{
  "abstract": "Background Patients with metastatic castration-resistant prostate cancer (mCRPC) are generally refractory to immune checkpoint inhibitors (ICIs), 1 2 with those having bone metastases, the most common metastatic site (70-90%),3 4 showing even poorer responses.1 5 The bone marrow (BM), as a primary hematopoietic organ, serves as a key reservoir for immune cells,6 with myeloid cells comprising >70% of the hematopoietic compartment.7 8 We have shown that this ICI resistance is mediated by immunosuppressive myeloid cells within human prostate tumors.9 Although neutrophils, known drivers of ICI resistance in multiple malignancies, are the most prevalent myeloid cell type in the BM, their immunosuppressive role in mCRPC bone metastases, particularly in humans, remains poorly characterized. Therefore, we hypothesize that single-cell characterization of immunosuppressive neutrophil subsets and their molecular mechanisms can find new therapeutic opportunities to enhance immunotherapeutic efficacy.Methods We performed single-cell profiling of biopsies from prostate cancer patients across various disease stages and tumor sites, including the prostate, bone, lymph nodes, and liver, as well as bone biopsies from tumor-free patients who underwent hip replacement surgery for isolated osteoarthritis. We also utilized a murine intratibial model of CRPC to validate our findings with multi-omic single-cell analyses, functional assays, in vivo ICI efficacy tests, and mechanistic studies.Results Single-cell profiling of patients revealed abundant neutrophil populations in the bone compared to other soft tissues, with several subsets showing developmental heterogeneity. Notably, mature neutrophils (mNeu) were significantly more abundant in mCRPC bone metastases compared to tumor-free bone biopsies, expressing elevated immunosuppressive transcriptional programs. 10–13 Multi-omic profiling of an intratibial mouse CRPC model revealed an analogous neutrophil subset capable of suppressing CD8+ T cell function in co-culture. Adoptive transfer of mNeu into CRPC-bearing mice promoted resistance to ICIs and reduced survival. Pathway analyses implicated proinflammatory signaling as a key mechanism.14 Consistent with this, both human and murine mNeu exhibited elevated IL1B expression. Pharmacologic inhibition of IL-1 receptor significantly diminished mNeu-mediated CD8+ T cell suppression in culture and improved the responses to combined PD-1 and CTLA-4 blockade in vivo.Conclusions Our studies reveal a distinct mediator of immunotherapeutic resistance in bone metastases. Not only is there an increased mNeu prevalence in human mCRPC metastatic to bone, but we find that these neutrophils drive ICI resistance through proinflammatory signaling pathways. Targeting these cells and/or their signaling axes may represent promising therapeutic strategies for mCRPC.References Beer TM, Kwon ED, Drake CG, Fizazi K, Logothetis C, Gravis G, et al. Randomized, double-blind, phase III trial of ipilimumab versus placebo in asymptomatic or minimally symptomatic patients with metastatic chemotherapy-naive castration-resistant prostate cancer. J Clin Oncol. 2017;35:40–7.Powles T, Yuen KC, Gillessen S, Kadel EE, Rathkopf D, Matsubara N, et al. Atezolizumab with enzalutamide versus enzalutamide alone in metastatic castration-resistant prostate cancer: a randomized phase 3 trial. Nat Med. 2022;28:144–53.Koo KC, Park SU, Kim KH, Rha KH, Hong SJ, Yang SC, et al. prognostic impacts of metastatic site and pain on progression to castrate resistance and mortality in patients with metastatic prostate cancer. Yonsei Méd J. 2015;56:1206–12.Halabi S, Kelly WK, Ma H, Zhou H, Solomon NC, Fizazi K, et al. Meta-analysis evaluating the impact of site of metastasis on overall survival in men with castration-resistant prostate cancer. J Clin Oncol. 