Functional Genetic Screens Reveal Key Pathways Instructing the Molecular Phenotypes of Tumor-Associated Macrophages

Abstract Tumor-associated macrophages (TAM) display remarkable functional heterogeneity, yet the molecular mechanisms driving their diverse phenotypes remain elusive. Using CRISPR screens in primary macrophages, we identified tumor-derived factors, including lactic acid, prostaglandin E2, and GM-CSF, as key modulators of TAM polarization. These factors interacted cooperatively and antagonistically to shape distinct TAM phenotypes that were highly conserved across human cancers. Mechanistically, lactic acid and PGE2 jointly induced angiogenic gene programs while suppressing GM-CSF–driven MHC-II expression at the chromatin level, creating mutually exclusive distributions of proangiogenic and MHC-II+ TAMs, which were differentially localized to specific spatial niches in the tumor microenvironment. Furthermore, we showed that shifting TAMs to an interferon-responsive phenotype, triggered by Adar inactivation, significantly promoted the infiltration of effector CD8+ T cells through specific receptor–ligand interactions. These findings uncover a conserved mechanism of TAM polarization and offer insights into therapeutic strategies for TAM reprogramming to potentiate cancer immunotherapy.