{
  "abstract": "Background Immunosuppressive tumor-associated macrophages (TAMs) dampen the anti-tumor immune response and drive resistance to immune checkpoint blockade (ICB). 1 Previous studies have identified folate receptor β (FRβ) as a biomarker of activated TAMs typically associated with worse prognosis.2–6 FRβ (gene: Folr2) is a high affinity folate receptor expressed uniquely on myeloid cells.7 Folate is a requisite coenzyme for one-carbon (1C) metabolism, which is critical for the maintenance of cellular redox balance, nucleotide synthesis, and global methylation reactions.8 9 The importance of FRβ-mediated folate uptake for pro-tumor TAM biology is unknown. Here we show FRβ-mediated 1C metabolism supports immunosuppressive macrophage functions through the control of reactive oxygen species (ROS).Methods We generated Folr2 -/- mice on the C57BL/6J genetic background. B16-OVA melanoma tumors were subcutaneously implanted into mice and immune infiltrates were characterized via flow cytometry. Bulk RNA-seq was conducted on flow-sorted TAMs. For in vitro experiments, bone-marrow derived macrophages (BMDMs) were cultured in M-CSF and physiological folate concentrations found in tumor-interstitial fluid. BMDMs were functionally profiled using intracellular cytokine staining, ELISA, bulk RNA-seq, ROS dye staining (CellRox, MitoSox), metabolomics, and CD8+ T-cell coculture studies. Analysis of single-nuclei RNA-seq and spatial transcriptomics was performed on myeloid cells from metastatic melanoma patient biopsies.Results FOLR2 + TAMs were enriched in melanoma biopsies from ICB-unresponsive patients, supporting an immunosuppressive role for FOLR2+ TAMs. In Folr2-/- mice, melanoma tumor growth was reduced by 70% compared with controls, with increased pro-inflammatory TAM differentiation and tumor infiltration by cytotoxic CD8+ T-cells. In vitro, Folr2 -/- BMDMs produced more pro-inflammatory cytokines and were unable to inhibit T cell activation compared to wild-type BMDMs. These phenotypes can be rescued by increasing folate in the media, suggesting Folr2 is required for immunosuppressive macrophage function due to increased folate uptake. Interestingly, Folr2 -/- BMDMs had increased intracellular ROS, were enriched for oxidative stress gene signatures, and possessed lower reduced glutathione metabolite pools, indicating a dysregulation of redox homeostasis. Transcriptionally, Folr2 -/- BMDMs upregulated NFkB signaling and other pro-inflammatory pathways that are positively regulated by ROS accumulation.10 Inhibiting ROS via antioxidant supplementation reversed the increased pro-inflammatory cytokine production and decreased T-cell suppression phenotypes observed in Folr2 -/- BMDMs.Conclusions Together, these data demonstrate that FRβ-mediated 1C metabolism is critical for the immunosuppressive functions of TAMs through mitigation of oxidative stress ( figure 1). Our studies underscore the importance of 1C metabolism as a pivotal regulator of TAM biology and potential target for TAM-directed cancer immunotherapies.Acknowledgements We would like to thank the following core facilities for their expertise and experimental assistance: UNC-CH Flow Cytometry Core, UNC-CH Animal Models Core, UNC-CH LCCC Translational Genomics Lab, and UNC-CH Microscopy Services Laboratory. This work was supported by Burroughs Wellcome Fund Career Award for Medical Scientists, NIH K08CA248960, V Foundation Scholar Award, and Melanoma Research Alliance Young Investigator Award.References Guerriero J. Macrophages: the road less traveled, changing anticancer therapy. Trends Mol Med. 2018;24(5):472-489.Puig-Kröger A, Sierra-Filardi E, Domínguez-Soto A, et al. Folate receptor β is expressed by tumor-associated macrophages and constitutes a marker for M2 anti-inflammatory/regulatory macrophages. Cancer Res. 2009;69(24):9395-9403.Tie Y, Zheng H, He Z, et al. Targeting folate receptor β positive tumor-associated macrophages in lung cancer with a folate-modified liposomal complex. Sig Transduct Target Ther. 2020;5(1):6.Rodriguez-Garcia A, Lynn R, Poussin M, et al. CAR-T cell-mediated depletion of immunosuppressive tumor-associated macrophages promotes endogenous antitumor immunity and augments adoptive immunotherapy. Nat Commun. 