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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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.
McNutt, M. Cancer immunotherapy. Science 342, 1417 (2013).
Mellman, I., Coukos, G. & Dranoff, G. Cancer immunotherapy comes of age. Nature 480, 480–489 (2011).
Devalaraja, S. et al. Tumor-derived retinoic acid regulates intratumoral monocyte differentiation to promote immune suppression. Cell 180, 1098–1114 (2020).
Lake, R. A. & Robinson, B. W. Immunotherapy and chemotherapy–a practical partnership. Nat. Rev. Cancer 5, 397–405 (2005).
Ngwa, W. et al. Using immunotherapy to boost the abscopal effect. Nat. Rev. Cancer 18, 313–322 (2018).
Min, Y. et al. Antigen-capturing nanoparticles improve the abscopal effect and cancer immunotherapy. Nat. Nanotechnol. 12, 877–882 (2017).
Palucka, K. & Banchereau, J. Cancer immunotherapy via dendritic cells. Nat. Rev. Cancer 12, 265–267 (2012).
Zou, W. Regulatory T cells, tumour immunity and immunotherapy. Nat. Rev. Immunol. 6, 295–307 (2006).
Bennaceur, K. et al. Dendritic cells dysfunction in tumour environment. Cancer Lett. 272, 186–196 (2008).
Wculek, S. K. et al. Dendritic cells in cancer immunology and immunotherapy. Nat. Rev. Immunol. 20, 7–24 (2020).
Barker, H. E., Paget, J. T., Khan, A. A. & Harrington, K. J. The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence. Nat. Rev. Cancer 15, 409–425 (2015).
Dougan, M. & Dougan, S. K. Programmable bacteria as cancer therapy. Nat. Med. 25, 1030–1031 (2019).
Merad, M. & Salmon, H. Cancer: a dendritic-cell brake on antitumour immunity. Nature 523, 294–295 (2015).
Weichselbaum, R. R., Liang, H., Deng, L. & Fu, Y. X. Radiotherapy and immunotherapy: a beneficial liaison? Nat. Rev. Clin. Oncol. 14, 365–379 (2017).
Bell, D. et al. In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J. Exp. Med. 190, 1417–1426 (1999).
Vermi, W. et al. Recruitment of immature plasmacytoid dendritic cells (plasmacytoid monocytes) and myeloid dendritic cells in primary cutaneous melanomas. J. Pathol. 200, 255–268 (2003).
Broz, M. L. et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell 26, 638–652 (2014).
Lavin, Y. et al. Innate immune landscape in early lung adenocarcinoma by paired single-cell analyses. Cell 169, 750–765.e17 (2017).
Jardim, J. F., Gondak, R., Galvis, M. M., Pinto, C. A. L. & Kowalski, L. P. A decreased peritumoral CD1a+ cell number predicts a worse prognosis in oral squamous cell carcinoma. Histopathology 72, 905–913 (2018).
Low, K. B. et al. Construction of VNP20009: a novel, genetically stable antibiotic-sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods Mol. Med. 90, 47–60 (2004).
Zhou, S., Gravekamp, C., Bermudes, D. & Liu, K. Tumour-targeting bacteria engineered to fight cancer. Nat. Rev. Cancer 18, 727–743 (2018).
Toley, B. J. & Forbes, N. S. Motility is critical for effective distribution and accumulation of bacteria in tumor tissue. Integr. Biol. 4, 165–176 (2012).
Bhattacharjee, T. & Datta, S. S. Bacterial hopping and trapping in porous media. Nat. Commun. 10, 2075 (2019).
Bottcher, J. P. et al. NK cells stimulate recruitment of cDC1 into the tumor microenvironment promoting cancer immune control. Cell 172, 1022–1037 (2018).
Jokerst, J. V., Lobovkina, T., Zare, R. N. & Gambhir, S. S. Nanoparticle PEGylation for imaging and therapy. Nanomedicine 6, 715–728 (2011).
Suh, S. et al. Nanoscale bacteria-enabled autonomous drug delivery system (NanoBEADS) enhances intratumoral transport of nanomedicine. Adv. Sci. 6, 1801309 (2019).
Fountoulakis, M., Tsangaris, G., Oh, J. E., Maris, A. & Lubec, G. Protein profile of the HeLa cell line. J. Chromatogr. A 1038, 247–265 (2004).
Pihlasalo, S., Auranen, L., Hanninen, P. & Harma, H. Method for estimation of protein isoelectric point. Anal. Chem. 84, 8253–8258 (2012).
Pan, J. et al. Antigen-directed fabrication of a multifunctional nanovaccine with ultrahigh antigen loading efficiency for tumor photothermal-immunotherapy. Adv. Mater. 30, 1704408 (2018).
Wculek, S. K. et al. Dendritic cells in cancer immunology and immunotherapy. Nat. Rev. Immunol. 20, 7–24 (2020).
