Abstract
Women diagnosed with metastatic triple negative breast cancer (mTNBC) have limited treatment options, are more prone to develop resistance and are associated with high mortality. A cold tumor immune microenvironment (TIME) characterized by low T cells and high tumor associated macrophages (TAMs) in mTNBC is associated with the failure of standard-of-care chemotherapy and immune checkpoint blockade (ICB) treatment. We demonstrate that the combination of immunomodulatory low-dose Cyclophosphamide (CTX) coupled with anti-CSF-1R antibody targeted therapy (SNDX-ms6352) and anti-PD-1 (ICB), was highly effective against aggressive metastatic Trp53 null TNBC transplantable syngeneic models that present with high macrophage infiltration. Mechanistically, CSF-1R inhibition along with CTX disrupted the M-CSF/CSF-1R axis which upregulated IL-17, IL-5 and type II interferon resulting in elevated B- and T cell infiltration. Addition of an anti-PD-1 maintenance dose helped overcome de novo PD-L1 intra-tumoral heterogeneity (ITH) associated recurrence in lung and liver mTNBC.
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Data availability
Raw and analyzed scRNA-seq data has been deposited in the NCBI GEO database accession number (GSE292908). Detailed information regarding the antibodies used for IMC is provided in Supplementary table 4, IMC single cell analysis provided in Supplementary dataset S15-17. All unique/stable reagents generated in this study are available from the Lead Contact following a completed Materials Transfer Agreement (MTA). Source data are provided with this paper. All illustrations were created with BioRender. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Jeffrey M. Rosen (jrosen@bcm.edu).
References
Cheang, M. C. et al. Defining breast cancer intrinsic subtypes by quantitative receptor expression. Oncologist 20, 474–482 (2015).
Schmid, P. et al. Atezolizumab and nab-paclitaxel in advanced triple-negative breast cancer. N. Engl. J. Med. 379, 2108–2121 (2018).
Emens, L. A. et al. First-line atezolizumab plus nab-paclitaxel for unresectable, locally advanced, or metastatic triple-negative breast cancer: IMpassion130 final overall survival analysis. Ann. Oncol. 32, 983–993 (2021).
Adams, S. et al. Pembrolizumab monotherapy for previously untreated, PD-L1-positive, metastatic triple-negative breast cancer: cohort B of the phase II KEYNOTE-086 study. Ann. Oncol. 30, 405–411 (2019).
Hoadley, K. A. et al. Tumor evolution in two patients with basal-like breast cancer: a retrospective genomics study of multiple metastases. PLoS Med. 13, e1002174 (2016).
Kim, I. S. et al. Immuno-subtyping of breast cancer reveals distinct myeloid cell profiles and immunotherapy resistance mechanisms. Nat. Cell Biol. 21, 1113–1126 (2019).
Yam, C. et al. Molecular characterization and prospective evaluation of pathologic response and outcomes with neoadjuvant therapy in metaplastic triple-negative breast cancer. Clin. Cancer Res. 28, 2878–2889 (2022).
Yam, C. et al. Immune phenotype and response to neoadjuvant therapy in triple-negative breast cancer. Clin. Cancer Res. 27, 5365-5375 (2021).
Pagani, O. et al. International guidelines for management of metastatic breast cancer: can metastatic breast cancer be cured?. J Natl Cancer Inst 102, 456–463 (2010).
Aftimos, P. et al. Genomic and transcriptomic analyses of breast cancer primaries and matched metastases in AURORA, the breast international group (BIG) molecular screening initiative. Cancer Discov. 11, 2796–2811 (2021).
Garcia-Recio, S. et al. Multiomics in primary and metastatic breast tumors from the AURORA US network finds microenvironment and epigenetic drivers of metastasis. Nat. Cancer 4, 128–147 (2023).
Guerriero, J. L. et al. Class IIa HDAC inhibition reduces breast tumours and metastases through anti-tumour macrophages. Nature 543, 428–432 (2017).
Sun, L. et al. Activating a collaborative innate-adaptive immune response to control metastasis. Cancer Cell 39, 1361–1374.e1369 (2021).
Willingham, S. B. et al. The CD47-signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc. Natl. Acad. Sci. USA 109, 6662–6667 (2012).
Singh, S. et al. Chemotherapy coupled to macrophage inhibition induces T-cell and B-cell infiltration and durable regression in triple-negative breast cancer. Cancer Res. 82, 2281–2297 (2022).