2021;12(1):877.Cresswell G, Wang B, Kischuk E, et al. Folate Receptor Beta Designates Immunosuppressive Tumor-Associated Myeloid Cells That Can Be Reprogrammed with Folate-Targeted Drugs. Cancer Res. 2021;81(3):671-684.Kurahara H, Takao S, Kuwahata T, et al. Clinical significance of folate receptor β-expressing tumor-associated macrophages in pancreatic cancer. Ann Surg Oncol. 2012;19(7):2264-2271.Samaniego R, Domínguez-Soto Á, Ratnam M, et al. Folate receptor β (FRβ) expression in tissue-resident and tumor-associated macrophages associates with and depends on the expression of PU.1. Cells 2020;9(6):1445.Ducker G, Rabinowitz J. One-Carbon Metabolism in Health and Disease. Cell Metab. 2017;25(1):27-42.Dang S, Jain A, Dhanda G, Bhattacharya N, Bhattacharya A, Senapati S. One carbon metabolism and its implication in health and immune functions. Cell Biochem and Funct. 2024;42(1):e3926.Morgan M, Liu Z. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 2011;21(1):103-115.Ethics Approval All mouse studies were approved by University of North Carolina at Chapel Hill Division for Comparative Medicine and Institutional Animal Care & Use Committee (Protocol #24-230). Human specimens were collected under IRB #LCCC13-2225.Abstract 799 Figure 1Folate receptor beta-mediated folate uptake suppresses pro-inflammatory macrophage functions",
  "authors": [
    {
      "affiliations": [
        "Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"
      ],
      "name": "Matthew P Zimmerman"
    },
    {
      "affiliations": [
        "The University of North Carolina, Chapel Hill, NC, USA"
      ],
      "name": "Vasyl Zhabotynsky"
    },
    {
      "affiliations": [
        "Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"
      ],
      "name": "Brian Fay"
    },
    {
      "affiliations": [
        "The University of North Carolina, Chapel Hill, NC, USA"
      ],
      "name": "Alex Bastian"
    },
    {
      "affiliations": [
        "The University of North Carolina, Chapel Hill, NC, USA"
      ],
      "name": "Emily K Cox"
    },
    {
      "affiliations": [
        "The University of North Carolina, Chapel Hill, NC, USA"
      ],
      "name": "Haiyang Wang"
    },
    {
      "affiliations": [
        "Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA"
      ],
      "name": "Wan Lin Chong"
    },
    {
      "affiliations": [
        "Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA"
      ],
      "name": "Katherine Vietor"
    },
    {
      "affiliations": [
        "Department of Nutrition, University of North Carolina at Chapel Hill, Nutrition Research Institute, Kannapolis, NC, USA"
      ],
      "name": "Susan Sumner"
    },
    {
      "affiliations": [
        "Department of Nutrition, University of North Carolina at Chapel Hill, Nutrition Research Institute, Kannapolis, NC, USA"
      ],
      "name": "Blake Rushing"
    },
    {
      "affiliations": [
        "Department of Nutrition, University of North Carolina at Chapel Hill, Nutrition Research Institute, Kannapolis, NC, USA"
      ],
      "name": "Sergey A Krupenko"
    },
    {
      "affiliations": [
        "Department of Medicine, Division of Dermatology, University of California Los Angeles, David Geffen School of Medicine, Los Angeles, CA, USA"
      ],
      "name": "Willy Hugo"
    },
    {
      "affiliations": [
        "Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA",
        "Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA"
      ],
      "name": "Jenny Ting"
    },
    {
      "affiliations": [
        "Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA",
        "Department of Medicine, Division of Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"
      ],
      "name": "Stergios J Moschos"
    },
    {
      "affiliations": [
        "Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA",
        "Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC, USA",
        "Department of Medicine, Division of Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA",
        "Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA"
      ],
      "name": "Brian C Miller"
    }
  ],
  "title": "799 Dissecting the role of folate receptor beta-mediated one-carbon metabolism in tumor-associated macrophages",
  "uid": "da625083-9760-5903-8693-eb7beca8ff58"
}