In the tumour microenvironment, critical drivers of immune escape include the oncogenic activity of the tumour cell-intrinsic osteopontin (OPN), the expression of programmed death ligand 1 (PD-L1) and the expansion of tumour-associated macrophages (TAMs). We investigated the feasibility of targeting these pathways as a therapeutic option in hepatocellular carcinoma (HCC) mouse models.

We analysed the number of tumour-infiltrating immune cells and the inflammatory immune profiles in chemically induced liver tumour isolated from wild-type and OPNknockout (KO) mice. In vitro cell cocultures were further conducted to investigate the crosstalk between TAMs and HCC cells mediated by OPN, colony stimulating factor-1 (CSF1) and CSF1 receptor (CSF1R). The in vivo efficacy of anti-PD-L1 and CSF1/CSF1R inhibition was evaluated in OPN overexpressing subcutaneous or orthotopic mouse model of HCC.

The numbers of TAMs, as well as the expression levels of M2 macrophage markers and PD-L1 were significantly decreased, but the levels of cytokines produced by T-helper 1 (Th1) cells were upregulated in tumour tissues from OPN KO mice compared with that from the controls. In addition, we observed a positive association between the OPN and PD-L1 expression, and OPN expression and TAM infiltration in tumour tissues from patients with HCC. We further demonstrated that OPN facilitates chemotactic migration, and alternative activation of macrophages, and promotes the PD-L1 expression in HCC via activation of the CSF1-CSF1R pathway in macrophages. Combining anti-PD-L1 and CSF1R inhibition elicited potent antitumour activity and prolonged survival of OPN^high tumour-bearing mice. Histological, flow cytometric and ELISA revealed increased CD8⁺ T cell infiltration, reduced TAMs and enhanced Th1/Th2 cytokine balance in multiple mouse models of HCC.

OPN/CSF1/CSF1R axis plays a critical role in the immunosuppressive nature of the HCC microenvironment. Blocking CSF1/CSF1R prevents TAM trafficking and thereby enhances the efficacy of immune checkpoint inhibitors for the treatment of HCC.