The proteomic landscape of stool-derived extracellular vesicles in patients with pre-cancerous lesions and colorectal cancer

Colorectal cancer (CRC) is the 2^nd most fatal cancer in the United States, but when detected early it is highly curable. Stool-derived extracellular vesicles (EVs) are a novel biomarker source that could augment the sensitivity for detection of CRC precursors. However, standardization of isolation methods for stool-derived EVs remains underexplored. We previously reported that size-exclusion chromatography (SEC) followed by ultrafiltration (UF-100kDa) was suitable for human stool supernatant EV isolation. In this study, we first assess alternative EV concentration methods (ultrafiltration [UF]; 10 kDa, 30 kDa, 50 kDa, 100 kDa and speed vacuum [SV]). Second, we investigate the host/bacterial EV proteomes by mass spectrometry. We report no difference in recovery, RNA and soluble protein contamination among concentration methods. Proteomic analysis reveals a diverse bacterial proteome, while human-derived proteins are more abundant. Specifically, pancreatic enzymes are among the most abundant proteins, further exploration revealed that zymogen granules are likely co-isolated in stool EV preparations. To enable discovery of EV-based molecular signatures of CRC precursors with high sensitivity, immunocapture strategies will likely be needed. Notably, we identified 10 surface proteins that may serve as candidates for the purification of colon-derived EVs. This work serves as framework for the future discovery and validation of EV-based biomarkers for CRC.