A multi-model study of extracellular vesicles in bowel cancer invasion and metastasis

Colorectal cancer (CRC) has one of the highest rates of cancer mortality worldwide. Despite progress in improving screening rates, approximately 35% of CRC patients are diagnosed with stage IV (metastatic) disease which has poor prognosis. In vitro 3D models have been developed which provide useful platforms to explore biomolecular processes in pre-clinical studies of cancer and metastasis, enabling the characterisation of mechanisms which can then be applied to the improvement of diagnosis, prognosis and disease treatment. Extracellular vesicles (EVs) are now known to play a critical role in cancer progression. Not only do they support cancer progression towards metastasis, but as carriers of bioactive cargo, they also show promise as potential cancer biomarkers. In this study, organotypic 3D models mimicking cancer invasion and pre-metastatic niche (PMN) in lung were developed to explore the role of EVs in advanced CRC stages and to evaluate their potential as prognostic markers. After validating a transwell system as an effective 3D model, this work establishes that CRC EVs induce the invasion of cancer cells in a stage-dependent manner, with metastatic SW620 EVs driving a more aggressive phenotype than SW480 EVs. Proteomic profiling of the PMN showed differences in protein content upon treatment and a list of potential m/z signals able to distinguish between SW480 and SW620 EV treatment was obtained by multivariate analysis. CRC EVs also induced α-smooth muscle actin (α-SMA) expression in fibroblasts, demonstrating activation of fibroblasts in the 3D model. Expression and activity of TG2, a multifunctional enzyme involved in cancer progression, was increased after CRC EVs treatment in the 3D model of invasion. This led to the exploration of the cell-specific expression of TG2 in a cancer cells/fibroblasts co-culture, which showed an EV-mediated increase of TG2 on the surface of cancer cells and a non-EV mediated decrease of TG2 expression in the fibroblast surface. TG2 activity was further explored in hepatic stellate cells, crucial cellular components of liver metastasis. Finally, a pilot biomarker study on a small cohort of patients was performed, using multivariate analysis of cell responses to plasma-derived EVs and was able to separate patients according to disease stage. The 3D models established and validated provided biomolecular information about the complex milieu of factors involved in CRC progression, including EVs, thus confirming the importance of 3D culture modelling in pre-clinical research. Moreover, this study was able to demonstrate key roles of cancer EVs on cellular activities which could then be translated for the development of alternative screening and prognostic approaches which may have application in improving the early diagnosis of patients with CRC.