EVs were purified from approximately 50 mL of cell culture medium collected from U87, A549, MIA PaCa-2 and HT-29 cell lines via differential ultracentrifugation, followed by lysis in a lysis buffer as previously described [18 (link)]. Protein lysates were separated by 10% sodium lauryl sulphate polyacrylamide (SDS-PAGE) gel, and then transferred onto apolyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich). The membranes were blocked andincubated at 4°C overnight with either an anti-EpcamAb (R&D systems, MAB960) or an anti-tsg101 Ab (Abcam, ab1337586), as tsg101 is reported to be widely expressed in EVs and could be used for normalization [19 ,20 (link)]. After being incubated with a secondary antibody (anti-mouse IgG (HAF007, R&D systems) for anti-Epcam, anti-rabbit IgG (ab205718, Abcam) for anti-tsg101) for 1 h at room temperature, the signals were visualized using the LI-COR Image Studio lite imaging system. The relative Epcam levels in EVs from different cell lines were normalized to the expression of tsg-101.