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Background Tumor-associated macrophages (TAMs) promote angiogenesis, metastasis, and immunosuppression in breast cancer (BC), contributing to resistance to immune checkpoint blockade (ICB). Current TAM-targeting strategies lack durability in solid tumors, highlighting a critical need for new approaches to enable ICB efficacy and promote anti-tumor immunity. Cellular therapies highlight a potential novel approach for TAM-targeting and have not been clinically tested. While CAR-T cells have transformed hematologic cancer treatment, their efficacy in solid tumors is limited by trafficking, barriers, and myeloid-driven immunosuppression. In contrast, monocytes home to tumors, overcome barriers, and differentiate into macrophages that persist in the tumor microenvironment. Leveraging monocyte biology, we have developed CAR-Monocytes (CAR-Mo) targeting the TAM-restricted receptor TREM2, enriched on immunosuppressive and lipid-associated TAMs, to overcome current limitations of TAM-targeting and CAR therapy in BC.Methods We utilized two publicly available single-cell RNAseq (scRNASeq) datasets of treatment naïve patients across all breast tumor subtypes (GSE161529, GSE176078) to identify the TAM subpopulations. Additionally, the TCGA breast (BRCA) cohort was used to correlate overall survival with TREM2 expression. Murine TREM2-CAR-Mo were established using a TREM2-41BBz retroviral CAR construct transduced into Trem2-/- ER-HoxB8 cells. Human TREM2-CAR-Mo was established using a 41BBz lentiviral CAR construct transduced into U-937 AML cells. Human and murine TREM2-CAR-Mac were established using PMA and M-CSF differentiation respectively. Syngeneic tumors were established using EO771 BC cells injected into the mammary fat pad of immunocompetent mice.Results We discovered that TREM2 is expressed across multiple TAM subsets in BC and identified that high TREM2 expression strongly associates with poor clinical outcomes and negatively correlates with T cell abundance. TREM2+ BC TAMs highly resemble lipid-associated TAM signatures identified in other tumor types. Both murine and human TREM2-CAR-Mo demonstrate significant selective phagocytosis of TREM2+ macrophages compared to non-targeting CAR controls in vitro. Furthermore, in vitro activated TREM2-CAR-Mo produce pro-inflammatory cytokines in response to engagement with TREM2. Murine TREM2-CAR-Mo significantly reduced the growth of syngeneic orthotopic EO771 tumors and enhanced intratumoral T cell accumulation in vivo. Additionally, anti-PD1 blockade further enhanced anti-tumor responses and significantly improved overall survival.Conclusions Murine and human TREM2-CAR-Mo selectively phagocytose TREM2+ macrophages in vitro, and significantly reduce breast tumor growth, increase intratumoral T cell accumulation, and enhance ICB sensitivity in vivo. These results suggest that TAM-targeting CAR-Mo offers a first-in-class platform to improve the effectiveness of current immunotherapy approaches in BC with high potential to overcome the challenges faced by cellular therapies in other solid tumors.Acknowledgements This work was supported by the BWH Innovation Fund, The Harvard Ludwig Center, NIH NCI R01/R37 CA269499, and NIH NCI F32 CA284543.