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 (pFLNASer1459) correlated with the Clinical Proteomic Tumor Analysis Consortium colon cancer dataset. Immunoblot analysis confirmed that sEV-derived pFLNASer1459 was significantly reduced in CRC patients compared with healthy subjects, whereas the highest levels were observed in HRA patients. Notably, sEV-derived pFLNASer1459, 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 pFLNASer1459 is a promising biomarker for colorectal neoplasm detection.