特莫唑胺（TMZ）是脑胶质瘤治疗的首选化疗策略。作为第二代烷基化剂，TMZ具有卓越的口服生物利用度。然而，受限的应答率（低于50％）和高发生率的药物抵抗严重限制了TMZ的应用，仍缺乏增加化疗敏感性的策略。本研究利用Luci-GL261胶质瘤原位异种移植模型结合生物发光成像评估TMZ的抗肿瘤效应，并区分TMZ敏感（S）/非敏感（NS）个体。应用综合微生物组和代谢组学分析，以解开肠道细菌在TMZ敏感性中的作用。应用Spearman的相关性分析测试粪菌水平和药效学指标之间的关联。抗生素治疗结合TMZ治疗用于确认肠道微生物群在TMZ应答中的作用。流式细胞术分析、ELISA和组织病理学分析用于探究肠道微生物通过免疫调节对TMZ的应答作用。首先，肠道细菌组成在胶质瘤发展和TMZ治疗期间显着改变。与此同时，体内的抗癌评估表明，在TMZ施用后，化疗疗效有显着差异。此外，16s rRNA基因测序和非靶向代谢组学分析揭示了TMZ敏感和非敏感小鼠之间不同的肠道微生物和免疫浸润状态，而差异肠道菌群和相关代谢物的丰度与TMZ药效学指标显著相关。进一步验证表明，抗生素治疗的肠道微生物群删减可加速胶质瘤的发展，削弱TMZ的疗效，并抑制免疫细胞（巨噬细胞和CD8α + T细胞）的招募。本研究证实了肠道微生物群通过免疫调节参与了胶质瘤的发展和个体化TMZ效力，因此肠道细菌可能作为TMZ临床应用的预测生物标志物和治疗靶点。 © 2023. 作者（们）

Temozolomide (TMZ) is the preferred chemotherapy strategy for glioma therapy. As a second-generation alkylating agent, TMZ provides superior oral bio-availability. However, limited response rate (less than 50%) and high incidence of drug resistance seriously restricts TMZ's application, there still lack of strategies to increase the chemotherapy sensitivity.Luci-GL261 glioma orthotopic xenograft model combined bioluminescence imaging was utilized to evaluate the anti-tumor effect of TMZ and differentiate TMZ sensitive (S)/non-sensitive (NS) individuals. Integrated microbiomics and metabolomics analysis was applied to disentangle the involvement of gut bacteria in TMZ sensitivity. Spearman's correlation analysis was applied to test the association between fecal bacteria levels and pharmacodynamics indices. Antibiotics treatment combined TMZ treatment was used to confirm the involvement of gut microbiota in TMZ response. Flow cytometry analysis, ELISA and histopathology were used to explore the potential role of immunoregulation in gut microbiota mediated TMZ response.Firstly, gut bacteria composition was significantly altered during glioma development and TMZ treatment. Meanwhile, in vivo anti-cancer evaluation suggested a remarkable difference in chemotherapy efficacy after TMZ administration. Moreover, 16s rRNA gene sequencing and non-targeted metabolomics analysis revealed distinct different gut microbiota and immune infiltrating state between TMZ sensitive and non-sensitive mice, while abundance of differential gut bacteria and related metabolites was significantly correlated with TMZ pharmacodynamics indices. Further verification suggested that gut microbiota deletion by antibiotics treatment could accelerate glioma development, attenuate TMZ efficacy and inhibit immune cells (macrophage and CD8α+ T cell) recruitment.The current study confirmed the involvement of gut microbiota in glioma development and individualized TMZ efficacy via immunomodulation, hence gut bacteria may serve as a predictive biomarker as well as a therapeutic target for clinical TMZ application.© 2023. The Author(s).