Single-cell transcriptomic characterization of the immunosuppressive tumor immune microenvironment in hepatocellular carcinoma: implications for SBRT-based radio-immunotherapy
TL;DR
The HCC TIME is profoundly immunosuppressive, driven by myeloid dominance and multi-checkpoint T cell exhaustion, providing direct mechanistic rationale for combining macrophage reprogramming, dual checkpoint blockade, and anti-angiogenic strategies with SBRT.
Abstract
Background Hepatocellular carcinoma (HCC) carries dismal prognosis, and the tumor immune microenvironment (TIME) critically determines the efficacy of SBRT-based radio-immunotherapy; yet its single-cell architecture remains undefined. Methods We analyzed scRNA-seq data (GEO: GSE149614; single patient HCC07, stage IIIB; normal liver, primary tumor, PVTT) using a standardized pipeline including dimensionality reduction, clustering, immune signature scoring, and checkpoint profiling (FDR <0.05). In vitro, HepG2 and Huh-7 cells (ATCC) were treated with M2-conditioned medium for RT-qPCR/ELISA validation of immunosuppressive mediators, and irradiated (Cs-137; 0–12 Gy) to assess radiation-induced immunogenic cell death modulation. Results scRNA-seq identified six major cell types across 25 clusters: myeloid cells expanded from 22.4% (normal) to 46.5% (tumor), while T/NK cells declined from 66.5% to 20.0%. SPP1, CXCL8, APOE, TGF-β1, IL-10, and VEGFA were upregulated in tumor/PVTT, with multi-checkpoint T cell exhaustion (PDCD1, LAG3, TIGIT, CTLA4) dominating tumor-infiltrating lymphocytes. RT-qPCR/ELISA confirmed M2-conditioned medium upregulated SPP1 (∼3.4-fold) and CXCL8 (∼4.1-fold; p < 0.001) in Hepatocellular carcinoma cells, and attenuated radiation-induced calreticulin exposure by ∼40% (p < 0.05). Conclusion The HCC TIME is profoundly immunosuppressive, driven by myeloid dominance and multi-checkpoint T cell exhaustion. The immunosuppressive microenvironment attenuates SBRT-induced ICD, providing direct mechanistic rationale for combining macrophage reprogramming, dual checkpoint blockade, and anti-angiogenic strategies with SBRT.