Complex interaction of tumor-derived factors instructs the spatially distinct phenotypes of tumor-associated macrophages [scRNA-Seq]

Despite the importance of tumor-associated macrophages (TAMs) in modulating anti-tumor immunity, the molecular determinants of their functional phenotypes remain elusive. Through a large-scale CRISPR screen, we discovered that tumor-derived lactic acid, PGE2, and GM-CSF collaboratively shape the highly conserved but mutually exclusive TAM phenotypes: MHC-II+ and angiogenic TAMs. Mechanistically, the dichotomous nature of these two phenotypes is driven by the antagonistic interactions between lactic acid/PGE2 and GM-CSF. Lactic acid and PGE2 coordinately induce the angiogenic gene program while suppressing the GM-CSF-induced MHC-II program at chromatin level. This mechanism leads to distinct spatial distribution of TAMs, with angiogenic TAMs in lactate-rich hypoxic regions and MHC-II+ TAMs outside these areas. Furthermore, in vivo genetic perturbation of TAMs showed that shifting TAMs to an interferon responsive program, triggered by Adar inactivation, substantially potentiates anti-tumor immunity. Our findings suggest a conserved mechanism of TAM polarization and a potential approach for reprogramming TAMs in immunotherapy. Overall design: To investigate how ISG15+ tumor-associated macrophages(TAM)-based on Adar knockout affect the whole tumor microenvironment, we isolated CD117+ hematopoietic stem cells(HSCs) from Loxp-Stop-Loxp-Cas9, Lyz2-Cre mice and generated control(sgNon-targeting control) and Adar myeloid specific conditonal knockout mice(mKO) by HSC recosntituion in lethally irradiated mice. Total tumor infiltrating immune cells(DAPI-CD45+) were isolated by Fluorescence-activated cell sorting(FACS) from Control and Adar mKO mice.