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Updated: Jun 20, 2025
An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue
Published on: February 3, 2023
Optimizing of a suitable protocol for isolating tissue-derived extracellular vesicles and profiling small RNA
Wenjing Yang1, Yu Liu1, Jiyan Wang1
1Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
Background:
Extracellular vesicles (EVs) facilitate cell-cell interactions in the tumour microenvironment. However, standard and efficient methods to isolate tumour tissue-derived EVs are lacking, and their biological functions remain elusive.
Methods:
To determine the optimal method for isolating tissue-derived EVs, we compared the characterization and concentration of EVs obtained by three previously reported methods using transmission electron microscopy, nanoparticle tracking analysis, and nanoflow analysis (Nanoflow). Additionally, the differential content of small RNAs, especially tsRNAs, between hepatocellular carcinoma (HCC) and adjacent normal liver tissues (ANLTs)-derived EVs was identified using Arraystar small RNA microarray. The targets of miRNAs and tsRNAs were predicted, and downstream functional analysis was conducted using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, non-negative matrix factorization and survival prediction analysis.
Results:
A differential centrifugation-based protocol without cell cultivation (NC protocol) yielded higher EV particles and higher levels of CD9+ and CD63+ EVs compared with other isolation protocols. Interestingly, the NC protocol was also effective for isolating frozen tissue-derived EVs that were indistinguishable from fresh tissue. HCC tissues showed significantly higher EV numbers compared with ANLTs. Furthermore, we identified different types of small RNAs in HCC tissue-derived EVs, forming a unique multidimensional intercellular communication landscape that can differentiate between HCC and ANLTs. ROC analysis further showed that the combination of the top 10 upregulated small RNAs achieved better diagnostic performance (AUC = .950 [.895-1.000]). Importantly, most tsRNAs in HCC tissue-derived EVs were downregulated and mitochondria-derived, mainly involving in lipid-related metabolic reprogramming.
Conclusion:
The NC protocol was optimal for isolating EVs from HCC, especially from frozen tissues. Our study emphasized the different roles of small-RNA in regulating the HCC ecosystem, providing insights into HCC progression and potential therapeutic targets.
