Fig. 4

Antitumor immune effects of US in combination with checkpoint blockade. a Schematic illustration of our experiment design. Mice with 4T1 tumors on both sides were used. Tumors on the right side were designated as “primary tumors” for US, and those on the left side were designated as “distant tumors” without direct exposure to US. Growth curves and excised tumors at the end of treatments for b, c primary tumors and d, e distant tumors on mice after various treatments indicated. (n = 5). Statistical significances were calculated via one-way ANOVA Tukey's multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001. f, g Representative flow cytometry plots showing different types of T cells in secondary tumors from different groups of mice, and h, i proportions of tumor-infiltrating CD4 + and CD8 + T cells among all cancer cells. Proportions of Foxp3 + Tregs among CD4 + T cells. (n = 3) Statistical significance was calculated by one-way ANOVA Dunnett's multiple comparisons test. P-value: *, P < 0.05;**, P < 0.01; ***, P < 0.001. j In vivo apoptosis and/or necrosis of the tumour induced by different treatment, as shown by H&E staining and TUNEL assay; representative CLSM images of the mimic distant tumours after immunofluorescence staining (scale bar = 50 μm)