Abstract P103: The introduction of a single strain of Bacillus into a germ-free environment did not impact the anti-PD-1 efficacy in a MC38 syngeneic model
Authors: Yang Tao , Chen Bonnie Xiaobo , Lu Rongfei , An Xiaoyu , Zang Mingfa , Li Jingjun , Guo Sheng , Qian Wubin , Fei Jian , Hao Tongyang , Xu Edward , Li Henry 
Published: December 2021

Background. Although immunotherapy has led to exceptional and durable clinical response, the majority of patients respond poorly to the current immunotherapies. Growing evidence has linked some of the poor responsiveness to the gut microbiota, and the modulation of gut microbiome composition is becoming a promising new strategy to enhance immune checkpoint inhibitor (ICI) treatment outcome. Mouse tumor modelling under germ-free (GF) conditions combined with introduction of defined bacterial strains could be a useful approach to investigate the impact of microbiota on ICI efficacy, as well as understanding the underlying mechanisms. We previously demonstrated that GF mice exhibited a significantly poor response to anti-PD-1 therapy when compared to the specific pathogen free (SPF) mice in a subcutaneous MC38 colorectal cancer model, which is consistent with other reports. Methods. Introduction of a single strain of Bacillus in the GF-environment was assessed for its impact on the anti-mouse PD-1 monoclonal antibody (mAb) therapy in GF-mice and SPF mice, both for efficacy and pharmacodynamics tumor infiltrating lymphocytes (TILs) profiling. C57BL/6 mice were inoculated subcutaneously with MC38 tumor cells and when the tumors were reached 80-120mm3, the mice were randomized for isotype or anti-PD-1 mAb treatment. Fecal sample 16S rRNA analysis (NGS) was used to confirm the gut bacteria status. Results. The MC38 tumor in GF mice has significantly fast baseline growth kinetics, even with the introduction of Bacillus under GF conditions compared to SPF mice, suggesting tumor immunity was not enhanced by Bacillus. Despite the introduction of Bacillus, GF mice also showed reduced response to anti-PD-1 treatment when compared to the SPF mice as previously reported, further confirming that introduction of Bacillus had minimal effects on the efficacy of anti-PD-1 therapy. Moreover, the SPF mice and GF mice with Bacillus exhibited distinct profiles of TILs, consistent with distinct efficacies as observed. GF free mice showed a lower frequency of CD45+ TILs in comparison to SPF mice. In addition, GF mice exhibited lower frequency of CD8+ TILs and TIL- NKT when compared to the SPF mice, both of which are consistent with the stronger efficacy seen in SPF mice. Meanwhile, GF mice also exhibited higher granulocytic myeloid derived suppressor cells (gMDSC) and lower M1/M2 ratio, both of which imply a more suppressive tumor microenvironment in GF mice. Fecal sample analysis using 16S rRNA analysis confirmed a single strain of Bacillus was indeed introduced into the guts of all the GF mice. Conclusions. The GF conditions provide a useful environment for the investigation of specific microbiota strains on the impact on ICI treatment outcome. In summary, the introduction of Bacillus in GF mice did not impact the efficacy of anti-PD-1, thus suggesting that other strain(s) of gut microbiota in SPF mice may impact this and need to be investigated.

Citation Format: Tao Yang, Bonnie Xiaobo Chen, Rongfei Lu, Xiaoyu An, Mingfa Zang, Jingjun Li, Sheng Guo, Wubin Qian, Jian Fei, Tongyang Hao, Edward Xu, Henry Li. The introduction of a single strain of Bacillus into a germ-free environment did not impact the anti-PD-1 efficacy in a MC38 syngeneic model [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P103.