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

Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.