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Abstract：
BACKGROUND: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: Our study reveals the TME of cholestasis-associated liver metastasis and proposes a new strategy for such patients by targeting bile acid anabolism.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: Our study reveals the TME of cholestasis-associated liver metastasis and proposes a new strategy for such patients by targeting bile acid anabolism.
摘要：
背景：胆汁淤积是肝转移进展过程中常见但严重的并发症。然而,胆汁淤积如何影响发育，治疗,肝转移的肿瘤微环境（TME）仍有待阐明。
方法：建立肝外和肝内胆汁淤积小鼠肝转移模型，检测不同基因的差异表达水平，免疫细胞的浸润和胆汁酸相关代谢物的变化，通过使用RNA测序，流式细胞术,以及液相色谱和质谱。在体外原代胆汁酸（BA）刺激下对中性粒细胞进行Westernblot，以研究表型改变的机制。进行BA处理的嗜中性粒细胞与CD8T细胞的体外共培养，以研究表型改变的嗜中性粒细胞的免疫抑制作用。从具有肝转移和胆汁淤积的结直肠癌患者收集的临床样品应用于RNA-Seq。
结果：与非胆汁淤积小鼠相比，胆汁淤积小鼠肝转移的进展明显加快，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志物，进一步表明在胆汁淤积期间诱导了免疫抑制性肿瘤微环境。尽管通过抗Ly6G抗体的中性粒细胞缺失部分阻碍了肝转移进展，它降低了小鼠的总体存活率。牛磺酸-β-胞嘧啶酸(Tβ-MCA)和甘氨胆酸(GCA),两个最丰富的胆汁淤积相关的主要BAs，p38MAPK信号通路显著促进中性粒细胞Arg1和iNOS的表达。此外,BAs预处理的中性粒细胞显着抑制CD8+T细胞的激活和细胞毒性作用，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,在胆汁淤积下靶向奥贝胆酸（OCA）的BA合成代谢可有效抑制肝转移进展，增强了免疫检查点阻断的功效，和延长小鼠的存活时间。
结论：我们的研究揭示了胆汁淤积相关肝转移的TME，并通过靶向胆汁酸合成代谢为此类患者提出了新的策略。
方法：建立肝外和肝内胆汁淤积小鼠肝转移模型，检测不同基因的差异表达水平，免疫细胞的浸润和胆汁酸相关代谢物的变化，通过使用RNA测序，流式细胞术,以及液相色谱和质谱。在体外原代胆汁酸（BA）刺激下对中性粒细胞进行Westernblot，以研究表型改变的机制。进行BA处理的嗜中性粒细胞与CD8T细胞的体外共培养，以研究表型改变的嗜中性粒细胞的免疫抑制作用。从具有肝转移和胆汁淤积的结直肠癌患者收集的临床样品应用于RNA-Seq。
结果：与非胆汁淤积小鼠相比，胆汁淤积小鼠肝转移的进展明显加快，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志物，进一步表明在胆汁淤积期间诱导了免疫抑制性肿瘤微环境。尽管通过抗Ly6G抗体的中性粒细胞缺失部分阻碍了肝转移进展，它降低了小鼠的总体存活率。牛磺酸-β-胞嘧啶酸(Tβ-MCA)和甘氨胆酸(GCA),两个最丰富的胆汁淤积相关的主要BAs，p38MAPK信号通路显著促进中性粒细胞Arg1和iNOS的表达。此外,BAs预处理的中性粒细胞显着抑制CD8+T细胞的激活和细胞毒性作用，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,在胆汁淤积下靶向奥贝胆酸（OCA）的BA合成代谢可有效抑制肝转移进展，增强了免疫检查点阻断的功效，和延长小鼠的存活时间。
结论：我们的研究揭示了胆汁淤积相关肝转移的TME，并通过靶向胆汁酸合成代谢为此类患者提出了新的策略。
