Fig. 6: EV-TβRII antagonizes the anti-tumor immunity and thus elevates tumor growth.

a–j Experimental analysis in vivo: BALB/c mice were nipple injectied with 4T1 cells (2 × 10⁵ cells per mouse) expressing control shRNA or doxycycline-inducible shRNA targeting TβRII (shTβRII) and tumors were grown for 3 weeks, followed by the administration of doxycycline (Dox) (a left panel). Primary tumor formation in each group at day 21 after implantation is shown (n = 6 mice per group); Normalized BLI signals (a right panel). Percentage of TβRII⁺ crEVs in plasma from mice b. Immunoblot analysis of TβRII in primary tumor, metastatic tumor and circulating EVs from plasma in mice c. Representative BLI signals of primary tumor d. Normalized BLI signals (left panel) and representative bioluminescent view of isolated lung (right panel) e; Scale bar, 2 mm. FACS analysis and quantification of the percentage of CD8⁺ or CD4⁺ cells from lymph nodes f and spleen g; FACS analysis and quantification of IFNγ⁺ h or GZMB⁺ i of CD8⁺ cells in tumor-infiltrating lymphocytes (TIL) populations from mice. FACS analysis and quantification of the percentage of tregs j. k Experimental analysis in vivo: BALB/c mice were nipple injected with 4T1 cells (5 × 10⁵ cells per mouse), followed by tail vein-injection of EVs derived from control 4T1 cells (TβRII⁺) or TβRII knock-out 4T1 cell (TβRII⁻) (50 μg per mouse every other day) for 3 weeks (left) (n = 5 mice per group). Tumor volume measured in time (right). l Quantification of the percentage of GZMB⁺ of CD8⁺ cells (left panel), IFNγ⁺ of CD8⁺ cells (middle panel) from draining lymph node (DLN) and tumor-infiltrating lymphocyte (TIL) and PD1⁺, TIM3⁺ of CD8⁺ cells from TIL (right panel). m BLI signals of lung metastasis (left panel), number of lung metastasis nodules (middle panel) and percentage of TβRII⁺ crEVs in plasma (right panel) of all mice in each experimental group at week 5 were shown. n Pearson correlation analysis of the ELISA-detected levels of IFN-γ and TβRII⁺ crEVs in patients with breast cancer (n = 46 samples). ns, not significant (p > 0.05) and *p < 0.05 (unpaired two-tailed Student’s t test b, e, f–j, l, m or two-way ANOVA a, k, n). Data are analyzed from three independent experiments and shown as mean + SD f–j, l or as means ± SD a, b, e, k, m. Source data are provided as a Source Data file.