Complex interaction of tumor-derived factors instructs the niche specific phenotypes of tumor-associated macrophages [CROP-Seq, in vitro and in vivo pool]

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. To characterize the whole transcriptional profiles targeted by our CRISPR screen candidates, we extended our tumor educated macrophage(TEM) system to CROP-seq, which is able to pair transcriptiome with sgRNA information. To validate the effect of these hits in vivo, we isolated CD117+ hematopoietic stem cells(HSCs) from Loxp-Stop-Loxp-Cas9, Lyz2-Cre mice and delivered CROP-seq pool into HSCs at MOI<0.3. Library cells were transplantated in lethally irradiated mice. After 6 weeks' reconstituion and LLC tumor inoculation, tumor associated macrophages(DAPI-CD45.2+CD11b+Ly6C-) were isolated for CROP-seq sequencing.