Trophoblast derived extracellular vesicles trigger secondary vesicle release from endometrial epithelial cells

Extracellular vesicles (EVs) are emerging mediators of cellular communication, carrying bioactive molecules that can alter the function of recipient cells. Yet, how these signals are recognized and interpreted by recipient cells remains unclear. For communication to be effective, signals must reach the right cells at the right time, suggesting that EVs likely target specific cells, an aspect often overlooked in mechanistic studies. In this study, we used an in vitro embryo implantation model consisting of JAr (trophoblast) and RL95-2 (endometrial) cells to explore targeted communication in a physiologically relevant context. Our previous findings showed that JAr-derived EVs specifically induce rapid secretion of milk fat globule epidermal growth factor-8 (MFGE8) from RL95-2 cells. Using MFGE8 as a functional readout, we further investigated the underlying mechanisms of EV mediated targeted cell communication. We found that RL95-2 responses to JAr EVs depend on clathrin-mediated endocytosis and endosomal acidification. Moreover, Rab27a-dependent secondary vesicle release contributed to increased MFGE8 secretion in endometrial cells. Importantly, optimal EV activity required precise pairing between EVs and recipient cells, indicating that EV-triggered vesicle release is not merely a recycling process. Finally, recipient cell stress disrupted vesicle release and impaired responses to EV signals. By combining this in vitro model with MFGE8 as a rapid functional readout, we highlight a natural mechanism of targeted EV communication.