Figure 5.

M-HIFU and PD-L1 blockade synergize to reject local tumor. (A) Established MM3MG-HER2 tumors in the legs of BALB/c mice were treated with M-HIFU on day 7. Anti-PD-L1 antibody (100 µg /100 µL) or isotype control IgG (100 µg/100 µL) was injected intraperitoneally 5, 8, and 11 days after M-HIFU treatment. Survival curves are shown and log-rank test was performed. n=13 mice (isotype control), 19 (monotherapy groups) or 18 (combination group), 50 mice in total. (B) Individual tumor growth curves are shown for each treatment group. The numbers in each plot show mice with tumor eradication/mice in the group. (C) Nine days after the initiation of M-HIFU treatment with/without anti-PD-L1 antibody, spleens were collected for immune assays. Induction of HER2 antigen-specific cellular response was analyzed by IFN-γ ELISpot assay using harvested splenocytes and HER2 peptide mix as a stimulating antigen. n=4 per each group. (D) The percentages of CD4⁺, CD8⁺, and CD49b⁺ cells in tumor-infiltrating CD45⁺ cells were analyzed by flow cytometry analysis. n=4 (treatment groups) or 3 (control group). (E, F) Mice were treated with the combination of M-HIFU and anti-PD-L1 antibody as in figure 4A, with or without administration of depleting antibody for CD4⁺, CD8a⁺ and NK cells on days 6, 9, 14, and every 5 days until the end of the experiment. n=5 mice (isotype control) or 6 (other groups), 29 mice in total. (E) Survival curves are shown and log-rank test was performed. (F) Individual tumor growth curves are shown for each cell depletion group. (C, D) Error bars represent SD. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. HER2, human ErbB-2; IFN-γ, interferon gamma; M-HIFU, mechanical high-intensity focused ultrasound; PD-L1, programmed cell death ligand 1.