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Elevated infiltration of immunosuppressive alternatively polarized (M2) macrophages is associated with poor prognosis in patients with cancer. The tumor microenvironment remarkably orchestrates molecular mechanisms that program these macrophages. Here we identify a novel role for oncogenic Hedgehog (Hh) signaling in programming signature metabolic circuitries that regulate alternative polarization of tumor-associated macrophages. Two immunocompetent orthotopic mouse models of mammary tumors were used to test the effect of inhibiting Hh signaling on tumor-associated macrophages. Treatment with the pharmacologic Hh inhibitor vismodegib induced a significant shift in the profile of tumor-infiltrating macrophages. Mass spectrometry-based metabolomic analysis showed Hh inhibition induced significant alterations in metabolic processes, including metabolic sensing, mitochondrial adaptations, and lipid metabolism. In particular, inhibition of Hh in M2 macrophages reduced flux through the UDP-GlcNAc biosynthesis pathway. Consequently, O-GlcNAc-modification of STAT6 decreased, mitigating the immune-suppressive program of M2 macrophages, and the metabolically demanding M2 macrophages shifted their metabolism and bioenergetics from fatty acid oxidation to glycolysis. M2 macrophages enriched from vismodegib-treated mammary tumors showed characteristically decreased O-GlcNAcylation and altered mitochondrial dynamics. These Hh-inhibited macrophages are reminiscent of inflammatory (M1) macrophages, phenotypically characterized by fragmented mitochondria. This is the first report highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. These data describe a novel immunometabolic function of Hh signaling that can be clinically exploited.Significance:These findings illustrate that Hh activity regulates a metabolic and bioenergetic regulatory program in tumor-associated macrophages that promotes their immune-suppressive polarization.

In the United States, a woman has a 12% chance of developing breast cancer, and current treatments offer little relief to patients diagnosed with metastatic disease. Tumorigenesis and successful establishment of metastases depend upon tumor cell interactions with the surrounding immune microenvironment. Elevated tumor infiltration of immunosuppressive (M2) macrophages correlates with poor prognosis of breast cancer patients. The tumor microenvironment remarkably orchestrates molecular mechanisms that program these macrophages toward the M2 phenotype. Also, metabolic programming is instrumental in orchestrating the polarization of macrophages to assume an M1 (tumor-eradicating) or an M2 (tumor-promoting) phenotype. Aberrant activation of Hedgehog (Hh) signaling in breast cancer cells enables them to survive, proliferate, and metastasize, thus making it a promising target for breast cancer treatment. Hh signaling also enables a crosstalk between breast cancer cells and cells in their milieu, thus contributing to M2 macrophage polarization. We used two immunocompetent orthotopic mouse models of mammary tumors to test the effect of inhibiting Hh signaling on tumor-associated macrophages, and discovered that treatment with the pharmacologic Hh inhibitor, Vismodegib, induced a significant shift in the profile of tumor-infiltrating macrophages. We hypothesized that Hh activity calibrates the metabolism in macrophages, leading to enhanced M2 phenotype and function within the tumor microenvironment. Using a mass spectrometry-enabled untargeted metabolomics approach, we identified that inhibiting Hh signaling reduces flux through the hexosamine biosynthetic pathway, resulting in reduced cellular O-GlcNAcylation in M2 macrophages. This impinges upon diminished STAT6 O-GlcNAcylation, which consequently decreases fatty acid oxidation and ultimately enacts a metabolic cascade including lipid utilization, cellular bioenergetics, and mitochondrial dynamics. As such, inhibiting Hh activity mitigates the metabolomic and bioenergetic underpinnings of the immunosuppressive program of M2 macrophages, resulting in macrophages that are functionally and phenotypically reminiscent of inflammatory, anti-tumor macrophages. In conclusion, we discovered a novel role for Hh signaling in promoting polarization of tumor-associated macrophages to the M2 type through recalibrating their metabolic circuitries, ultimately leading to diminished M2 phenotype and function within the tumor microenvironment. This is the first evidence highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. This knowledge will help us to better understand how to target and diminish the pro-tumorigenic functions of tumor-infiltrating macrophages. Citation Format: Dominique C. Hinshaw, Ann Hanna, Tshering Lama-Sherpa, Brandon Metge, Sarah C. Kammerud, Gloria A. Benavides, Atul Kumar, Heba A. Alsheikh, Mateus Mota, Dongquan Chen, Scott Ballinger, Jeffrey C. Rathmell, Selvarangan Ponnazhagan, Victor Darley-Usmar, Rajeev S. Samant, Lalita A. Shevde. Hedgehog signaling regulates metabolism and polarization of mammary tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2103.