Tumor microenvironment and transcriptional analysis of CD45+ leukocytes isolated from syngeneic MC38 tumors from mice fed ketogenic diet or control diet

Tumor microenvironment (TME) has a dynamic composition that affects targeted therapies for colorectal cancer (CRC). Cancer associated fibroblasts (CAFs) direct and induce infiltration of immunosuppressive cells through secreted cytokines such as CXCL12. Here, we show that decreased ketogenesis is a signature of CRCs and that an increase in ketogenesis, using a ketogenic diet (KD), decreases CXCL12 in CAFs, tumors, serum, livers, and lungs. Increase in ketogenesis results in the decreased expression of KLF5, which binds to the CXCL12 promoter and induces CXCL12 expression. KD decreases intratumoral accumulation of M2 macrophages and myeloid-derived suppressor cells (MDSCs), increases infiltration of NK and cytotoxic T cells and enhances the anticancer effects of PD-1 blockade in murine-derived CRCs. Moreover, an increase in ketogenesis inhibits CRC migration, invasion, and metastasis in vitro and in vivo. Importantly, our studies demonstrate that downregulation of de novo ketogenesis in the TME is a critical step in CRC progression. Single-cell transcriptional analysis of CD45+ leukocytes isolated 14 days post-implantation from subcutaneous MC38 tumors in C57BL6/J mice fed control or ketogenic diet. Each sample contains CD45+ cells combined from 5 mice.

肿瘤微环境（Tumor microenvironment, TME）具有动态可变的组成特征，可影响结直肠癌（colorectal cancer, CRC）的靶向治疗效果。癌症相关成纤维细胞（Cancer associated fibroblasts, CAFs）可通过分泌CXCL12等细胞因子，介导并诱导免疫抑制细胞的浸润。本研究证实，生酮作用减弱是结直肠癌的标志性分子特征；通过生酮饮食（ketogenic diet, KD）增强机体生酮作用，可降低癌症相关成纤维细胞、肿瘤组织、血清、肝脏及肺组织中CXCL12的表达水平。生酮作用增强可下调转录因子KLF5的表达，而KLF5可直接结合CXCL12基因启动子并诱导其转录激活。生酮饮食可减少小鼠结直肠癌模型的瘤内M2型巨噬细胞与髓系来源抑制细胞（myeloid-derived suppressor cells, MDSCs）的聚集，增加自然杀伤（NK）细胞与细胞毒性T细胞的肿瘤浸润，并增强PD-1阻断疗法的抗肿瘤效应。此外，增强生酮作用可在体外与体内实验中抑制结直肠癌细胞的迁移、侵袭与转移过程。尤为关键的是，本研究表明肿瘤微环境中从头生酮作用的下调是结直肠癌进展的核心环节。本研究对喂食正常对照或生酮饮食的C57BL/6J小鼠皮下接种MC38肿瘤后14天分离的CD45+白细胞进行了单细胞转录组分析；每个样本由5只小鼠的CD45+细胞混合制备而成。