a–c Unloaded EVs, CDN2 (20 µg), and exoCDN2 (0.1 µg) were injected intratumorally at Day 1 and Day 4 (n = 3 animals per group) into B16F10 tumors. Tumors were collected at 4 and 24 h after first dose and second doses. a Tumor sections were stained with H&E, or for IFN-β mRNA, CD8, and F4/80 expression. IFN-β positive area (b) and co-localization of F4/80 (green) and IFN-β (red) after free CDN2 (20 µg) and exoCDN2 (0.1 µg) treatment (c). CD8 positive cells (d) were measured. e, f Percentage of CD8 T cells in live CD45+ cells in tumors (e) and CD86 expression on dendritic cells (f) were measured by flow cytometry after two doses of PBS, CDN2 (20 µg), and exoCDN2 (0.2 µg) into B16F10 tumors (n = 5 animals per group). Data are presented as means ± s.e.m from replicate samples as indicated. *P < 0.05; **P < 0.01; ****P < 0.0001 by one-way ANOVA with Tukey’s multiple comparison test; n.s. non-significant.