Spatiotemporal dynamics of the tumor microenvironment in hepatocellular carcinoma during combination immunotherapy

Atezolizumab plus bevacizumab (ATZ/BEV) is a standard first-line therapy for advanced hepatocellular carcinoma (HCC); however, many patients do not achieve meaningful tumor regression. The temporal and spatial immune remodeling associated with ATZ/BEV remains poorly understood.

We performed single-cell RNA sequencing of paired hepatectomy specimens obtained before and after ATZ/BEV from one patient and of tumor center and margin samples from another patient after ATZ/BEV. Cell composition, subclusters, and cell-cell communication were analyzed. In addition, candidate molecules identified by transcriptomic analysis were further assessed using serum-based assays and immunohistochemistry.

These single-cell analyses suggested that ATZ/BEV was associated with a shift toward an immune-active tumor microenvironment, with increased CD8+ T cells together with reduced endothelial cells. CD8+ T cells showed increased effector and exhaustion signatures, indicating coexistence of activation and dysfunction. CellChat analysis demonstrated selective activation of the TIGIT-PVR/NECTIN2 axis after treatment. Spatial analysis showed that the tumor margin was enriched for CD8+ T cells and exhibited stronger effector and exhaustion activity than the tumor center. Immunoregulatory signaling was also more prominent at the margin. Serum TIGIT levels were significantly higher after ATZ/BEV than in upfront resection cases (p=0.0325). Immunohistochemistry showed greater margin-to-center differences in TIGIT (p=0.0019) and PVR (p=0.0008) in the tumor after ATZ/BEV.

Our exploratory findings suggest that ATZ/BEV may remodel the HCC microenvironment toward a state characterized by concurrent CD8+ T-cell activation and inhibitory signaling through the TIGIT-PVR/NECTIN2 axis, particularly at the tumor margin.