Single-vesicle heterogeneity and uptake of extracellular vesicles affected by oncogenic KRAS and BRAF revealed by proteomics and nano-flow cytometry

Extracellular vesicles (EVs) facilitate intercellular communication through the transfer of bioactive molecules that modulate tumor behavior. In this study, we demonstrate that oncogenic KRAS- and BRAF-mutations alter EV composition, modify surface properties, and impact intracellular trafficking. Through comparative proteomics, uptake assays, and nano-flow cytometry, we found that mutant EVs display increased levels of CD44, LSR, ITGB4, and CSPG4, leading to enhanced uptake and preferential trafficking to the endoplasmic reticulum and nucleus, while avoiding lysosomal degradation. Disruption of CD44 and CSPG4-mediated interactions with hyaluronic acid or chondroitin sulfate, respectively, reduced EV internalization and lysosomal trafficking, verifying their roles in mutation-dependent uptake. Notably, doxorubicin-loaded mutant EVs showed improved nuclear delivery and increased cytotoxicity compared to wild-type EVs, indicating that mutation-specific EVs function as efficient drug delivery vehicles. Collectively, these results reveal a direct association between oncogenic signaling and EV diversity, highlighting the potential of mutation-adapted EVs for precise delivery of therapeutics to specific subcellular compartments.