Jacob Gillett-Woodley

Abstract

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.

Malignant melanoma has one of the lowest 5‐year survival rates of any cancer and is characterised by its high invasiveness and metastatic potential, with especially poor outcomes in patients who develop brain metastases. Crosstalk between melanoma cells and cells of the tumour microenvironment (TME), including cancer‐associated fibroblasts (CAFs), is a central driver of disease progression. While the role of melanoma‐derived small extracellular vesicles (sEVs) in reprogramming stromal cells has been well documented, the reciprocal effects of CAF‐derived sEVs remain less clear. Here, using an in vitro model of melanoma CAFs, we show that CAF sEVs alter melanoma cells and fibroblasts to promote oncogenic traits and remodel endothelial cells, including brain microvascular cells, in ways consistent with early pre‐metastatic niche (PMN) changes. Multi‐omics cargo profiling revealed significant differential expression of proteins and RNAs linked to extracellular matrix organisation, vascular remodelling, and patient outcomes, with functional validation identifying THBS1 as an EV cargo that restrains endothelial sprouting while potentially promoting barrier destabilisation. Together, these findings suggest that CAF‐derived sEVs contribute to local and distal PMN remodelling, highlight their potential as therapeutic targets, and identify EV cargoes with promise as circulating biomarkers in melanoma.