Lin Bingqian, Jiao Zhenna, Dong Shouquan, Yan Weikai, Jiang Jinting, Du Yanfang, Weng Xiaocheng, Wang Hongling, Hu Zhiyuan, Liu Yibin, Zhou Xiang
State Key Laboratory of Metabolism and Regulation in Complex Organisms, College of Chemistry and Molecular Sciences, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan, China.
Reproductive Medicine Center, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China.
Nat Commun. 2025 Aug 29;16(1):8084. doi: 10.1038/s41467-025-63435-w.
Extracellular vesicles (EVs) are promising biomarkers for cancer diagnosis and prognosis due to their ability to carry specific biomolecular cargo, including DNA. However, the clinical utility of DNA methylation-based liquid biopsies using EV-DNA remains underexplored. The low quantity and relatively long length of EV-DNA complicate whole-genome methylation profiling. To address this, we develop Tn5-assisted Enzymatic Methyl-sequencing with Post-conversion Tailing (TEMPT), a bisulfite-free whole-genome profiling method for EV-DNA. TEMPT employs single-adapter Tn5 tagmentation, enzymatic conversion of unmodified cytosines, and post-conversion tailing to generate high-depth whole-genome EV-DNA methylomes. We apply TEMPT to EV-DNA from 58 gastric cancer and polyp samples, generating methylomes from sub-nanogram inputs and identifying differentially methylated regions (DMRs) that distinguish cancer from controls. We identify potential cancer biomarkers through DMR-associated genes, highlighting the roles of EVs in cellular communication. Our findings suggest that immune cells may serve as an alternative source of EV-DNA. This approach holds significant promise for advancing EV-DNA research and its applications in early disease diagnosis.
细胞外囊泡（EVs）因其能够携带包括DNA在内的特定生物分子货物，有望成为癌症诊断和预后的生物标志物。然而，使用EV-DNA进行基于DNA甲基化的液体活检的临床应用仍未得到充分探索。EV-DNA的低含量和相对较长的长度使全基因组甲基化分析变得复杂。为了解决这个问题，我们开发了一种用于EV-DNA的无亚硫酸氢盐全基因组分析方法——Tn5辅助的转换后加尾酶促甲基测序（TEMPT）。TEMPT采用单适配器Tn5转座标签、未修饰胞嘧啶的酶促转化和转换后加尾，以生成高深度的全基因组EV-DNA甲基化组。我们将TEMPT应用于58个胃癌和息肉样本的EV-DNA，从亚纳克级输入生成甲基化组，并识别区分癌症与对照的差异甲基化区域（DMRs）。我们通过与DMR相关的基因识别潜在的癌症生物标志物，突出了EVs在细胞通讯中的作用。我们的研究结果表明免疫细胞可能是EV-DNA的另一个来源。这种方法在推进EV-DNA研究及其在疾病早期诊断中的应用方面具有重大前景。
