FIGURE 1.

Schematic overview of the experimental workflow. Tissue samples were collected from colorectal cancer patients during surgery. Each patient yielded one tumour tissue sample and one non‐tumour ‘mucosa’ tissue sample to serve as the control, and these were immediately subjected to vesicle extraction after excision. The extraction was performed by a combination of both mechanical and enzymatic means. By cutting the tissue into smaller pieces, EVs were allowed to more readily diffuse into the medium. Collagenase D digestion further aided in this by degrading the extracellular matrix, while DNase 1 prevented the formation of a sticky pellet further downstream in the isolation protocol (Crescitelli et al., 2020). Following the release of EVs into the medium, filtration and a sequential centrifugation protocol were applied to eliminate large tissue pieces, free cells, and cell debris. Lastly, EVs were isolated through an ultracentrifugation protocol with a density cushion flotation as the last step, where buoyant EVs could be collected at the intersection of 1.078 and 1.175 g/mL Optiprep. Protein extraction, digestion, and TMT labelling preceded the final step of mass spectrometry analysis.