EV-Tracer enables lineage-resolved detection and molecular profiling of extracellular vesicle-associated signals in cancer–fibroblast co-culture
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Extracellular vesicles (EVs) facilitate intercellular communication by transferring diverse bioactive molecules from donor to recipient cells. 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 affect EV-associated molecular profiles. To address this, EV-Tracer, a CD63-based dual-fluorescence tracing and capture system for detecting, isolating, and profiling lineage-associated EV fractions, was developed. 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.
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Brief abstract
EV-Tracer, in which Achilles or mScarlet fluorescent proteins were inserted into the small extracellular loop of CD63, enabled tracer-specific detection and affinity capture of lineage-associated EV fractions in mixed-cell culture conditions. The platform connects ExoCounter quantification, live-cell imaging and downstream RNA profiling. In a proof-of-concept application, exploratory RNA sequencing and independent qRT-PCR indicated that direct cell contact was associated with interferon-related EV RNA signals. In summary, our approach provides a useful platform for dissecting lineage-associated EV communication.
