Exploring the Spatial Limits of Extracellular Vesicles‐Mediated Intercellular Communication

ABSTRACT Extracellular vesicles (EVs) are biological nanovectors that retain molecular signatures of their cells of origin and mediate intercellular communication, resulting in ideal platforms for the development of diagnostic tools and bio‐inspired drug delivery technologies. Despite their potential, the mechanisms underlying EV release, uptake and distribution remain unclear. Here, we leverage high‐resolution live‐cell imaging, quantitative analytical methods and in vivo mouse models to define the major determinants of EV diffusion. Our findings reveal that cell density plays a crucial role in EV dissemination. Specifically, sparsely distributed cells exhibit higher EV release rates into the supernatant due to increased surface exposure in vitro. In contrast, densely packed cells promote EV internalization and degradation by adjacent cells, effectively restricting EV diffusion in vivo and in vitro. As a result, EVs travel only limited distances, with the majority being internalized by adjacent cells. These findings challenge the prevailing assumption that any EV can act as long‐range messengers and instead highlight their function as short‐range communication agents primarily confined within their tissue or organ of origin. This study provides fundamental insights into EV‐mediated intercellular signalling and has important implications for their use in diagnostic and therapeutic applications.