Spatiotemporal control of engineered bacteria to express interferon-γ by focused ultrasound for tumor immunotherapy

Abstract

Bacteria-based tumor therapy has recently attracted wide attentions due to its unique capability in targeting tumors and preferentially colonizing the core area of the tumor. Various therapeutic genes are also harbored into these engineering bacteria to enhance their anti-tumor efficacy. However, it is difficult to spatiotemporally control the expression of these inserted genes in the tumor site. Here, we engineer an ultrasound-responsive bacterium (URB) which can induce the expression of exogenous genes in an ultrasound-controllable manner. Owing to the advantage of ultrasound in tissue penetration, an acoustic remote control of bacterial gene expression can be realized by designing a temperature-actuated genetic switch. Cytokine interferon-γ (IFN-γ), an important immune regulatory molecule that plays a significant role in tumor immunotherapy, is used to test the system. Our results show that brief hyperthermia induced by focused ultrasound promotes the expression of IFN-γ gene, improving anti-tumor efficacy of URB in vitro and in vivo. Our study provides an alternative strategy for bacteria-mediated tumor immunotherapy.

Fig. 1. Schematic diagram of URB in controlling IFN-γ expression by focused ultrasound and their mechanisms for cancer immunotherapy.

Upon systemic administration of URB that contains IFN-γ gene inserted in the temperature-sensitive genetic circuit, these genetically engineered bacteria would deliver into the tumors due to their tumor-targeting capability. Then, the tumor was irradiated by focused ultrasound to heat these intratumoral bacteria to 42–45°C for inducing the expression of IFN-γ gene. The production and secretion of IFN-γ not only can promote the apoptosis of cancer cells, but also induce the macrophage polarization from M2 to M1 phenotype and the activation of CD4⁺ and CD8⁺ T cells (Right panel).In addition, high levels of IFN-γ can also activate macrophages and T cells in the spleen to inhibit lung metastasis and distant tumor growth through immune memory responses (Left panel).

Fig. 2. The gene expression of URB trigged by hyperthermia and ultrasound.

a Mechanism of mCherry/IFN-γ expression based on pBV220 plasmid in the bacteria. b Fluorescence image of mCherry protein in URB under different temperature. Images were representative of three experiments. c Fluorescence image of mCherry protein in URB under 45 °C with different time. Images were representative of three experiments. d Quantification of temperature of bacteria solution irradiated with the different acoustic pressures by focused ultrasound. Experiments were performed three times independently with similar results. e Quantification of fluorescence signals of bacteria solution after ultrasound irradiation. n = 3 biologically independent samples at each time point. Data were presented as mean ± S.D. f Fluorescence image of mCherry protein of URB in the center of the gel phantom under ultrasound irradiation. Images were representative of three experiments. g Fluorescence image of mice injected with 1 × 10⁷ CFU of URB under liver-targeted ultrasound irradiation. Images were representative of three experiments. h Quantification of fluorescence signals of major organs of mice injected with 1 × 10⁷ CFU of URB under liver-targeted ultrasound irradiation. The fluorescent intensity was normalized by organ size. n = 3 biologically independent 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. 3. In vitro characterization of URB.

a Representative SDS-PAGE image of IFN-γ protein expressed in URB under different temperature. Images were representative of three experiments. b Quantitative analysis of IFN-γ 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-γ 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-γ 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-γ 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-γ levels bacterial culture medium treatment. g Flow cytometry analysis of M2 phenotype macrophages (CD206⁺) after different IFN-γ 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-γ concentrations. n = 5 biologically independent cells samples per group. i Cytotoxicity of macrophages stimulated with different IFN-γ 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. 4. Tumor targeting of URB.

a In vivo fluorescence imaging of tumor-bearing mice different time after intravenous injection of 1 × 10⁷ CFU of DiR-stained live URB or heating-inactivated URB. Images were representative of three experiments. b, c The intensities of fluorescence imaging (b) and corresponding fluorescence signal (c) of tumors and major organs of tumor-bearing mice 48 h after intravenous injection of 1 × 10⁷ CFU of DiR-stained live URB or heating-inactivated URB. n = 3 biologically independent animals per group. (Statistical analysis was calculated by using independent T test.) d Fluorescence microscopy images of tumors and major organs of tumor-bearing mice 48 h after intravenous injection of 1 × 10⁷ CFU of DiR-stained URB (scale bar = 25 μm). Images were representative of three experiments (n = 3 animals per group). e Immunofluorescence staining of tumor slices from peritumoral region (left) and central region (right). DAPI stands for the nuclei of tumor cells, red for DiR-stained live URB and green for HIF-1α highly expressed cells. Scale bar = 50 μm. Images were representative of three experiments (n = 3 animals per group). f Photographs of solid LB agar plates of bacterial colonization in tumors and major organs collected from tumor-bearing mice at different time points after intravenous administration of 1 × 10⁷ CFU of URB. g Quantification of bacterial colonization in major organs and tumors collected from tumor-bearing mice at different time points after administration of 1 × 10⁷ CFU of live URB (Asterisks: Tumor vs every other organs). n = 3 biologically independent animals per group at each time point. 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-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-α⁺CD4⁺) in tumor. o, p Flow cytometric analysis and quantification of functional CD8⁺ T cells (TNF-α⁺CD8⁺) in tumor. q, t IFN-γ (q), TNF-α (r), IL-1β (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. 6. 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.

Fig. 7. Growth inhibition effect of orthotopically transplanted liver tumor.

a Schematic showing the treatment schedule in the orthotopically transplanted liver tumor model (n = 8). b Representative bioluminescence images of liver tumor-bearing mice in different groups. c Bioluminescence quantification of liver tumor-bearing mice treated with different treatments. n = 8 biologically independent animals per group (Asterisks: URB + US group vs every other group). d Survival curves of tumor-bearing mice for different treatment groups. e Weight change of mice treated with different treatments. 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.