 139 phosphorylation sites on 52 proteins were identified, among which 16 phosphorylation sites on 12 sEV proteins showed significant changes (≥ 2-fold) with 90% confidence in site localization. Web-based validation demonstrated that the phosphorylation level of sEV-derived filamin-A at serine 1459 (pFLNA Ser1459 ) correlated with the Clinical Proteomic Tumor Analysis Consortium colon cancer dataset. Immunoblot analysis confirmed that sEV-derived pFLNA Ser1459 was significantly reduced in CRC patients compared with healthy subjects, whereas the highest levels were observed in HRA patients. Notably, sEV-derived pFLNA Ser1459 , alone or in combination with FLNA, CD9, and TSG101, showed superior diagnostic performance in distinguishing HRA patients from CRC patients and healthy subjects. These findings suggest that plasma sEV-derived pFLNA Ser1459 is a promising biomarker for colorectal neoplasm detection.