Small Extracellular Vesicles Isolation

HCT116 and HT29 cells were plated in T75 flasks (5 × 10⁴ cells/cm²) and grown in a complete medium for 48 h until they reached approximately 85–95% confluence. After, cells were washed three times with 0.22-μm filtered PBS and incubated with McCoy’s 5A culture medium without FBS for the following 24 h. After that, the conditioned medium was collected. Conditioned medium was precleared by centrifugation at 4,000 × g for 30 min to remove cell debris. The supernatant was filtered through 0.22-μm filtered PBS and concentrated 30× using a Vivaspin Turbo 15 10,000 MWCO (Sartorius, United Kingdom) filter capsule by centrifugation at 4,000 × g for 15 min. After that, the concentrates were centrifuged at 15,000 × g for 20 min to remove microvesicles (Patel et al., 2019). Next, 0.5 v:v of Total Exosome Reagent (Thermo Fisher Scientific, MA, United States) was added to the concentrated medium and incubated overnight at 4°C under agitation. Next, samples were centrifuged at 10,000 × g, 4°C for 1 h. The fraction of pelleted sEVs was washed in 0.22-μm filtered PBS followed by the second step of 10,000 × g centrifugation at 4°C for 1 h. Pelleted sEVs were resuspended in 100 μl of 0.22-μm filtered PBS. The remaining cells (EVs donor cells) were scraped and lysed for Western blotting, as described in the following section. We have submitted all relevant data to the EV-TRACK knowledgebase (EV-TRACK ID: {"type":"entrez-nucleotide","attrs":{"text":"EV210162","term_id":"151310125","term_text":"EV210162"}}EV210162; Van Deun et al., 2017).