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Oncolytic mineralized bacteria as potent locally administered immunotherapeutics

Nature Biomedical Engineering (2024)Cite this article

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Abstract

Oncolytic bacteria can trigger innate immune activity. However, the antitumour efficacy of inactivated bacteria is poor, and attenuated live bacteria pose substantial safety risks. Here we show that intratumourally injected paraformaldehyde-fixed bacteria coated with manganese dioxide potently activate innate immune activity, modulate the immunosuppressive tumour microenvironment and trigger tumour-specific immune responses and abscopal antitumour responses. A single intratumoural administration of mineralized Salmonella typhimurium suppressed the growth of multiple types of subcutaneous and orthotopic tumours in mice, rabbits and tree shrews and protected the cured animals against tumour rechallenge. We also show that mineralized bacteria can be administered via arterial embolization to treat orthotopic liver cancer in rabbits. Our findings support the further translational testing of oncolytic mineralized bacteria as potent and safe antitumour immunotherapeutics.

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Fig. 1: Preparation, characterization and immune activation of oncolytic mineralizing bacteria.
Fig. 2: Mineralized bacteria for in vivo tumour treatment in multiple tumour models.
Fig. 3: Mineralized bacteria activates multiple immune responses in vivo.
Fig. 4: Systemic immune response activation and immune protection induced by mineralized bacteria.
Fig. 5: Oncolytic mineralized bacteria for optimized treatment.
Fig. 6: Oncolytic mineralized bacteria-based TAIE therapy for orthotopic liver tumours.

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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 available for research purposes from the corresponding authors on reasonable request. Source data for the figures are provided with this paper.

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Acknowledgements

This work was partially supported by the National Research Programs of China (2021YFF0701800), the National Natural Science Foundation of China (T2321005, 52032008, 32071382, 21927803), the New Cornerstone Science Foundation through the XPLORER PRIZE, Program for Changjiang Scholars and Innovative Research Team in University (number IRT_15R13), Suzhou Science and Technology Development Project—Science and Technology Innovation in Medicine and Health Care (SKY2021033), the Suzhou Key Laboratory of Nanotechnology and Biomedicine, Collaborative Innovation Center of Suzhou Nano Science and Technology, and the 111 Program from the Ministry of Education of China.

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  1. These authors contributed equally: Chenya Wang, Liping Zhong.

Authors and Affiliations

  1. Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based Functional Materials and Devices, Soochow University, Suzhou, China

    Chenya Wang, Qi Zhuang, Fei Gong, Xiaojing Chen, Nailin Yang, Junyan Li, Qi Zhao, Qian Chen, Rui Peng & Zhuang Liu

  2. National Center for International Research of Biotargeting Theranostics, Guangxi Key Laboratory of Biotargeting Theranostics, Collaborative Innovation Center for Targeting Tumor Diagnosis and Therapy, Guangxi Medical University, Nanning, China

    Liping Zhong, Cong Hu, Fuquan Huang, Xinjun Sun, Yu Huo & Yongxiang Zhao

  3. Department of Interventional Radiology, The First Affiliated Hospital of Soochow University, Suzhou, China

    Jiachen Xu

  4. InnoBM Pharmaceuticals Co. Ltd., Suzhou, China

    Huiquan Tao, Qi Zhao & Zhuang Liu

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Contributions

Z.L., R.P., Y.Z., C.W. and L.Z. conceived and designed the study. C.W., L.Z., J.X., Q. Zhuang, F.G., X.C., H.T., C.H., F.H., N.Y., J.L., Q. Zhao, X.S., Y.H. and Q.C. performed the experiments. C.W., L.Z., Q. Zhuang, F.G. and C.H. participated in data analysis. The manuscript was written by C.W., Z.L., L.Z. and R.P.

Corresponding authors

Correspondence to Yongxiang Zhao, Rui Peng or Zhuang Liu.

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Competing interests

Soochow University has filed patent applications related to this work which are licensed to InnoBM Pharmaceuticals, listing Z.L., R.P., C.Y.W., Q. Zhuang and Q. Zhao as inventors. The application numbers are 2021112844294 (substantive examination) and PCT/CN2022/097190 (public), respectively. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Guangjun Nie, Jinhui Wu and Xian-Zheng Zhang for their contribution to the peer review of this work

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Extended data

Extended Data Fig. 1 Mineralized F.S bacteria activate multiple immune responses in vivo 1 day post treatments.

The statistical data of CD45+CD11b+ leukocytes (a), TNF-α (b), IL-1β (c), IL-6 (d) and IFN-β (e) levels in the tumor microenvironment on Day 1 post treatments in Fig. 3 (n = 6). Replicates are biological (n = 6) and the data are expressed as mean ± s.e.m. Significance calculated using a two-tailed Student’s t-test.

Source data

Extended Data Fig. 2 Mineralized F.S bacteria activate multiple immune responses in vivo 5 days post treatments.

(a) CD45+CD11b+ leukocytes in the tumor microenvironment on Day 5 post treatments in Fig. 3. (b&c) Mean fluorescence intensities of CD80 (M1-like macrophages) and CD206 (M2-like macrophages) gated on F4/80+CD11b+CD45+ cells in Fig. 3K. (d-g) Cytokine levels including IFN-γ (D), TNF-α (E), IL-1β (F) and IL-6 (G) in the tumor microenvironment on Day 5 post treatments in Fig. 3. Replicates are biological (n = 6) and the data are expressed as mean ± s.e.m. Significance calculated using a two-tailed Student’s t-test.

Source data

Extended Data Fig. 3 In vivo immune responses triggered by mineralized F.S bacteria in B16F10 melanoma tumor-bearing mice.

Flow cytometry analysis of the mice on Day 1 (a-d) and Day 3 (e-h) post-treatment with MnO2, F.S or mineralized F.S, at the corresponding MnO2 dose of 20 μg and F.S dose of 3.6*108 bacteria cell. (n = 5). (a&e) Mature DCs in lymph nodes. (b) CD45+CD11b+F4/80+ macrophages in TME. (c&g) CD45+CD3-NK1.1+ NK cells in TME. (d&h) CD45+CD3+ T cells in TME. (f) The ratio of M1-like macrophages (CD80hi) to M2-like macrophages (CD206hi) gating on CD45+CD11b+F4/80+ cells in TME. Replicates are biological (n = 5) and the data are expressed as mean ± s.e.m. Significance calculated using a two-tailed Student’s t-test.

Source data

Extended Data Fig. 4 Mineralized F.S bacteria immunotherapy achieved systemic antitumor responses after localized treatment in CT26 tumor model.

(a) Schematic illustration to show the timeline for bilateral CT26 tumor treatment. Mice were post various treatments as indicated: normal saline (Blank), 15 μg anti-PD-1 (i.v.), 20 μg mineralized F.S (i.t.) and 15 μg anti-PD-1 (i.v.) plus 20 μg mineralized F.S (i.t.) (b&c) The average tumor growth curves (b) and individual tumor growth curves (c) of primary tumors with mineralized F.S injection as well as distant tumors without direct treatment. The mouse is deemed dead and the average tumor growth curve is stopped when the volume of either side of the bilateral tumor exceeds 1500 mm3. Replicates are biological (n = 6) and the data are expressed as mean ± s.e.m.

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Full-length unprocessed western blot data for Supplementary Fig. 10.

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Wang, C., Zhong, L., Xu, J. et al. Oncolytic mineralized bacteria as potent locally administered immunotherapeutics. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01191-w

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