GNG4 associated with tumor immune exclusion and compromised CD8+ T cell infiltration via microenvironmental crosstalk in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality globally, with resistance to immune checkpoint inhibitors posing a major clinical challenge. The intrinsic mechanisms by which tumors restrict CD8⁺ T cell infiltration and function are not fully understood. This study aimed to systematically identify molecular programs associated with immune exclusion in HCC and explore their spatial and functional microenvironmental associations. We employed an integrative analytical approach combining single-cell RNA sequencing, spatial transcriptomics, and multiplex immunofluorescence to dissect immunosuppressive patterns in HCC tissues. Functional relevance was assessed using shRNA-mediated knockdown of GNG4, assessing its effects on tumor cell behavior in vitro and in vivo. Microenvironmental associations were further investigated in human HCC samples via multiplex immunofluorescence. GNG4 was identified as a malignant hepatocyte-enriched gene strongly associated with poor patient prognosis and high-risk scores. Its expression was spatially enriched in tumor regions non-responsive to neoadjuvant immunotherapy. Knockdown of GNG4 impaired HCC cell proliferation, migration, and invasion in vitro and significantly suppressed tumor growth in vivo. Mechanistically, Gng4 deficiency driven tumor restriction in vivo is characterized by a dual mechanism, encompassing both a tumor-cell-autonomous growth inhibition and an enhanced microenvironmental immune control, the latter manifesting as increased CD8 + T cell infiltration and elevated cytotoxic potential (IFN-γ + and Granzyme B + ). Computational cellular communication models and spatial transcriptomics revealed that GNG4 expression in malignant cells is spatially accompanied by an altered expression state of the signature molecule MID1 in neighboring CD8 + T cells. Furthermore, an in vitro rescue assay utilizing primary murine CD8 + T cells confirmed that the functional restoration of T cells elicited by shGng4 malignant conditioned medium is functionally correlated with the recovery of Mid1 expression within T cells. In human HCC samples, multiplex immunofluorescence confirmed an inverse spatial correlation between GNG4 expression in malignant cells and the localized density of CD8 + MID1 + T cells. Our study uncovers that GNG4 expression in malignant cells is spatially and functionally correlated with a specialized multi-cellular microenvironmental crosstalk that correlates with reduced CD8 + T cell spatial infiltration in HCC. Recognizing these boundaries offers potential prognostic and translational insights into the immune exclusion landscape of advanced hepatocellular carcinoma.