Isolation Strategy Shapes the Matrisome Landscape of Cancer-Associated Fibroblast Extracellular Vesicles

Cancer-associated fibroblasts (CAFs) secrete small extracellular vesicles (sEVs) that mediate stromal remodelling, tumour progression, and pre-metastatic niche formation. A foundational assumption in EV research is that MISEV-compliant preparations from the same conditioned medium are biologically equivalent. Here, we directly challenged this assumption through a side-by-side comparison of ultracentrifugation (UC), size exclusion chromatography (SEC), and the EXODUS nanofiltration platform using breast CAF-conditioned media, characterised in accordance with MISEV2023 guidelines using nanoparticle tracking analysis, cryogenic transmission electron microscopy (Cryo-TEM), and quantitative proteomics. EXODUS and SEC recovered approximately 7-fold more particles per mL than UC. While Cryo-TEM confirmed intact vesicle morphology across all methods, UC preparations exhibited substantial non-vesicular background, with gene ontology analysis revealing significant enrichment of ribosomal, mitochondrial, and ER-derived proteins absent from EXODUS and SEC. Matrisome profiling further uncovered method-dependent divergence in the composition of core versus matrisome-associated proteins, highlighting differences extending beyond standard purity metrics. These findings demonstrate that MISEV2023 compliance is necessary but insufficient for methodological equivalence. The isolation method should be treated as a biological variable and selected according to the EV subpopulation or cargo class under investigation.