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Background Macrophages are highly specialized immune cells capable of dynamic functional shifts, or ‘plasticity,’ in response to environmental cues. Tumor-associated macrophages (TAMs) are the most abundant immune subset within the tumor microenvironment (TME). While proinflammatory TAMs facilitate anti-tumor responses, TME-derived signals drive the accumulation of anti-inflammatory, tumor-supporting TAMs. Despite extensive in vitro research, whether macrophage plasticity occurs in tumors in vivo, and its mechanisms and kinetics, remain unknown, limiting the advancement of macrophage-targeted immunotherapies for cancer. Our working hypothesis is that in vivo plasticity is the driving force for the loss of anti-tumor functions in TAMs.Methods We developed a novel fate-mapping mouse model (IL-12bBFP-creERT2 x Rosa26LSL-tdTomato) allowing the in vivo fate-mapping of IL-12-producing proinflammatory (BFP+) macrophages when they stop producing IL-12b (BFP-, tdTomato+). In vitro validation was performed by assessing the coordination between IL-12 production and BFP expression in bone marrow-derived macrophages following various stimulations. For in vivo analyses, MC-38 colon carcinoma-bearing mice were treated with R848-loaded nanoparticles and anti-PD1 therapy, in the presence of tamoxifen to fate-map pro-inflammatory TAMs. TAMs were profiled by flow cytometry and single-cell RNA sequencing 4 hours, 2 days, and 7 days after therapy. Functional readouts included the frequency and phenotype of IL-12b fate-mapped TAMs, temporal gene expression dynamics, and phenotypic profiling after loss of IL-12b expression due to a polarizing TME.Results In vitro, combined CpG/IFNγ and R848/IFNγ stimulation robustly induced BFP+ macrophages coordinated with IL-12 production. 4OH-tamoxifen stably induced tdTomato expression. In vivo, TAMs transiently expressed IL-12b, peaking between 4-6hrs post-R848 administration. Fate-mapped tdTomato+ TAMs appeared at 24hrs and reached a maximum at 48hrs post-therapy. Notably, tdTomato+ TAMs remained in tumors for 7 days post-R848 therapy. Single-cell RNAseq analysis revealed a proinflammatory shift in TAMs at the 4hr time point characterized by the expression of IL12b, Cd40, Nos2, and Tnf. However, this state is transient and lost at the 48hrs and 7-day time points, suggesting the TME overcomes this proinflammatory phenotype and induces TAM plasticity towards a more lipid-laden, immunosuppressive state.Conclusions This study characterizes in vivo fate-mapping of TAM polarization and plasticity in response to immunotherapy. Our novel mouse model reveals that immunotherapy instructs rapid, transient proinflammatory TAMs followed by plasticity towards immunosuppressive functions. These findings advance the understanding of TAM functional dynamics, provide insight into putative determinants of proinflammatory function and plasticity, and pave the way for rationally designing interventions that sustain TAM activity and overcome immunosuppressive switch in cancers.