Pan-cancer epigenetic landscape of human tumor-associated macrophages reveals crucial enhancers governing their heterogenous formation by Pol II pausing modulation

INTRODUCTION: Tumor-associated macrophages (TAMs) are a functionally heterogeneous population within the tumor microenvironments, exhibiting both anti-tumor and pro-tumor functions. Pro-tumorigenic TAMs can derive from peripheral monocytes or tissue-resident macrophages (TRMs). However, the epigenetic mechanisms driving the differentiation of these distinct cellular sources into specific TAM subtypes remain unclear. OBJECTIVES: Here, we sought to define the epigenetic mechanisms that drive the divergent differentiation of monocyte and TRM-derived TAMs. Through a pan-cancer analysis, we map the regulatory cascade, from cis-regulatory elements and key transcription factors to the release of RNA Polymerase II (Pol II) pausing, that specifies TAM heterogeneity. METHODS: We performed single-cell epigenetic profiling of macrophages derived from various human cancers, precancerous lesions, and healthy adult and fetal tissues using publicly available datasets, coupled with single-cell multi-omics integration. Key findings were validated through multi-omics analysis of mouse bone marrow-derived macrophage and CRISPR-based functional assays using in vitro human cell culture models. RESULTS: TAM trajectories, we identified PPARG, NFAT5 and MECP2 as pivotal regulators that promote pro-tumorigenic polarization. Conversely, the differentiation of TRMs to C1QC + TAMs was associated with regulators MAF, HES1 and PRDM1. Furthermore, we discovered a core transcriptional signature regulated by SPP1+ TAM-specific super-enhancers that is significantly associated with poor prognosis across 18 cancer types. Mechanistically, we demonstrated that these enhancers, such as HIPK2 enhancers, establish the pro-tumorigenic TAM state primarily by modulating Pol II pausing rather than by altering promoter accessibility of their target genes. CONCLUSION: Our work delineates the epigenetic and transcriptional circuitry that establishes TAM heterogeneity as potential therapeutic levers to enhance cancer immunotherapy.