Integrated Single-Cell Analysis Dissects Regulatory Mechanisms Underlying Tumor-Associated Macrophage Plasticity in Hepatocellular Carcinoma

<b>Background</b>: Tumor-associated macrophages (TAMs) are critical regulators of the hepatocellular carcinoma (HCC) microenvironment, yet their epigenetic heterogeneity and regulatory programs remain poorly defined. <b>Methods</b>: We performed integrative analysis on single-cell RNA-seq and ATAC-seq profiling of HCC patients to dissect TAM subtypes at high resolution. By correlating chromatin accessibility with gene expression, we identified cell-type-specific candidate <i>cis</i>-regulatory elements (CREs). TAM subsets with prognostic significance were determined through integration with HCC clinical cohorts. Pseudotime and multi-regional analyses were used to uncover regulatory trajectories underlying macrophage phenotypic transitions. The identification framework of a super-enhancer (SE) was constructed, and potential therapeutic targets were prioritized using drug-gene interaction data. <b>Results</b>: We delineated the regulatory landscape of TAMs in HCC, revealing cell-type-specific chromatin accessibility patterns underlying TAM heterogeneity. The 65,342 CREs linked to gene expression were identified, with distal CREs contributing most to cell-type-specific regulation. Notably, SPP1⁺ TAMs were found to be enriched in tumor cores and associated with poor prognosis in HCC. Liver-resident Kupffer cells showed progressive loss of the core transcription factors SPIC and MAFB, suggesting a potential transition into SPP1⁺ TAMs under tumor pressure. We identified 133 SPP1⁺ TAM-specific SEs and constructed a TF-SE-target gene regulatory network. Notably, 13 target genes showed higher drug-gene interaction effects, highlighting their therapeutic potential. <b>Conclusions</b>: This study provides the chromatin accessibility map of TAMs in HCC and reveals how distal CRE-driven transcriptional programs shape TAM states. Our findings lay the foundation for understanding the epigenetic regulation of TAM heterogeneity and nominate potential targets for TAM-directed immunotherapy in HCC.