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Systemic immune responses to irradiated tumours via the transport of antigens to the tumour periphery by injected flagellate bacteria

Abstract

Because the tumour microenvironment is typically immunosuppressive, the release of tumour antigens mediated by radiotherapy or chemotherapy does not sufficiently activate immune responses. Here we show that, following radiotherapy, the intratumoural injection of a genetically attenuated strain of Salmonella coated with antigen-adsorbing cationic polymer nanoparticles caused the accumulation of tumour antigens at the tumour’s periphery. This enhanced the crosstalk between the antigens and dendritic cells, and resulted in large increases in activated ovalbumin-specific dendritic cells in vitro and in systemic antitumour effects, and extended survival in multiple tumour models in mice, including a model of metastasis and recurrence. The antitumour effects were abrogated by the antibody-mediated depletion of CD8+ T cells, indicating that systemic tumour regression was caused by adaptive immune responses. Leveraging flagellate bacteria to transport tumour antigens to the periphery of tumours to potentiate the activation of dendritic cells may open up new strategies for in situ cancer vaccination.

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Fig. 1: Characterization of the movement of antigen-capturing bacteria in vitro.
Fig. 2: Movement of antigen-capturing bacteria in vivo, and enhanced immune responses and abscopal effects in B16-OVA tumour-bearing mice.
Fig. 3: Analysis of proteins captured by antigen-capturing bacteria after incubation with CT26-tumour-tissue-derived proteins.
Fig. 4: Antigen-capturing bacteria improve immune responses and trigger the abscopal effect in CT26-tumour-bearing mice.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, but they are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

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Acknowledgements

This research was supported by the National Key R&D Program of China (2017YFA0205400), the National Natural Science Foundation of China (No. 32171372, 31872755 and 81872811) and Jiangsu Outstanding Youth Funding (BK20190007). Transgenic OT-1 mice were generously gifted by Prof. H. Tang (Shandong First Medical University, China) and VNP20009 were kindly provided by Prof. Z. Hua (Nanjing University, China).

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Authors and Affiliations

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Contributions

W.W. and J.W. conceived and designed the experiments. W.W., H.X., Q.Y. and F.T. performed the experiments. H.X., Q.Y., F.T., Y.H. and J.W. assisted in the data analysis. J.W., W.W. and I.W. prepared the manuscript. J.W., A.Y. and Y.H. supervised the project.

Corresponding authors

Correspondence to Yiqiao Hu or Jinhui Wu.

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The authors declare no competing interests.

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Nature Biomedical Engineering thanks Jeffrey Hubbell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Quantitative analysis of tumour-infiltrating CD103+ DCs, and ex vivo immunofluorescence staining of OVA+ CD103+ DCs in tumour marginal tissue.

a, Fold change of the intratumoural MHCII+CD11c+CD103+ DCs compared with RT + dB+ treatment 72 h post-injection of antigen-capturing bacteria (bacteria, 5·106 CFU per mouse; OVA-FITC, 5 mg·kg−1; n = 5, biologically independent animals). b, Ex vivo immunofluorescence staining of OVA+ CD103+ DCs in distal tumour marginal tissue. The images of tumour marginal tissue were captured 12 h after intratumoural injection of the antigen-capturing bacteria (5·106 CFU per mouse; OVA-FITC, 2.5 mg·kg−1). OVA, DCs, and tumour nuclei were stained with FITC (green), anti-CD11c-PE antibody (yellow), anti-CD103-APC antibody (red), and DAPI (blue), respectively. Scale bar, 30 μm. c, Semiquantitative analysis of the OVA distribution in distal tumour margin (n = 5, biologically independent animals).

Source data

Extended Data Fig. 2 Antigen-capturing bacteria improve immune responses and trigger abscopal effects in CT26-tumour-bearing mice.

a, Schematic of the therapeutic treatments for inhibiting the growth of primary and re-challenged CT26 tumours. Irradiated tumours are indicated as ‘primary tumour’ and were treated with antigen-capturing bacteria, and the re-challenged tumours are labelled as ‘secondary tumour’ and were not treated. b,c, Average tumour growth curves of primary (b, n = 10, biologically independent animals) and secondary CT26 tumours (c) post-treatment. d, Representative photographs of the CT26 tumours 32 days after the corresponding treatments. For the experiments in b and c, data are the mean ± s.e.m. Statistical significance was determined by a two-way ANOVA with Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 3 Enhanced therapeutic and abscopal effects by the combination of antigen-capturing bacteria and αPD-L1 antibodies in bilateral CT26-tumour-bearing mice.

