25-Hydroxycholesterol Regulates Lysosome AMPK Activation and Metabolism Reprograming to Educate Immunosuppressive Macrophages

Abstract Different macrophage subsets exhibit anti-tumor or immunosuppression to regulate tumor progression. Cholesterol metabolism is vital to manipulate macrophage function. Here, we screened cholesterol enzymes and metabolites in three types of immunosuppressive macrophages, which all upregulated cholesterol-25-hydroxylase (Ch25h) expression to cause 25-hydroxycholesterol (25HC) accumulation. scRNA-seq data confirmed that MARCO+ and LYVE1+ macrophage subsets from various human cancers were Ch25hhigh subsets, correlated to lower survival rates. TEM including lactic Acid, Acidic pH and IL4/IL13 enhanced Ch25h expression, and targeting Ch25h abrogated macrophage immunosuppressive function to convert cold tumor into hot tumor, which also synergized with anti-PD1 therapy to improve antitumor efficacy. Critically, 25HC accumulation in lysosome acted as a novel AMPKα agonist, which enhances mitochondrial function and oxidative phosphorylation. We therefore propose Ch25h as an immunometabolic checkpoint and a drug target for tumor immunotherapy, which manipulates macrophage polarization and reshapes TME.