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Background Glioblastoma (GBM) is the most aggressive primary brain tumor, with poor prognosis despite standard treatment. 1 It remains resistant to immunotherapy,2–7 primarily due to exclusion and dysfunction of CD8+ T cells,8 9 largely driven by immunosuppressive tumor-associated macrophages (TAMs),10–13 that promote T cell exhaustion.12–15 TAMs are key targets for reprogramming anti-glioma immunity. To advance immunotherapeutic strategies, novel regulators of anti-GBM immunity must be identified. Here, we reveal dural nociceptors—pain-sensing sensory neurons16—as previously unrecognized drivers of immune suppression in GBM.Methods Nociceptors were ablated using resiniferatoxin. The myeloid-intrinsic role of the neuropeptide, calcitonin gene-related peptide (CGRP) was assessed using RAMP1 fl/flLysMCre+ mice, which lack the expression of receptor activity-modifying protein 1 (RAMP1), the CGRP co-receptor, in myeloid cells. Syngeneic orthotopic murine GBM models (GL261, CT2A, M005) were used. Survival was assessed by Kaplan-Meier analysis. Flow cytometry and scRNA-seq were used to profile tumor-infiltrating immune cells. To test CD8+ T cell dependence, a cohort received depleting anti-CD8 antibody; another received anti-CTLA-4 antibody to assess sensitization to immune checkpoint blockade therapy (ICBT). Trigeminal ganglion nociceptors were cultured with GBM-associated cerebrospinal fluid (CSF) and analyzed by immunofluorescence. Publicly available human GBM scRNA-seq datasets17–20 were analyzed for CGRP pathway relevance.Results Nociceptor ablation prolonged survival, reduced immunosuppressive features of TAMs, enhanced CD8 + T cell infiltration and effector function, and reduced T cell exhaustion in GBM-bearing mice. It also sensitized previously unresponsive GBM to ICB. GBM-associated CSF activated nociceptors and elevated secretion of CGRP. In human GBM datasets,17–20 RAMP1 was differentially expressed on TAM subsets. A CGRP-specific gene signature (CGRPsig)21 identified CGRPsighi TAMs as highly immunosuppressive. GBM-bearing RAMP1fl/flLysMCre+ mice mirrored the effects of nociceptor ablation—prolonging survival, remodeling the immune landscape, and sensitizing tumors to ICB. This survival benefit was abrogated by CD8+ T cell depletion suggesting CGRP-driven TAM-T cell crosstalk drives immune suppression and therapeutic resistance in GBM. Mechanistically, we uncover reciprocal antagonism between CGRP and IFN-γ signaling in macrophages: CGRP inhibits STAT1 activation, while IFN-γ suppresses CGRP-driven cAMP levels and RAMP1 expression. This crosstalk shapes TAM phenotype and positions CGRP as a key regulator of macrophage-mediated immunosuppression in GBM.Conclusions This study defines a previously unrecognized neuro-immune circuit in GBM, where dural nociceptors and the CGRP-RAMP1 axis actively drive immune suppression ( figure 1). With CGRP antagonists already approved and nociceptor-targeted therapies in trials, these findings offer a translational strategy to reprogram the GBM microenvironment and enhance immunotherapy response.References Osuka S, Van Meir EG. Overcoming therapeutic resistance in glioblastoma: The way forward. Journal of Clinical Investigation 2017;127. Preprint at https://doi.org/10.1172/JCI89587Wu W, et al. Glioblastoma multiforme (GBM): An overview of current therapies and mechanisms of resistance. Pharmacological Research 2021;171. Preprint at https://doi.org/10.1016/j.phrs.2021.105780Bausart M, Préat V, Malfanti A. Immunotherapy for glioblastoma: the promise of combination strategies. Journal of Experimental and Clinical Cancer Research 2022;41. Preprint at https://doi.org/10.1186/s13046-022-02251-2Daubon T, Hemadou A, Romero Garmendia I, Saleh M. Glioblastoma immune landscape and the potential of new immunotherapies. Frontiers in Immunology 11. Preprint at https://doi.org/10.3389/fimmu.2020.585616 (2020).Lim M, Xia Y, Bettegowda C,Weller M. Current state of immunotherapy for glioblastoma. Nature Reviews Clinical Oncology 15. Preprint at https://doi.org/10.1038/s41571-018-0003-5 (2018).Muftuoglu Y, Liau LM. Results from the CheckMate 143 clinical trial: stalemate or new game strategy for glioblastoma immunotherapy? JAMA Oncology 2020;6. Preprint at https://doi.org/10.1001/jamaoncol.2020.0857Jackson CM, Choi J, Lim M. Mechanisms of immunotherapy resistance: lessons from glioblastoma. Nature Immunology 2019;20. Preprint at https://doi.org/10.1038/s41590-019-0433-y (2019).Woroniecka KI, Rhodin KE, Chongsathidkiet P, Keith KA, Fecci PE. T-cell dysfunction in glioblastoma: applying a new framework. Clinical Cancer Research 2018;24. Preprint at https://doi.org/10.1158/1078-0432.CCR-18-0047 (2018).Watowich MB, Gilbert MR, Larion M. T cell exhaustion in malignant gliomas. Trends in Cancer 2023;9. Preprint at https://doi.org/10.1016/j.trecan.2022.12.008 (2023).Quail DF, Joyce JA. The microenvironmental landscape of brain tumors. Cancer Cell 2017;31. Preprint at https://doi.org/10.1016/j.ccell.2017.02.009Engblom C, Pfirschke C, Pittet MJ. The role of myeloid cells in cancer therapies. Nature Reviews Cancer 2016;16. Preprint at https://doi.org/10.1038/nrc.2016.54 (2016).Pyonteck SM, et al. CSF-1R inhibition alters macrophage polarization and blocks glioma progression. Nat Med 2013;19.Yan D, et al. Inhibition of colony stimulating factor-1 receptor abrogates microenvironment-mediated therapeutic resistance in gliomas. Oncogene 2017;36.Waibl Polania J, et al. Antigen presentation by tumor-associated macrophages drives T cells from a progenitor exhaustion state to terminal exhaustion. Immunity 2024. doi:10.1016/j.immuni.2024.11.026.Kilian M, et al. MHC class II-restricted antigen presentation is required to prevent dysfunction of cytotoxic T cells by blood-borne myeloids in brain tumors. Cancer Cell 2023;41.Dubin AE, Patapoutian A. Nociceptors: the sensors of the pain pathway. Journal of Clinical Investigation 2010;120. Preprint at https://doi.org/10.1172/JCI42843Abdelfattah N, et al. Single-cell analysis of human glioma and immune cells identifies S100A4 as an immunotherapy target. Nat Commun 2022;13.Xiong A, Zhang J, Chen Y, Zhang Y, Yang F. Integrated single-cell transcriptomic analyses reveal that GPNMB-high macrophages promote PN-MES transition and impede T cell activation in GBM. EBioMedicine 2022;83.Jain S, et al. Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects. Journal of Clinical Investigation 2023;133.Pombo Antunes AR, et al. Single-cell profiling of myeloid cells in glioblastoma across species and disease stage reveals macrophage competition and specialization. Nat Neurosci 2021;24:595–610.Pinho-Ribeiro FA, et al. Bacteria hijack a meningeal neuroimmune axis to facilitate brain invasion. Nature 2023;615.Ethics Approval All mouse studies were conducted under a protocol approved by the Institutional Animal Care and Use Committee of the Ohio State University (2022A00000009-R2)Abstract 801 Figure 1Nociceptors as immune gatekeepers in glioblastoma