Cholestasis-induced phenotypic transformation of neutrophils contributes to immune escape of colorectal cancer liver metastasis
Authors: Sun Li , Yang Nanyan , Liu Zhihong , Ye Xiandong , Cheng Mengting , Deng Lingjun , Zhang Junhao , Wu Jingjing , Shi Min , Liao Wangjun 
Published: July 2024

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.

Graphical Abstract
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.