Cholestasis is a common yet severe complication that occurs during the advancement of liver metastasis. However, how cholestasis impacts the development, treatment, and tumor microenvironment (TME) of liver metastasis remains to be elucidated. Extrahepatic and intrahepatic cholestatic mouse models with liver metastasis were established to detect the differential expression levels of genes, infiltration of immune cells and change in bile acid-associated metabolites by using RNA-Sequencing, flowcytometry, and liquid chromatography and mass spectrometry. Western blot was applied to neutrophils under the stimulation of primary bile acids (BAs) in vitro to study the mechanism of phenotypic alteration. In vitro coculture of BA-treated neutrophils with CD8+ T cells were performed to study the immune-suppressive effect of phenotypic-altered neutrophils. Clinical samples collected from colorectal cancer patients with liver metastasis and cholestasis were applied to RNA-Seq. Compared to non-cholestatic mice, the progression of liver metastasis of cholestatic mice was significantly accelerated, which was associated with increased neutrophil infiltration and T-cell exclusion. Both neutrophils and T cells expressed higher immunosuppressive markers in the cholestatic mouse model, further indicating that an immunosuppressive tumor microenvironment was induced during cholestasis. Although neutrophils deletion via anti-Ly6G antibody partially hindered liver metastasis progression, it reduced the overall survival of mice. Tauro-β-muricholic acid (Tβ-MCA) and Glycocholic acid (GCA), the two most abundant cholestasis-associated primary BAs, remarkably promoted the expression of Arg1 and iNOS on neutrophils via p38 MAPK signaling pathway. In addition, BAs-pretreated neutrophils significantly suppressed the activation and cytotoxic effects of CD8+ T cells, indicating that the immunosuppressive phenotype of neutrophils was directly induced by BAs. Importantly, targeting BA anabolism with Obeticholic acid (OCA) under cholestasis effectively suppressed liver metastasis progression, enhanced the efficacy of immune checkpoint blockade, and prolonged survival of mice. Our study reveals the TME of cholestasis-associated liver metastasis and proposes a new strategy for such patients by targeting bile acid anabolism. Schematic model depicting the proposed mechanism of cholestasis-mediated progression of colorectal liver metastasis. As cholestasis progresses, excessive primary bile acids that accumulate in the liver intoxicates hepatocytes, which lead to exacerbated release of chemokines, particularly CXCL2 and CXCL5. Neutrophils are then accumulated by CXCL2 and CXCL5 and undergo an immunosuppressive-phenotypic alteration induced by direct stimulation of BAs via activating the p38 MAPK signaling pathway, which eventually led to the dysfunction of T cells and progression of LM. Targeting bile acid anabolism can effectively restore the immune-activated microenvironment and prevent the progression of LM.

中文翻译：

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胆汁淤积诱导的中性粒细胞表型转变有助于结直肠癌肝转移的免疫逃逸


胆汁淤积是肝转移进展过程中常见但严重的并发症。然而，胆汁淤积如何影响肝转移的发展、治疗和肿瘤微环境（TME）仍有待阐明。建立肝外和肝内胆汁淤积肝转移小鼠模型，利用RNA测序、流式细胞仪、液相色谱和质谱技术检测基因的差异表达水平、免疫细胞的浸润以及胆汁酸相关代谢物的变化。将蛋白质印迹应用于体外初级胆汁酸（BA）刺激下的中性粒细胞，以研究表型改变的机制。将 BA 处理的中性粒细胞与 CD8+ T 细胞进行体外共培养，以研究表型改变的中性粒细胞的免疫抑制作用。从患有肝转移和胆汁淤积的结直肠癌患者收集的临床样本应用于RNA-Seq。与非胆汁淤积小鼠相比，胆汁淤积小鼠的肝转移进展明显加速，这与中性粒细胞浸润和T细胞排斥增加有关。在胆汁淤积小鼠模型中，中性粒细胞和 T 细胞均表达较高的免疫抑制标记物，进一步表明胆汁淤积期间诱导了免疫抑制肿瘤微环境。尽管通过抗 Ly6G 抗体删除中性粒细胞部分阻碍了肝转移进展，但它降低了小鼠的总体存活率。牛磺-β-鼠胆酸 (Tβ-MCA) 和甘胆酸 (GCA) 是两种最丰富的胆汁淤积相关初级 BA，可通过 p38 MAPK 信号通路显着促进中性粒细胞 Arg1 和 iNOS 的表达。 此外，BAs预处理的中性粒细胞显着抑制CD8+T细胞的活化和细胞毒性作用，表明中性粒细胞的免疫抑制表型是由BAs直接诱导的。重要的是，在胆汁淤积状态下用奥贝胆酸（OCA）靶向BA合成代谢可有效抑制肝转移进展，增强免疫检查点阻断的功效，并延长小鼠的生存期。我们的研究揭示了胆汁淤积相关肝转移的 TME，并通过针对胆汁酸合成代谢为此类患者提出了新策略。示意图模型描绘了所提出的胆汁淤积介导的结直肠肝转移进展机制。随着胆汁淤积的进展，肝脏中积累的过量初级胆汁酸会使肝细胞中毒，从而导致趋化因子（尤其是 CXCL2 和 CXCL5）的加剧释放。然后，中性粒细胞通过 CXCL2 和 CXCL5 积累，并通过激活 p38 MAPK 信号通路直接刺激 BA 诱导免疫抑制表型改变，最终导致 T 细胞功能障碍和 LM 进展。靶向胆汁酸合成代谢可以有效恢复免疫激活的微环境并阻止LM的进展。