PURPOSE The role that gut microbiota plays in determining the efficacy of the anti-tumor effect of immune checkpoint inhibitors is gaining increasing attention, and fecal bacterial transplantation has been recognized as a promising strategy for improving or rescuing the effect of immune checkpoint inhibition. However, techniques for the precise monitoring of in vivo bacterial behaviors after transplantation are limited. In this study, we aimed to use metabolic labeling and subsequent positron emission tomography (PET) imaging to track the in vivo behaviors of gut bacteria that are responsible for the efficacy of anti-PD-1 therapy in living mice. METHODS The antitumor effect of anti-PD-1 blockade was tested in a low-response 4T1 syngeneic mouse model with or without fecal transplantation and with or without broad-spectrum antibiotic imipenem treatment. High-throughput sequencing analyses of 16S rRNA gene amplicons in feces of 4T1 tumor-bearing mice pre- and post-anti-PD-1 treatment were performed. The identified bacteria, Bacteroides fragilis (B. fragilis), were labeled with 64Cu and fluorescence dye by the metabolic labeling of N3 followed by click chemistry. In vivo PET and optical imaging of B. fragilis were performed in mice after oral gavage. RESULTS The disturbance of gut microbiota reduced the efficacy of anti-PD-1 treatment, and the combination of B. fragilis gavage and PD-1 blockade was beneficial in rescuing the antitumor effect of anti-PD-1 therapy. Metabolic oligosaccharide engineering and biorthogonal click chemistry resulted in successful B. fragilis labeling with 64Cu and fluorescence dye with high in vitro and in vivo stability and no effect on viability. PET imaging successfully detected the in vivo behaviors of B. fragilis after transplantation. CONCLUSION PET tracking by metabolic labeling is a powerful, noninvasive tool for the real-time tracking and quantitative imaging of gut microbiota. This strategy is clinically translatable and may also be extended to the PET tracking of other functional cells to guide cell-based adoptive therapies.

中文翻译：

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活体小鼠移植后肠道菌群的无创PET追踪。

目的肠道菌群在确定免疫检查点抑制剂的抗肿瘤作用的功效中的作用越来越受到关注，粪便细菌移植已被认为是改善或拯救免疫检查点抑制作用的有前途的策略。但是，用于精确监测移植后体内细菌行为的技术是有限的。在这项研究中，我们旨在使用代谢标记和随后的正电子发射断层扫描（PET）成像来追踪肠道细菌的体内行为，这些行为负责抗PD-1治疗活小鼠的功效。方法在有或没有粪便移植以及有或没有广谱抗生素亚胺培南治疗的低应答4T1同系小鼠模型中测试了抗PD-1阻断剂的抗肿瘤作用。抗PD-1治疗前后，对4T1荷瘤小鼠粪便中的16S rRNA基因扩增子进行高通量测序分析。鉴定出的细菌，脆弱的拟杆菌（Bacteroides fragilis）（B。fragilis）用Nu3的代谢标记，然后单击化学法，用64Cu和荧光染料标记。口服管饲法在小鼠体内进行了脆弱类芽孢杆菌的体内PET和光学成像。结果肠道菌群紊乱降低了抗PD-1治疗的疗效，脆性芽孢杆菌灌胃和PD-1阻滞相结合有利于挽救抗PD-1治疗的抗肿瘤作用。代谢寡糖工程技术和双正交点击化学技术成功地使用64Cu和荧光染料成功标记了脆弱的B. fragilis，具有较高的体内和体外稳定性，并且对生存能力没有影响。PET成像成功地检测了脆弱类芽孢杆菌在移植后的体内行为。结论通过代谢标记进行PET跟踪是一种强大的，无创的工具，可用于实时跟踪和定量分析肠道菌群。该策略可在临床上翻译，也可扩展到其他功能性细胞的PET跟踪，以指导基于细胞的过继疗法。脆弱后的移植。结论通过代谢标记进行PET跟踪是一种强大的，无创的工具，可用于实时跟踪和定量分析肠道菌群。该策略可在临床上翻译，也可扩展到其他功能性细胞的PET跟踪，以指导基于细胞的过继疗法。脆弱后的移植。结论通过代谢标记进行PET跟踪是一种强大的，无创的工具，可用于实时跟踪和定量分析肠道菌群。该策略可在临床上翻译，也可扩展到其他功能性细胞的PET跟踪，以指导基于细胞的过继疗法。