Comparative evaluation of methods for isolating extracellular vesicles from ICC cell culture supernatants: Insights into proteomic and glycomic analysis

作者：Linlin Wu, Jiao Wei, Yueping Zhan, Xiao Xiao, Xuewen Xu, Chenjun Huang, Long Tian, Yueyue Li, Bin Fu, Mengmeng Wang, Chunfang Gao · 发表于：Cell Communication and Signaling · 年份：2025 · DOI：10.1186/s12964-025-02207-x · 被引用次数：5 · 研究领域：Extracellular vesicles in disease、Cholangiocarcinoma and Gallbladder Cancer Studies、Protease and Inhibitor Mechanisms

BACKGROUND: Extracellular vesicles (EVs) are nanoscale structures involved in intercellular communication and play a key role in cancer pathology. Intrahepatic cholangiocarcinoma (ICC) is a highly invasive malignancy marked by abnormal sialylated glycosylation. Analyzing proteins and glycans in EVs provides insights into ICC molecular subtyping and mechanisms. Optimizing EV isolation methods for ICC-derived EVs enables comprehensive proteomic and glycomic analysis. METHODS: We systematically evaluated five EV isolation methods-Ultracentrifugation (UC), exoEasy, Total Exosome Isolation (TEI), EVtrap, and ÄKTA-by analyzing the biophysical properties, proteomic profiles, and glycomic structures of EVs. Subsequently, we applied TMT-based quantitative proteome and light/heavy methylamine labeling for the quantification of sialylated N-glycan linkage isomers to investigate alterations in proteins and N-glycans within EVs secreted by HuCCT1 and HCCC-9810 cells with overexpressing ST6 β‑galactoside α2,6‑sialyltransferase 1 (ST6GAL1). RESULTS: By evaluating the biophysical properties, proteome, and N-glycome of EVs extracted using five different methods, UC was identified as the optimal approach for this study, as it offered a balance between operational complexity, cost-effectiveness, and the preservation of EVs activity. In this study, a total of 1,928 high-confidence proteins and over 84 high-confidence glycans were quantified. EVs secreted by HuCCT1 and HCCC-9810 cells overexpress...