EV-Tracer enables lineage-resolved detection and molecular profiling of extracellular vesicle-associated signals in cancer–fibroblast coculture.

However, EVs released by distinct cellular lineages become difficult to distinguish when mixed in multicellular experimental models, limiting the analysis of how cell–cell interactions influence EV-associated molecular profiles. Here, we developed EV-Tracer, a CD63-based dual-fluorescence tracing and capture system for detecting, isolating, and profiling lineage-associated EV fractions.

Achilles or mScarlet was inserted into the small extracellular loop of CD63, enabling tracer-specific EV detection by digital counting, antibody-based isolation, and live-cell visualisation. Exploratory EV RNA sequencing suggested that physical cell–cell contact was associated with distinct EV RNA profiles, including interferon-related signals, which were supported by targeted cellular and EV-associated RNA analyses. EV-Tracer provides a practical framework for investigating lineage-associated EV dynamics and molecular signals in mixed-cell systems.