2016;34:1652–9.Joseph GJ, Johnson DB, Johnson RW. Immune checkpoint inhibitors in bone metastasis: Clinical challenges, toxicities, and mechanisms. J Bone Oncol. 2023;43:100505.Lucas D. Structural organization of the bone marrow and its role in hematopoiesis. Curr Opin Hematol. 2021;28:36–42.Pang WW, Price EA, Sahoo D, Beerman I, Maloney WJ, Rossi DJ, et al. Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age. Proc National Acad Sci. 2011;108:20012–7.Chen X, Deng H, Churchill MJ, Luchsinger LL, Du X, Chu TH, et al. bone marrow myeloid cells regulate myeloid-biased hematopoietic stem cells via a histamine-dependent feedback loop. Cell Stem Cell. 2017;21:747–760.e7.Lyu A, Fan Z, Clark M, Lea A, Luong D, Setayesh A, et al. Evolution of myeloid-mediated immunotherapy resistance in prostate cancer. Nature. 2025;637:1207–17.Coffelt SB, Kersten K, Doornebal CW, Weiden J, Vrijland K, Hau C-S, et al. IL-17-producing γδ T cells and neutrophils conspire to promote breast cancer metastasis. Nature. 2015;522:345–8.Trovato R, Fiore A, Sartori S, Canè S, Giugno R, Cascione L, et al. Immunosuppression by monocytic myeloid-derived suppressor cells in patients with pancreatic ductal carcinoma is orchestrated by STAT3. J Immunother Cancer. 2019;7:255.Gong Z, Li Q, Shi J, Li P, Hua L, Shultz LD, et al. Immunosuppressive reprogramming of neutrophils by lung mesenchymal cells promotes breast cancer metastasis. Sci Immunol. 2023;8:eadd5204.Gungabeesoon J, Gort-Freitas NA, Kiss M, Bolli E, Messemaker M, Siwicki M, et al. A neutrophil response linked to tumor control in immunotherapy. Cell. 2023;186:1448-1464.e20.Wang L, Sfakianos JP, Beaumont KG, Akturk G, Horowitz A, Sebra RP, et al. Myeloid cell-associated resistance to PD-1/PD-L1 blockade in urothelial cancer revealed through bulk and single-cell RNA sequencing. Clin cancer Res. 2020;27:4287–300.",
  "authors": [
    {
      "affiliations": [
        "University of California San Francisco, San Francisco, CA, USA",
        "Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA",
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Aram Lyu"
    },
    {
      "affiliations": [
        "University of California San Francisco, San Francisco, CA, USA"
      ],
      "name": "Zenghua Fan"
    },
    {
      "affiliations": [
        "University of California San Francisco, San Francisco, CA, USA"
      ],
      "name": "Matthew Clark"
    },
    {
      "affiliations": [
        "University of California San Francisco, San Francisco, CA, USA"
      ],
      "name": "Averey Lea"
    },
    {
      "affiliations": [
        "University of California San Francisco, San Francisco, CA, USA"
      ],
      "name": "Rachel Wolters"
    },
    {
      "affiliations": [
        "University of California San Francisco, San Francisco, CA, USA"
      ],
      "name": "Longhui Qiu"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Kiersten Tucker"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Lam Trieu"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Krutika Khinvasara"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Alex Tran"
    },
    {
      "affiliations": [
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Ryan Owens"
    },
    {
      "affiliations": [
        "Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA",
        "Dana-Farber Cancer Institute, Boston, MA, USA",
        "Broad Institute of Harvard and MIT, Cambridge, MA, USA"
      ],
      "name": "Eliezer Van Allen"
    },
    {
      "affiliations": [
        "University of California San Francisco, San Francisco, CA, USA",
        "Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA",
        "Fred Hutchinson Cancer Center, Seattle, WA, USA"
      ],
      "name": "Lawrence Fong"
    }
  ],
  "title": "463 Dissecting myeloid-driven mechanisms of immunotherapy resistance in prostate cancer bone metastases",
  "uid": "0d8f2299-47e1-5db6-90e0-ab628171d53a"
}