Extracellular Vesicles Drive Invasive Characteristics and Matrix Remodelling in Organotypic Models of Colorectal Cancer Progression

Colorectal cancer (CRC) remains a leading cause of cancer‐related mortality, with metastasis accounting for over 90% of CRC deaths. While the role of extracellular vesicles (EVs) in cancer progression is recognised, their impact on invasive behaviour and extracellular matrix (ECM) remodelling within physiologically relevant three‐dimensional (3D) microenvironments remains poorly understood. This study utilised quantitative organotypic 3D models to investigate how EVs derived from primary (SW480) and metastatic (SW620) CRC cell lines influence invasion, stromal activation, and ECM remodelling. We developed models incorporating CRC cells, fibroblasts, endothelial cells, and macrophages to mimic the tumour microenvironment (TME) and lung stroma. EVs isolated from SW480 and SW620 cells were isolated and characterized according to MISEV guidelines and introduced into the models. Treatment with metastatic SW620 EVs significantly enhanced depth and extent of CRC cell invasion compared to primary SW480 EVs or controls, and increased invasion of multicellular clusters. Immunofluorescence analysis revealed elevated expression of cadherin 2 (CADH2) and catenin delta 1 (CTNND1) in SW620 EV‐treated models, indicating involvement of epithelial‐mesenchymal transition (EMT). In lung stroma models, SW620 EVs reduced matrix stiffness, implying ECM remodelling. Mass spectrometry and multivariate analysis identified distinct proteomic signatures in SW620 EV‐treated models, with significant alterations in collagen type XI expression and unique mass‐to‐charge (m/z) peaks, indicating selective ECM remodelling. SW620 EVs also induced activation of stromal fibroblasts and endothelial cells, as evidenced by increased α‐smooth muscle actin (α‐SMA) and von Willebrand Factor (vWF) expression. These findings demonstrated that metastatic CRC‐derived EVs enhance invasive behaviour and remodel the ECM, creating a permissive microenvironment for metastasis. The differential effects of primary versus metastatic EVs underscore the importance of tumour stage‐specific vesicle signatures in CRC progression. This study provides a robust 3D model framework to quantify EV‐mediated mechanisms and identify therapeutic targets to disrupt pro‐metastatic communication in CRC.