Background: Early screening for colorectal cancer (CRC) is essential in order to improve prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to the low invasiveness and reproducibility. In addition, circulating exosomes (crExos) expressing tumor-specific proteins are potential biomarkers for various cancer types. Here, we developed an optimized DIA-MS-based diagnostic method in liquid biopsies, which can be potentially adapted as a rapid and non-invasive screening tool to detect early stages of CRC.
Methodologies: Exosomes were isolated from the culture supernatants of human CRC cells lines, and the plasma of CRC patients at different tumor stages, by prolonged ultracentrifugation combined with sucrose density gradient centrifugation. Tumor-specific exosomal proteins were identified using Tandem Mass Tag (TMT)-based shotgun proteomics and phosphoproteomics. The results were verified on a second independent cohort and a mouse tumor-bearing model using Western blotting, and the candidate biomarkers were further validated in a third CRC cohort by Data Independent Acquisition (DIA)-mass spectrometry (MS). Finally, the DIA-MS methodology was optimized to permit high-throughput detection of exosome biomarkers in another independent cohort of CRC patients and healthy controls.
Findings: High levels of total and phosphorylated exosomal fibronectin 1 (FN1), haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with tumor progression, and FGA was the most dominant biomarker. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crExos were likely released by the CRC cells. Furthermore, optimized DIA-MS validated that FGA+ crExos could distinguish colon adenoma and CRC patients from healthy individuals with higher specificity and sensitivity than the conventional tumor biomarkers.
Conclusion: DIA-MS detected proteome of crExos is a potential non-invasive screening tool to identify early stage of CRC.