Engelhardt, J. J. et al. Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells. Cancer Cell 21, 402–417 (2012).
Klarquist, J. S. & Janssen, E. M. Melanoma-infiltrating dendritic cells: limitations and opportunities of mouse models. Oncoimmunology 1, 1584–1593 (2012).
Gerner, M. Y. & Mescher, M. F. Antigen processing and MHC-II presentation by dermal and tumor-infiltrating dendritic cells. J. Immunol. 182, 2726–2737 (2009).
Raman, V., van Dessel, N., O’Connor, O. M. & Forbes, N. S. The motility regulator flhDC drives intracellular accumulation and tumor colonization of Salmonella. J. Immunother. Cancer 7, 44 (2019).
Roberts, E. W. et al. Critical role for CD103(+)/CD141(+) dendritic cells bearing CCR7 for tumor antigen trafficking and priming of T cell immunity in melanoma. Cancer Cell 30, 324–336 (2016).
Castle, J. C. et al. Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma. BMC Genomics 15, 190 (2014).
D’Alise, A. M. et al. Adenoviral vaccine targeting multiple neoantigens as strategy to eradicate large tumors combined with checkpoint blockade. Nat. Commun. 10, 2688 (2019).
Kreiter, S. et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520, 692–696 (2015).
Krysko, D. V. et al. Immunogenic cell death and DAMPs in cancer therapy. Nat. Rev. Cancer 12, 860–875 (2012).
Chen, Q. et al. Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy. Nat. Commun. 7, 13193 (2016).
Kuai, R., Ochyl, L. J., Bahjat, K. S., Schwendeman, A. & Moon, J. J. Designer vaccine nanodiscs for personalized cancer immunotherapy. Nat. Mater. 16, 489–496 (2017).
Luo, M. et al. A STING-activating nanovaccine for cancer immunotherapy. Nat. Nanotechnol. 12, 648–654 (2017).
Hugo, W. et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell 165, 35–44 (2016).
Lawrence, M. S. et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 499, 214–218 (2013).
Russo, M. et al. Adaptive mutability of colorectal cancers in response to targeted therapies. Science 366, 6472 (2019).
Barkal, A. A. et al. CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy. Nature 572, 392–396 (2019).
Chao, Y. et al. Combined local immunostimulatory radioisotope therapy and systemic immune checkpoint blockade imparts potent antitumour responses. Nat. Biomed. Eng. 2, 611–621 (2018).
Chen, Q. et al. In situ sprayed bioresponsive immunotherapeutic gel for post-surgical cancer treatment. Nat. Nanotechnol. 14, 89–97 (2019).
Chen, W. et al. Bacteria-driven hypoxia targeting for combined biotherapy and photothermal therapy. ACS nano 12, 5995–6005 (2018).
Felfoul, O. et al. Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions. Nat. Nanotechnol. 11, 941–947 (2016).
Zhou, S. et al. Suppression of pancreatic ductal adenocarcinoma growth by intratumoral delivery of attenuated Salmonella typhimurium using a dual fluorescent live tracking system. Cancer Biol. Ther. 17, 732–740 (2016).
Coutermarsh-Ott, S. L., Broadway, K. M., Scharf, B. E. & Allen, I. C. Effect of Salmonella enterica serovar Typhimurium VNP20009 and VNP20009 with restored chemotaxis on 4T1 mouse mammary carcinoma progression. Oncotarget 8, 33601–33613 (2017).
Farache, J. et al. Luminal bacteria recruit CD103+ dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation. Immunity 38, 581–595 (2013).
Yu, Y. A. et al. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat. Biotechnol. 22, 313–320 (2004).
Zhou, S. Synthetic biology: bacteria synchronized for drug delivery. Nature 536, 33–34 (2016).
Hu, Q. et al. Oncogenic lncRNA downregulates cancer cell antigen presentation and intrinsic tumor suppression. Nat. Immunol. 20, 835–851 (2019).
Ma, L. et al. Enhanced CAR-T cell activity against solid tumors by vaccine boosting through the chimeric receptor. Science 365, 162–168 (2019).
Melero, I., Castanon, E., Alvarez, M., Champiat, S. & Marabelle, A. Intratumoural administration and tumour tissue targeting of cancer immunotherapies. Nat. Rev. Clin. Oncol. 18, 558–576 (2021).
Wang, W. et al. Perfluorocarbon regulates the intratumoural environment to enhance hypoxia-based agent efficacy. Nat. Commun. 10, 1580 (2019).
Zheng, D. W. et al. Optically-controlled bacterial metabolite for cancer therapy. Nat. Commun. 9, 1680 (2018).
Hu, Q. L. et al. Engineering nanoparticle-coated bacteria as oral DNA vaccines for cancer immunotherapy. Nano Lett. 15, 2732–2739 (2015).
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).
The authors declare no competing interests.
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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).
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.
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.
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.
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).
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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