Abstract
Remodeling of the tumor microenvironment (TME) under therapeutic pressure is a critical determinant of treatment response and resistance in hepatocellular carcinoma (HCC). Triple-combination therapy integrating targeted agents, immune checkpoint inhibitors, and radiotherapy (T+I+R) has shown potential synergistic effects in intermediate to advanced HCC, particularly in patients with portal vein tumor thrombus (PVTT), yet the spatial and cellular mechanisms underlying its efficacy remain largely unknown. In this retrospective clinical cohort study, we compared T+I+R with targeted therapy plus radiotherapy (T+R) in advanced HCC, and further employed single-cell spatial transcriptomics and spatial proteomics to generate an integrated multi-omics atlas mapping tumor and stromal compartments, cellular compositions, and intercellular interactions with spatial resolution. Clinically, T+I+R achieved superior tumor shrinkage and disease control compared with T+R. Spatial multi-omics revealed marked region-specific remodeling, with myofibroblastic cancer-associated fibroblasts, angiogenic tip endothelial cells, and conventional dendritic cells enriched at the invasive margin and associated with therapeutic resistance, while CD8(+) effector T cells were redistributed away from immunosuppressive niches, a spatial configuration correlating with enhanced response. These findings identify spatial segregation between cytotoxic and suppressive immune elements as a potential hallmark of effective therapy, providing a high-resolution spatial framework for understanding T+I+R induced TME remodeling and offering mechanistic insights to guide biomarker discovery and the optimization of combination strategies in advanced HCC.