a, Schematic of the experimental design to inhibit the growths of bilateral CT26 tumours. The irradiated tumour (5 Gy) is indicated as ‘primary tumour’, and was treated with antigen-capturing bacteria (107 CFU per mouse) and the second tumour is designated as ‘secondary tumour’, and was not treated. b, Response rate of the primary and secondary tumours (saline, n = 8; RT, n = 9; RT + PD-L1, RT + B+ + PD-L1, RT + B + PD-L1, n = 10). c, Weight changes of tumour-bearing mice during the treatments (saline, n = 8; RT, n = 9; RT + PD-L1, RT + B+ + PD-L1, RT + B + PD-L1, n = 10). d-g, Flow cytometric analysis of CD3+ T cells (d; RT, RT + PD-L1, n = 7; Saline, RT + B+ + PD-L1, RT + B + PD-L1, n = 8), CD4+ T cells (e), CD8+ T cells (f) and regulatory T cells (Treg, CD4+CD25+; g). All of these infiltrated immune cells were collected from the secondary tumour on day 22. For the experiments in d-g, data are the mean ± s.e.m. Statistical significance was determined by analysis of one-way ANOVA with Tukey’s multiple comparisons test.

Source data

Extended Data Fig. 4 Enhanced therapeutic efficacy and abscopal effects, triggered by antigen-capturing bacteria in 4T1-tumour-bearing mice.

a, Schematic of the therapeutic treatment in female 4T1 tumour-bearing mice. The Irradiated tumour (5 Gy) is indicated as ‘primary tumour’ and was treated with antigen-capturing bacteria (107 CFU per mouse) and the re-challenged tumour (day 19, 5·104 cells per mouse) is labelled as ‘secondary tumour’, and was not treated. b-c, Averaged growth curves of primary (b, n = 10, biologically independent animals) and secondary tumours (c). d, Survival percentage of 4T1 tumour-bearing mice. e, Free percentage of the secondary 4T1 tumour. For the experiments in b and c, data are the mean ± s.e.m. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. Differences in survival were determined by using the Kaplan–Meier method, and the P value was determined via the log-rank test.

Source data

Extended Data Fig. 5 Enhanced immune responses by antigen-capturing bacteria in 4T1-tumour-bearing mice.

a-c, Flow cytometric analysis of CD4+ (a), CD8+ (b, gate: tumour cell) and Treg cell (CD25+Foxp3+ %, gate: CD4+ T cell, c) in secondary 4T1 tumour (RT + B, RT + B+, n = 4; RT + BmPEG, n = 5; biologically independent animals). d-e, The ratios of CD8+ to Treg (d) and CD4+ T cell to Treg (e). f, Flow cytometric analysis of effector memory T cells (TEM, CD3+CD8+cd44+CD62L) 30 days after treatments (n = 5, biologically independent animals) and their representative flow cytometry plot (g). For the experiments in a-f, data are the mean ± s.e.m., statistical significance was determined by analysis of one-way ANOVA with Tukey’s multiple comparisons test (a-e) and two-way ANOVA with Dunnett’s multiple comparisons test (f).

Source data

Extended Data Fig. 6 Inhibition of 4T1 tumour metastasis by antigen-capturing bacteria.

a, Schematic of the therapeutic treatment to inhibit 4T1-tumour metastasis. b, Representative images of lung tissues fixed by Bouin’s solution. c, Panoramic scanning of H&E sections of lung metastases and the images of H&E slices in the liver. The black arrow indicates metastatic lesions in the lung and liver tissues. Scale bar, 1,000 μm. d, Quantification of metastatic lesions in lung tissues. e, Survival percentage of 4T1 tumour bearing mice after treatments. For the experiments in d, data are the mean ± s.e.m. (RT, RT + dB+, RT + B, n = 7; Saline, n = 8; RT + B+, n = 6; biologically independent animals, two-tailed Student’s t-test).

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Wang, W., Xu, H., Ye, Q. et al. Systemic immune responses to irradiated tumours via the transport of antigens to the tumour periphery by injected flagellate bacteria. Nat Biomed Eng 6, 44–53 (2022). https://doi.org/10.1038/s41551-021-00834-6

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