Novel retinoic acid receptor-related orphan receptor gamma inverse agonist enhances checkpoint blockade in hepatocellular carcinoma: a preclinical mouse study

Background and Aims: Neutrophil-associated inflammation is associated with poor outcomes in hepatocellular carcinoma (HCC), but tumour-intrinsic regulators of neutrophil recruitment and checkpoint response remain unclear. We evaluated whether inhibiting retinoic acid receptor-related orphan receptor gamma (RORgamma) alters neutrophil recruitment, programmed death-ligand 1 (PD-L1) expression, and response to PD-L1 blockade. Approach and Results: Survival associations were analysed in The Cancer Genome Atlas HCC cohort. Neutrophil migration toward HCC cells and PD-L1 expression were assessed after RORC silencing or TF-S10 treatment. Immunocompetent mice bearing orthotopic Hep-55.1c tumours received vehicle, TF-S10, anti-PD-L1, or combination treatment. Higher S100A9 and CXCL8 expression was associated with shorter survival (P = 0.000459 and P = 0.0004413, respectively), and tumour-bearing mice had higher neutrophil-to-lymphocyte ratios (P < 0.0001). RORgamma targeting reduced neutrophil migration and intratumoural Ly6G+; infiltration while increasing tumour-cell PD-L1 expression in vitro. Combination treatment reduced endpoint liver tumour volume versus anti-PD-L1 monotherapy (P < 0.01). Half of combination-treated mice had tumour volumes less than or equal to 0.10 cm^3 and none exceeded 1.00 cm^3, compared with 62% of controls exceeding 1.00 cm^3. Histologically, 85% of combination-treated mice had less than or equal to 10% liver tumour involvement, versus no controls. Combination treatment also reduced tumour-associated neutrophil populations and peripheral neutrophil-to-lymphocyte ratios. All active treatments reduced pulmonary metastatic burden versus controls, without significant differences among treatment groups; 53% of combination-treated mice had no detectable lung metastases. Conclusions: RORgamma inhibition reduces neutrophil recruitment and increases tumour-cell PD-L1 expression. Combined RORgamma inhibition and PD-L1 blockade improves hepatic tumour control in preclinical HCC and warrants further investigation.