Wesolowski, R. et al. Phase Ib study of the combination of pexidartinib (PLX3397), a CSF-1R inhibitor, and paclitaxel in patients with advanced solid tumors. Ther. Adv. Med. Oncol. 11, 1758835919854238 (2019).
Ries, C. H. et al. Targeting tumor-associated macrophages with anti-CSF-1R antibody reveals a strategy for cancer therapy. Cancer Cell 25, 846–859 (2014).
Tu, M. M. et al. Inhibition of the CCL2 receptor, CCR2, enhances tumor response to immune checkpoint therapy. Commun. Biol. 3, 720 (2020).
Wang, H. & Yee, D. I-SPY 2: a Neoadjuvant adaptive clinical trial designed to improve outcomes in high-risk breast cancer. Curr. Breast Cancer Rep. 11, 303–310 (2019).
Cassetta, L. et al. Human tumor-associated macrophage and monocyte transcriptional landscapes reveal cancer-specific reprogramming, biomarkers, and therapeutic targets. Cancer Cell 35, 588–602.e510 (2019).
Beck, A. H. et al. The macrophage colony-stimulating factor 1 response signature in breast carcinoma. Clin. Cancer Res. 15, 778–787 (2009).
Wolff, D. et al. Axatilimab in recurrent or refractory chronic graft-versus-host disease. N. Engl. J. Med. 391, 1002–1014 (2024).
Herschkowitz, J. I. et al. Comparative oncogenomics identifies breast tumors enriched in functional tumor-initiating cells. Proc. Natl. Acad. Sci. USA 109, 2778–2783 (2012).
Olivier, M. et al. The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer. Clin. Cancer Res. 12, 1157–1167 (2006).
Ellis, M. J. et al. Whole-genome analysis informs breast cancer response to aromatase inhibition. Nature 486, 353–360 (2012).
Razavi, P. et al. The genomic landscape of endocrine-resistant advanced breast cancers. Cancer Cell 34, 427–438.e426 (2018).
Ma, S., Caligiuri, M. A. & Yu, J. Harnessing IL-15 signaling to potentiate NK cell-mediated cancer immunotherapy. Trends Immunol. 43, 833–847 (2022).
Huntington, N. D. et al. IL-15 trans-presentation promotes human NK cell development and differentiation in vivo. J. Exp. Med. 206, 25–34 (2009).
Zhang, X., Sun, S., Hwang, I., Tough, D. F. & Sprent, J. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity 8, 591–599 (1998).
Martínez-Sabadell, A., Arenas, E. J. & Arribas, J. IFNγ signaling in natural and therapy-induced antitumor responses. Clin. Cancer Res. 28, 1243–1249 (2022).
Szabo, S. J. et al. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science 295, 338–342 (2002).
House, I. G. et al. Macrophage-Derived CXCL9 and CXCL10 Are Required for Antitumor Immune Responses Following Immune Checkpoint Blockade. Clin. Cancer Res. 26, 487–504 (2020).
Bill, R. et al. macrophage polarity identifies a network of cellular programs that control human cancers. Science 381, 515–524 (2023).
Nagai, S. & Toi, M. Interleukin-4 and breast cancer. Breast Cancer 7, 181–186 (2000).
Zhao, J., Chen, X., Herjan, T. & Li, X. The role of interleukin-17 in tumor development and progression. J. Exp. Med. 217, e20190297(2020).
Murugaiyan, G. & Saha, B. Protumor vs antitumor functions of IL-17. J. Immunol. 183, 4169–4175 (2009).
Güç, E. & Pollard, J. W. Redefining macrophage and neutrophil biology in the metastatic cascade. Immunity 54, 885–902 (2021).
Swierczak, A. et al. The promotion of breast cancer metastasis caused by inhibition of CSF-1R/CSF-1 signaling is blocked by targeting the G-CSF receptor. Cancer Immunol. Res. 2, 765–776 (2014).
Huang, R. et al. CCL5 derived from tumor-associated macrophages promotes prostate cancer stem cells and metastasis via activating β-catenin/STAT3 signaling. Cell Death Dis. 11, 234 (2020).
Jin, L. et al. Breast cancer lung metastasis: Molecular biology and therapeutic implications. Cancer Biol. Ther. 19, 858–868 (2018).
Vishnoi, M. et al. The identification of a TNBC liver metastasis gene signature by sequential CTC-xenograft modeling. Mol. Oncol. 13, 1913–1926 (2019).
