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Fig. 3 WT EV-polarized macrophage supernatant increases cancer cell migration. Primary macrophages were polarized with EVs isolated with the TFF system at a ratio of 1:20,000 (cell:EV), and TCM (TAM-like) for 24 h. The supernatant from the non-polarized M0 and polarized macrophages was added to the HCT116 cells, and the wound closure was assessed over 72 h with an Incucyte live cell imaging system. A Representative images of cancer cells at times 0 and 72 h after the addition of the supernatants. B Statistical analysis was performed for the last time point using one-way ANOVA followed by Bonferroni's post-hoc test. Data are presented as mean ± SD, and p < 0.05 was considered statistically significant (n = 3 individual donors, triplicate). * indicates a significant difference between treatments and M0. # shows a significant difference between WT and KO EVs