Abstract Objective Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. Design Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. Results Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoral XCR1 + dendritic cell activation and CD8 + T cell numbers which are associated with anti-tumoral immunity, this was confirmed by loss of therapeutic effect upon genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8 + T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a pro-tumor to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8 + T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. Conclusion CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC. Significance of this study What is already known on this subject? Immune checkpoint inhibition therapy is emerging as a promising new therapy for the treatment of advanced hepatocellular carcinoma (HCC). Only a minority of HCC patients will respond to immune checkpoint inhibition (ICI) therapy and recent data suggest that HCC on the background of NASH may have reduced sensitivity to this treatment strategy. Neutrophils are a typical myeloid component of the liver in NASH and are found either within the HCC tumour microenvironment or in a peritumoural location. Neutrophils have considerable phenotypic plasticity and can exist in both tumour promoting and tumour suppressing states. Neutrophils may have the ability to influence ICI therapy. What are the new findings? CXCR2 + neutrophils are found in human NASH and within the tumour of both human and mouse models of NASH-HCC. The resistance of NASH-HCC to anti-PD1 therapy is overcome by co-treatment with a CXCR2 small molecule inhibitor, with evidence of reduced tumour burden and extended survival. Anti-PD1 and CXCR2 inhibitor combine to selectively reprogramme tumour-associated neutrophils (TANs) from a pro- to an anti-tumour phenotype. Reprogrammed TANs proliferate locally within Granzyme B + immune clusters that contain physically associating CD8 + T cells and antigen presenting cells. Conventional XCR1 + dendritic cells (cDC1s) are found to be elevated in anti-PD1 and CXCR2 inhibitor treated HCCs and together with CD8 + T cells are required for therapeutic benefit. How might it impact on clinical practice in the foreseeable future? TANs can be selectively manipulated to adopt an anti-tumour phenotype which unlocks their potential for cancer therapy. The ability of CXCR2 antagonism to combine with ICI therapy to bring about enhanced therapeutic benefit in NASH-HCC (and potentially in HCC of other aetiologies) warrents clinical investigation.

CXCR2 inhibition enables NASH-HCC immunotherapy