Steinbauer, M. et al. GFP-transfected tumor cells are useful in examining early metastasis in vivo, but immune reaction precludes long-term tumor development studies in immunocompetent mice. Clin Exp Metastasis 20, 135–141 (2003).
Stripecke, R. et al. Immune response to green fluorescent protein: implications for gene therapy. Gene Ther. 6, 1305–1312 (1999).
Grzelak, C. A. et al. Elimination of fluorescent protein immunogenicity permits modeling of metastasis in immune-competent settings. Cancer Cell 40, 1–2 (2022).
Pedroza, D. A., Gao, Y., Zhang, X. H. & Rosen, J. M. Leveraging preclinical models of metastatic breast cancer. Biochim. Biophys. Acta Rev. Cancer 1879, 189163 (2024).
Black, M. et al. Activation of the PD-1/PD-L1 immune checkpoint confers tumor cell chemoresistance associated with increased metastasis. Oncotarget 7, 10557–10567 (2016).
Núñez Abad, M. et al. Programmed Death-Ligand 1 (PD-L1) as Immunotherapy Biomarker in Breast Cancer. Cancers (Basel) 14 (2022).
Hong, M., Kim, J. W., Kim, M. K., Chung, B. W. & Ahn, S. K. Programmed cell death-ligand 1 expression in stromal immune cells is a marker of breast cancer outcome. J. Cancer 11, 7246–7252 (2020).
Zhang, M. et al. Expression of PD-L1 and prognosis in breast cancer: a meta-analysis. Oncotarget 8, 31347–31354 (2017).
Cha, J. H., Chan, L. C., Li, C. W., Hsu, J. L. & Hung, M. C. Mechanisms controlling PD-L1 expression in cancer. Mol. Cell 76, 359–370 (2019).
Yuan, X. et al. CREB-binding protein/P300 bromodomain inhibition reduces neutrophil accumulation and activates antitumor immunity in triple-negative breast cancer. JCI Insight 9, e182621 (2024).
Deng, L. et al. Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J. Clin. Invest. 124, 687–695 (2014).
Tumeh, P. C. et al. PD-1 blockade induces responses by inhibiting adaptive immune resistance. Nature 515, 568–571 (2014).
Iwase, T. et al. Maintenance pembrolizumab therapy in patients with metastatic HER2-negative breast cancer with prior response to chemotherapy. Clin. Cancer Res. 30, 2424–2432 (2024).
Zhang, L. et al. A prognostic model for triple-negative breast cancer patients with liver metastasis: a population-based study. Heliyon 10, e27837 (2024).
Xie, J. & Xu, Z. A Population-Based Study on Liver Metastases in Women with Newly Diagnosed Breast Cancer. Cancer Epidemiol. Biomarkers Prev. 28, 283–292 (2019).
Yu, J. et al. Liver metastasis restrains immunotherapy efficacy via macrophage-mediated T cell elimination. Nat. Med. 27, 152–164 (2021).
Goddard, E.T., Fischer, J. & Schedin, P. A portal vein injection model to study liver metastasis of breast cancer. J. Vis. Exp. 26, 54903 (2016).
Sevmis, M. et al. Splenectomy-induced leukocytosis promotes intratumoral accumulation of myeloid-derived suppressor cells, angiogenesis and metastasis. Immunol. Invest. 46, 663–676 (2017).
Crittenden, M. R. et al. The peripheral myeloid expansion driven by murine cancer progression is reversed by radiation therapy of the tumor. PLoS ONE 8, e69527 (2013).
Cannarile, M. A. et al. Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer therapy. J Immunother Cancer 5, 53 (2017).
Xu, Y. et al. Immunosuppressive tumor-associated macrophages expressing interlukin-10 conferred poor prognosis and therapeutic vulnerability in patients with muscle-invasive bladder cancer. J. Immunother. Cancer 10, e003416 (2022).
Nakamura, K. & Smyth, M. J. TREM2 marks tumor-associated macrophages. Signal Transduct. Target Ther. 5, 233 (2020).
Mathiesen, H. et al. Prognostic value of CD163. Immunol. Lett. 272, 106970 (2025).
Lauwers, Y. et al. Imaging of tumor-associated macrophage dynamics during immunotherapy using a CD163-specific nanobody-based immunotracer. Proc. Natl. Acad. Sci. USA 121, e2409668121 (2024).
Wang, H. et al. Integrative analysis identifies CXCL11 as an immune-related prognostic biomarker correlated with cell proliferation and immune infiltration in multiple myeloma microenvironment. Cancer Cell Int. 22, 187 (2022).
