Li Zichun, Liu Yifan, Liu Feiran, Sun Guochen, Zhang Xiaoling, Jing Jing
School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, 100081, PR China.
Department of Neurosurgery, The First Medical Center, PLA General Hospital, Beijing, 100853, PR China.
Anal Chim Acta. 2025 Feb 15;1339:343623. doi: 10.1016/j.aca.2025.343623. Epub 2025 Jan 6.
Exosomes, which are known to transport diverse proteins from parent cells to recipient cells, consequently influence the biological activities of the recipient cells. Among those proteins, the epithelial cell adhesion molecule (EpCAM), plays a crucial role as it is implicated in cell adhesion and signaling processes. As exosomal EpCAM potentially affects the migration of recipient cells, direct visualization with high spatial resolution is essential to better understand this impact and the role of exosomal EpCAM in recipient cells. Such understanding may provide valuable insights into the mechanisms underlying various diseases and potential treatment strategies. (94) RESULTS: This work focus on the selective labeling and fluorescent imaging of glycosylated EpCAM on tumor-derived exosomes using bioorthogonal click chemistry and aptamer-targeting strategies. To commence, exosomes with EpCAM overexpression, EpCAM N-glycosylation mutation, EpCAM silencing, or wildtype, were obtained by genetic manipulation. Subsequently, the glycosylation of exosomal EpCAM was directly visualized by capitalizing on the intramolecular fluorescence resonance energy transfer (FRET) that takes place between fluorescent EpCAM aptamers and fluorescent tags bound to glycans. As a result, this approach demonstrated its efficacy in investigating both the existence and the glycosylation state of exosomal EpCAM. Importantly, we proceeded to observe the uptake of tumor-derived exosomes by their recipient cells. It was then remarkably found that the expression and glycosylation levels of EpCAM in the co-cultured exosomes have a significant and substantial impact on the migratory ability of the recipient immune cells. (139) SIGNIFICANCE: We set up a novel labeling strategy for exosomal glycosylated EpCAM. This approach enabled us to realize the direct observation of exosomal EpCAM and its glycosylation with high spatial resolution. Based on this method, we find a significant role that the expression and the glycosylation of exosomal EpCAM in recipient cell adhesion. (52).
外泌体能够将多种蛋白质从亲代细胞转运至受体细胞，进而影响受体细胞的生物学活性。在这些蛋白质中，上皮细胞黏附分子（EpCAM）发挥着关键作用，因为它参与细胞黏附和信号传导过程。由于外泌体EpCAM可能影响受体细胞的迁移，因此高空间分辨率的直接可视化对于更好地理解这种影响以及外泌体EpCAM在受体细胞中的作用至关重要。这种理解可能为各种疾病的潜在机制和治疗策略提供有价值的见解。（94）结果：本研究聚焦于利用生物正交点击化学和适配体靶向策略对肿瘤来源外泌体上糖基化的EpCAM进行选择性标记和荧光成像。首先，通过基因操作获得了过表达EpCAM、EpCAM N-糖基化突变、EpCAM沉默或野生型的外泌体。随后，利用荧光EpCAM适配体与结合聚糖的荧光标签之间发生的分子内荧光共振能量转移（FRET），直接对外泌体EpCAM的糖基化进行可视化。结果表明，该方法在研究外泌体EpCAM的存在及其糖基化状态方面具有有效性。重要的是，我们进而观察了肿瘤来源外泌体被其受体细胞摄取的情况。结果显著发现，共培养外泌体中EpCAM的表达和糖基化水平对受体免疫细胞的迁移能力有重大且实质性的影响。（139）意义：我们建立了一种针对外泌体糖基化EpCAM的新型标记策略。这种方法使我们能够以高空间分辨率直接观察外泌体EpCAM及其糖基化。基于该方法，我们发现外泌体EpCAM的表达和糖基化在受体细胞黏附中发挥着重要作用。（52）
