Surface or Luminal? Decoding Tumor Extracellular Vesicle Logic for Functional Insight and Therapeutic Reprogramming

Abstract Extracellular vesicles (EVs) hold great promise for diagnostics and therapeutics, but clinical translation is hindered by their heterogeneity and the lack of tools for spatially resolved, subpopulation‐specific functional analysis. Here, the EV bimodal functional regulator (eBFR), an integrated platform combining: (1) CLEAR (Cargo Luminal Elimination to Attenuate Risk) for selective luminal depletion while preserving surface integrity; (2) SWITCHER (SWITCHable EV Releaser) for modular EV subpopulation enrichment; and (3) eSimoa (EV single‐molecule array) for ultrasensitive, compartment‐specific protein quantification. Using tumor‐derived EVs, CLEAR removed pro‐tumor luminal cargo—attenuating their pro‐tumorigenic activity—while preserving preferential uptake by autologous tumor cells, enhancing drug loading by 8430‐fold and improving anticancer efficacy by 37%. Omics‐guided functional assays reveal a spatial “division of labor,” with surface proteins driving uptake and luminal cargo regulating proliferation and migration. These insights inform a 3D EV Functional Decision Matrix linking spatial molecular signatures to phenotypic outcomes. Applied to SARS‐CoV‐2 Spike EVs, eBFR uncovers dose‐, compartment‐, and cell type‐dependent effects, including a “Trojan Horse” mechanism for viral protein transfer and propagation via secondary EVs. eBFR offers a mechanistically informed framework for decoding EV biology, reprogramming vesicle function, and guiding rational design of biosafety‐enhanced, next‐generation EV‐based therapeutics in precision nanomedicine.