Fig 2: URB-mediated tumor immunotherapy.a Schematic illustration of URB-mediated tumor immunotherapy to inhibit tumor growth in a 4T1 subcutaneous tumor transplantation mouse model. b Tumor growth curve of tumor-bearing mice treated with different treatments. n = 9 biologically independent animals per group. (Asterisks: URB + US vs every other group). c Survival curves for different treatment groups. d H&E-stained, TUNEL-stained and Ki67-stained tumor slices for each group. Scale bar = 100 µm. Images were representative of three experiments (n = 3 animals per group). e, f Flow cytometric analysis and quantification of M1 macrophages (CD80+F4/80+) in tumor. g, h Flow cytometric analysis and quantification of M2 macrophages (CD206+F4/80+) in tumor. i Flow cytometric quantification of CD4+ T cell (CD4+CD3+) and CD8+ T cells (CD8+CD3+) in tumor. j, k Flow cytometric quantification of Ki67+ (j) and Tim-3+ (k) in CD4+ and CD8+ T cells in tumor. l Flow cytometric quantification of Treg (FOXP3+CD4+) cells in tumor. m, n Flow cytometric analysis and quantification of functional CD4+ T cells (TNF-a+CD4+) in tumor. o, p Flow cytometric analysis and quantification of functional CD8+ T cells (TNF-a+CD8+) in tumor. q, t IFN-? (q), TNF-a (r), IL-1ß (s) and IL-10 (t) levels in tumors at 1, 3, 5, 7 days after different treatments (Asterisks: URB + US vs every other group). For Fig. 5 (e–t), n = 4 biologically independent animals per group. Data were presented as mean ± S.D. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05;). Source data are provided as a Source Data file.

Fig 3: Arg2 TSB modulates surface markers and metabolic parameters in primary BMDM(A–D) Surface markers expression. Pro-inflammatory markers CD80 (A) and CD86 (B) and anti-inflammatory markers CD71 (C) and CD206 (D) surface levels in Arg2 vs NC TSB transfected BMDM (in presence and absence of LPS) were compared by flow cytometry (n = 3, in single). Histograms depict the results obtained in one representative experiment. The bar graphs represent the means of mean fluorescence intensity ± SEM of positive cells in three biological replicates. Data were compared by one-way ANOVA (Sidak’s multiple comparisons test, *p = 0.05, **p < 0.01, ****p < 0.0001). (E) Representative Seahorse metabolic flux trace of BMDM transfected with Arg2 vs NC TSB in absence (left) or presence (right) of LPS (10 ng/mL) (n = 5, 4–7 technical replicates). (F) Quantitative oxidative parameters changes in BMDM transfected with Arg2 vs NC TSB in absence or presence of LPS (n = 5, 4–7 technical replicates). Data were compared by two-way ANOVA (Tukey’s multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 4: In vitro characterization of URB.a Representative SDS-PAGE image of IFN-? protein expressed in URB under different temperature. Images were representative of three experiments. b Quantitative analysis of IFN-? protein content in bacterial culture medium under ultrasound irradiation with different time. n = 3 biologically independent samples per group. c Viability assay of cells treated with bacterial culture medium with different IFN-? concentrations. n = 5 biologically independent cells samples per group (Statistical analysis was calculated by using independent T test). d Fluorescence microscopy image of breast cancer cells stained with Calcein-AM and PI after different IFN-? levels bacterial culture medium treatment (scale bar = 20 µm). Images were representative of three experiments. e Flow cytometry analysis of M1 phenotype macrophages (CD80+) after different IFN-? levels bacterial culture medium treatment. n = 4 biologically independent cells samples per group. f Flow cytometry analysis of M1 phenotype macrophages (CD86+) after different IFN-? levels bacterial culture medium treatment. g Flow cytometry analysis of M2 phenotype macrophages (CD206+) after different IFN-? levels bacterial culture medium treatment. h Quantitative analysis of NO concentrations in the cell culture medium of macrophages RAW 264.7 treated with different bacterial IFN-? concentrations. n = 5 biologically independent cells samples per group. i Cytotoxicity of macrophages stimulated with different IFN-? concentrations to breast cancer cells. MOI ratio = 5:1 (tumor cells:macrophages). n = 5 biologically independent cells samples per group. Data were presented as mean ± S.D. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05;). Source data are provided as a Source Data file.

Fig 5: URB inhibited distant tumor growth and metastasis.a Schematic showing the treatment schedule in a distant tumor mouse model. b Primary tumor growth curve of mice treated with different treatments. n = 9 biologically independent animals per group (Asterisks: URB + US vs every other group). c Distant tumor growth curve of mice treated with different treatments (Asterisks: URB + US vs every other group). d Survival curves for different treatment groups. e Lung photographs and corresponding H&E-stained lung slices collected from the different group mice after treatment. f The number of metastasis foci in the lung of different groups (n = 5). g The weight of the lung of different groups (n = 5). h, i Flow cytometric analysis of M1 macrophages (CD80+F4/80+) in spleen. j, k Flow cytometric analysis of M2 macrophages (CD206+F4/80+) in spleen. l Flow cytometric quantification of CD4+ T cell (CD4+CD3+) and CD8+ T cell (CD8+CD3+) in spleen. m Flow cytometric quantification of Ki67+ in CD4+ and CD8+ T cells in spleen. n Flow cytometric quantification of Treg (FOXP3+CD4+) in spleen. o Flow cytometric quantification of central memory CD4+ and CD8+T cell in spleen. p, q Flow cytometric analysis and quantification of functional CD8+ T cells (TNF-α+CD8+) in spleen. r IFN-γ levels in serum at 1, 3, 5, 7 days after treatments. (Asterisks: URB + US group vs every other group). s TNF-α levels in serum at 1, 3, 5, 7 days after treatments (Asterisks: URB + US group vs every other group). t Flow cytometric quantification of effector memory CD4+ and CD8+T cell in distant tumor. u TGF-β levels in distant tumors at 1, 3, 5, 7 days after treatments (Asterisks: URB + US group vs every other group). For Fig. 6 (h–u), n = 4 biologically independent animals per group. Data were presented as mean ± S.D. Statistical analysis was calculated by using one-way analysis of variance with a Tukey’s test (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05;). Source data are provided as a Source Data file.