The role of neutrophils in MASLD associated hepatocellular carcinoma

Abstract

Background Neutrophils are key drivers of hepatocellular carcinoma (HCC) progression and immunotherapy resistance in the context of metabolic dysfunction-associated steatotic liver disease (MASLD). I aimed to investigate neutrophil heterogeneity and phenotype in both patients and pre-clinical models. I also aimed to investigate mechanisms of immune checkpoint inhibitor resistance in a pre-clinical model of MASLD-HCC. Methods I isolated neutrophils from HCC and chronic liver disease (CLD) patients, healthy volunteers and mice with MASLD and orthotopically implanted HCCs. I characterised neutrophils using flow cytometry and functional assays. To investigate immune checkpoint inhibitor effectiveness in MASLD-HCC I implanted MASLD or lean mice with intrahepatic or subcutaneous tumours then treated with anti-PDL1/anti-VEGF therapy. Results There was an increased frequency of circulating neutrophils in HCC and CLD patients as well as HCC-bearing mice with a preferential increase in low density (LD) neutrophils with an activated, degranulated and aged phenotype. Tumour neutrophils had a similar phenotype. These neutrophil populations had high reactive oxygen species (ROS) production and high PDL1 expression which may indicate immunosuppressive functions. Phosphorylated STAT3 expression was increased in tumour neutrophils, suggesting this as a driving pathway. Treatment with a JAK2 inhibitor reduced tumour burden and ameliorated neutrophil ageing, degranulation and activation. In MASLD mice anti-PDL1/anti-VEGF therapy had a poor treatment effect in intrahepatic tumours but a good treatment effect in subcutaneous tumours. This was likely due to an expansion of PD1+CXCR6+EOMES+ CD8 T cells, which were expanded in the MASLD liver. Conclusion My data demonstrates neutrophil heterogeneity in HCC with an expansion of circulating LD neutrophils. LD neutrophils and tumour neutrophils have an activated, degranulated and aged phenotype which may be therapeutically manipulated by inhibiting the JAK2-STAT3 pathway. In pre-clinical models of MASLD-HCC anti PDL1/anti-VEGF therapy resistance was driven by the intrahepatic microenvironment of MASLD. Specifically due to the expansion of PD1+CXCR6+EOMES+ CD8 T cells with an exhausted and auto-aggressive phenotype.