Torraca, V. et al. The CXCR3-CXCL11 signaling axis mediates macrophage recruitment and dissemination of mycobacterial infection. Dis. Model Mech. 8, 253–269 (2015).
Liu, M. et al. Colon cancer cells secreted CXCL11 via RBP-Jκ to facilitated tumour-associated macrophage-induced cancer metastasis. J. Cell Mol. Med. 25, 10575–10590 (2021).
Epping, M. T., Hart, A. A., Glas, A. M., Krijgsman, O. & Bernards, R. PRAME expression and clinical outcome of breast cancer. Br. J. Cancer 99, 398–403 (2008).
Chen, Y. L. Prognostic significance of tumor-associated macrophages in patients with nasopharyngeal carcinoma: a meta-analysis. Medicine (Baltimore) 99, e21999 (2020).
Yang, Z. et al. The prognostic and clinicopathological value of tumor-associated macrophages in patients with colorectal cancer: a systematic review and meta-analysis. Int. J. Colorectal Dis. 35, 1651–1661 (2020).
Shen, H. et al. Prognostic value of tumor-associated macrophages in clear cell renal cell carcinoma: a systematic review and meta-analysis. Front Oncol 11, 657318 (2021).
Wang, P. F. et al. Prognostic role of pretreatment circulating MDSCs in patients with solid malignancies: a meta-analysis of 40 studies. Oncoimmunology 7, e1494113 (2018).
Tsujikawa, T. et al. Quantitative multiplex immunohistochemistry reveals myeloid-inflamed tumor-immune complexity associated with poor prognosis. Cell Rep. 19, 203–217 (2017).
DeNardo, D. G. et al. Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov. 1, 54–67 (2011).
Mehta, A. K. et al. Targeting immunosuppressive macrophages overcomes PARP inhibitor resistance in BRCA1-associated triple-negative breast cancer. Nat. Cancer 2, 66–82 (2021).
Generali, D. et al. Immunomodulation of FOXP3+ regulatory T cells by the aromatase inhibitor letrozole in breast cancer patients. Clin. Cancer Res. 15, 1046–1051 (2009).
Fares, J. E. et al. Metronomic chemotherapy for patients with metastatic breast cancer: Review of effectiveness and potential use during pandemics. Cancer Treat Rev. 89, 102066 (2020).
Khan, K. A. et al. Immunostimulatory and anti-tumor metronomic cyclophosphamide regimens assessed in primary orthotopic and metastatic murine breast cancer. NPJ Breast Cancer 6, 29 (2020).
Scurr, M. et al. Low-dose cyclophosphamide induces antitumor T-cell responses, which associate with survival in metastatic colorectal cancer. Clin. Cancer Res. 23, 6771–6780 (2017).
Webb, E. R. et al. Cyclophosphamide depletes tumor infiltrating T regulatory cells and combined with anti-PD-1 therapy improves survival in murine neuroblastoma. iScience 25, 104995 (2022).
Pan, Y. et al. Survival of tissue-resident memory T cells requires exogenous lipid uptake and metabolism. Nature 543, 252–256 (2017).
Lin, R. et al. Fatty acid oxidation controls CD8. Cancer Immunol. Res. 8, 479–492 (2020).
Hwang, S. M. et al. Transgelin 2 guards T cell lipid metabolism and antitumour function. Nature 635, 1010–1018 (2024).
Rosenheim, M. L. Chronic pyelonephritis. Isr. J. Med. Sci. 3, 93–97 (1967).
Kesireddy, M. et al. Overall survival and prognostic factors in metastatic triple-negative breast cancer: a national cancer database analysis. Cancers (Basel) 16, 1791 (2024).
Pasha, N. & Turner, N. C. Understanding and overcoming tumor heterogeneity in metastatic breast cancer treatment. Nat Cancer 2, 680–692 (2021).
Chan, L. K., Tsui, Y. M., Ho, D. W. & Ng, I. O. Cellular heterogeneity and plasticity in liver cancer. Semin. Cancer Biol. 82, 134–149 (2022).
Klughammer, J. et al. A multi-modal single-cell and spatial expression map of metastatic breast cancer biopsies across clinicopathological features. Nat. Med. 30, 3236–3249 (2024).
Kuett, L. et al. Distant metastases of breast cancer resemble primary tumors in cancer cell composition but differ in immune cell phenotypes. Cancer Res. 85, 15–31 (2025).
Oberg, H. H., Wesch, D., Kalyan, S. & Kabelitz, D. Regulatory interactions between neutrophils, tumor cells and T cells. Front. Immunol. 10, 1690 (2019).
