Figure 3. Combined treatment with BiTN_HER and aCD47 induces antitumor immune responses in HER2/neu-expressing mouse tumor model.

a,b, Combination of BiTN_HER and aCD47 treatment inhibited the growth of HER2/neu^high TUBO breast cancer cells and prolonged the survival of BALB/c mice, compared with HER2/neu^low 4T1 breast cancer cells. ***P<0.001 by log-rank test (n=6). c, Combined treatment with BiTN_HER and aCD47 induced the greatest tumor-inhibition effect in HER2/neu-expressing TUBO tumors, with 3 of 6 mice remaining tumor-free (TF). d-g, Immune cell infiltration profiles within HER2/neu^high TUBO tumors and HER2/neu^low 4T1 tumors (n=3). (d) Combined treatment with BiTN_HER and aCD47 promoted the infiltration of dendritic cells (DCs) and macrophages within TUBO tumors relative to 4T1 tumors. (e) Combined treatment with BiTN_HER and aCD47 increased the infiltration of CD4⁺ T and CD8⁺ T cells into TUBO tumors. (f,g) Combined treatment with BiTN_HER and aCD47 increased the number of IFNγ-producing CD8⁺ T cells in TUBO tumors and decreased the number of intratumoral regulatory CD4⁺ T cells. h, Combined treatment with BiTN_HER and aCD47 increased the peripheral-blood cytokine levels in TUBO tumor-bearing mice but not in 4T1 tumor-bearing mice (n=3). For all figures, the data are presented as mean±s.e.m.; *P<0.05, **P<0.01, ****P<0.0001 by one-way ANOVA with a Bonferroni post hoc correction; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by unpaired Student’s t-test for the indicated comparisons; n.s., not significant.