Northrop-Albrecht Emmalee J, Kim Yohan, Taylor William R, Majumder Shounak, Kisiel John B, Lucien Fabrice
Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA.
Department of Urology, Mayo Clinic, Rochester, MN, USA.
Commun Biol. 2025 Feb 13;8(1):228. doi: 10.1038/s42003-025-07652-5.
Colorectal cancer (CRC) is the 2 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.
结直肠癌（CRC）是美国第二大致命癌症，但如果早期发现，其治愈率很高。粪便衍生的细胞外囊泡（EVs）是一种新型生物标志物来源，可提高CRC前体检测的灵敏度。然而，粪便衍生EVs分离方法的标准化仍未得到充分探索。我们之前报道过，尺寸排阻色谱法（SEC）结合超滤法（UF-100kDa）适用于人粪便上清液中EVs的分离。在本研究中，我们首先评估了替代的EV浓缩方法（超滤[UF]；10 kDa、30 kDa、50 kDa、100 kDa和旋转蒸发仪[SV]）。其次，我们通过质谱法研究宿主/细菌EV蛋白质组。我们报告说，浓缩方法在回收率、RNA和可溶性蛋白质污染方面没有差异。蛋白质组学分析揭示了多样化的细菌蛋白质组，而人源蛋白质更为丰富。具体而言，胰腺酶是最丰富的蛋白质之一，进一步探索发现，酶原颗粒可能在粪便EV制剂中共同分离出来。为了能够高灵敏度地发现基于EV的CRC前体分子特征，可能需要免疫捕获策略。值得注意的是，我们鉴定出10种表面蛋白，它们可能作为纯化结肠来源EVs的候选蛋白。这项工作为未来基于EV的CRC生物标志物的发现和验证提供了框架。