Stires, H. et al. Improving the odds together: a framework for breast cancer research scientists to include patient advocates in their research. NPJ Breast Cancer 8, 75 (2022).
Cortes, J. et al. Pembrolizumab plus chemotherapy versus placebo plus chemotherapy for previously untreated locally recurrent inoperable or metastatic triple-negative breast cancer (KEYNOTE-355): a randomised, placebo-controlled, double-blind, phase 3 clinical trial. Lancet 396, 1817–1828 (2020).
Emens, L.A. et al. Society for Immunotherapy of Cancer (SITC) clinical practice guideline on immunotherapy for the treatment of breast cancer. J. Immunother. Cancer 9, e002597 (2021).
Schmid, P. et al. Pembrolizumab for Early Triple-Negative Breast Cancer. N. Engl. J. Med. 382, 810–821 (2020).
Miles, D. et al. Primary results from IMpassion131, a double-blind, placebo-controlled, randomised phase III trial of first-line paclitaxel with or without atezolizumab for unresectable locally advanced/metastatic triple-negative breast cancer. Ann. Oncol. 32, 994–1004 (2021).
Kim, S. J., Garcia-Recio, S., Creighton, C. J., Perou, C. M. & Rosen, J. M. Alterations in Wnt- and/or STAT3 signaling pathways and the immune microenvironment during metastatic progression. Oncogene 38, 5942–5958 (2019).
Acknowledgements
We are grateful for the suggestions from both the Zhang and Rosen laboratory members. We thank Peter Ordentlich from Syndax for providing SNDX-ms6352 and Dr. Patricia Castro from the Human Tissue Acquisition and Pathology Core at BCM for IMC antibody validation. D.A.P. is supported by the American Cancer Society Postdoctoral Fellowship (PF-22-163-01-MM) and NIGMS MOSAIC (K99GM155594). A.J.S. is supported by Susan G Komen (SAC232150). N.Z. is supported by Cancer Prevention and Research Institute of Texas (CPRIT) (RP220468). C.M.P. was supported by funds from NCI Breast SPORE program (P50-CA058223) and U01-CA289839-01. X.H.-F.Z. is supported by US Department of Defense (DAMD W81XWH-16-1-0073 and DAMD W81XWH-20-1-0375), NIH (R01CA183878, R01CA227904, R01CA221946, R01CA251950 and 5P50CA186784-03) the Breast Cancer Research Foundation, and McNair Foundation. J.M.R. was supported by NIH (R01CA016303-46 and R01CA148761-13) and Susan G. Komen (SAC232150). This project was supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (CA125123 and RR024574) and the assistance of Joel M. Sederstrom. The scRNA-seq experiments were supported by the Single Cell Genomics Core at BCM, which is partially supported by NIH (S10OD025240) and CPRIT (RP200504). The Integrated Microscopy core supported by NIH (DK56338, CA125123, ES030285), and CPRIT (RP150578, RP170719). We would like to thank Texas Children’s Hospital for the use of the Small Animal Imaging facility (SAIF) for getting PET imaging. We thank the Pathology Core of Lester and Sue Smith Breast Center at BCM for tissue sectioning. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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D.A.P. conceptualized and designed the study, conducted the experiments, analyzed the data, and wrote the manuscript. X.Y., F.L., H.L.C., W.B. and Q.Z. provided bioinformatics support. C.Z., S.J.C., N.L., C.V.O., O.P. conducted experiments and analyzed data. W.W and P.P analyzed imaging mass cytometry experiments. A.J.S. and N.Z. designed the flow cytometry experiments and analyzed the data. P.S. and M.W.G. conducted PET/CT scans and analyzed the data. C.M.P. provided bioinformatics support and edited the manuscript. X.H.-F.Z. and J.M.R. conceived and supervised the study and edited the manuscript.
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C.M.P. is an equity stockholder of and consultant for BioClassifier LLC. C.M.P. is also listed as an inventor on a patent application (US9631239B2) for the Breast PAM50 Subtyping assay (not related to this work). The remaining authors declare no competing interests.
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Pedroza, D.A., Yuan, X., Liu, F. et al. Anti-CSF-1R therapy with combined immuno-chemotherapy coordinate an adaptive immune response to eliminate macrophage enriched triple negative breast cancers. Nat Commun (2026). https://doi.org/10.1038/s41467-025-67964-2
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DOI: https://doi.org/10.1038/s41467-025-67964-2
