Visualizing extracellular vesicle-mediated RNA transfer using a novel metabolic labeling approach

Extracellular vesicles (EVs) are key players in intercellular communication, facilitated by the transfer of RNA and other molecular cargo between cells. However, how EV-RNA is processed in recipient cells remains poorly understood, particularly the uptake and intracellular trafficking pathways leading to functional transfer remains elusive. Visualizing EV-RNA in recipient cells may help to address these knowledge gaps. Although EV labeling methods are well-established, tracking endogenous RNA cargo within EVs and in recipient cells is challenging due to its low abundance, requiring highly sensitive labeling methods. Existing RNA labeling methods often lack sensitivity, specificity for donor cell-derived RNA, or are limited to labeling long RNA species. In this study, we propose a novel metabolic labeling approach to track RNA in donor cells, EVs and recipient cells using 5-ethynyl uridine (5-EU). 5-EU is a nucleoside analog of uridine that is incorporated into nascent RNA and can be detected through click chemistry with a fluorescent azide. To enhance RNA labeling efficiency, we overexpressed Uridine-Cytidine Kinase 2 (UCK2) in MDA-MB-231 donor cells, which increased 5-EU incorporation into nascent RNA chains. This approach allowed visualization and quantification of RNA in isolated EVs. Furthermore, co-culturing MDA-MB-231 UCK2+ donor cells expressing a CD63-HaloTag fusion protein with HMEC1-eGFP+ recipient cells enabled visualization and quantification of intercellular transfer of EVs and their endogenous RNA cargo. This novel approach offers a sensitive and specific tool for studying physiological EV-RNA transfer, thereby advancing our understanding of the biological roles of EVs in intercellular communication.