Bioorthogonal click chemistry and aptamer-targeting enables highly selective fluorescence labeling of exosomal glycosylated EpCAM for super resolved imaging

Background: 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).