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Neuronal substance P drives metastasis through an extracellular RNA–TLR7 axis

Nature volume 633, pages 207–215 (2024)Cite this article

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Abstract

Tumour innervation is associated with worse patient outcomes in multiple cancers1,2, which suggests that it may regulate metastasis. Here we observed that highly metastatic mouse mammary tumours acquired more innervation than did less-metastatic tumours. This enhanced innervation was driven by expression of the axon-guidance molecule SLIT2 in tumour vasculature. Breast cancer cells induced spontaneous calcium activity in sensory neurons and elicited release of the neuropeptide substance P (SP). Using three-dimensional co-cultures and in vivo models, we found that neuronal SP promoted breast tumour growth, invasion and metastasis. Moreover, patient tumours with elevated SP exhibited enhanced lymph node metastatic spread. SP acted on tumoral tachykinin receptors (TACR1) to drive death of a small population of TACR1high cancer cells. Single-stranded RNAs (ssRNAs) released from dying cells acted on neighbouring tumoural Toll-like receptor 7 (TLR7) to non-canonically activate a prometastatic gene expression program. This SP- and ssRNA-induced Tlr7 gene expression signature was associated with reduced breast cancer survival outcomes. Therapeutic targeting of this neuro–cancer axis with the TACR1 antagonist aprepitant, an approved anti-nausea drug, suppressed breast cancer growth and metastasis in multiple models. Our findings reveal that tumour-induced hyperactivation of sensory neurons regulates multiple aspects of metastatic progression in breast cancer through a therapeutically targetable neuropeptide/extracellular ssRNA sensing axis.

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Fig. 1: Sensory neurons promote invasion, proliferation and metastasis in breast cancer.
Fig. 2: Neuronal SP promotes breast cancer metastasis.
Fig. 3: Neuronal SP promotes the release of metastatic ssRNAs from cancer cells.
Fig. 4: Sensory neurons signal through tumoural TLR7 to promote metastasis.
Fig. 5: The antiemetic aprepitant targets this neuro–cancer axis to inhibit metastasis.

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Data availability

Raw sequencing data and count tables for transcriptional profiling of 4T1-derived spheroids have been deposited at the Gene Expression Omnibus under accession number GSE267958. Reads were mapped to the mouse genome assembly GRCm38. Data for the METABRIC study are publicly available under EGA accession number EGAS00000000083. Data from the TCGA study are publicly available online (https://portal.gdc.cancer.gov). Source data are provided with this paper.

Code availability

All custom computer code is publicly available at GitHub (https://github.com/benostendorf/padmanaban_etal_2024).

References

  1. Ayala, G. E. et al. Cancer-related axonogenesis and neurogenesis in prostate cancer. Clin. Cancer Res. 14, 7593–7603 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Huang, D. et al. Nerve fibers in breast cancer tissues indicate aggressive tumor progression. Medicine 93, e172 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Oertel, H. Innervation and tumour growth: a preliminary report. Can. Med. Assoc. J. 18, 135–139 (1928).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Renz, B. W. et al. β2 adrenergic-neurotrophin feedforward loop promotes pancreatic cancer. Cancer Cell 34, 863–867 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Latil, A. et al. Quantification of expression of netrins, slits and their receptors in human prostate tumors. Int. J. Cancer 103, 306–315 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Osswald, M. et al. Brain tumour cells interconnect to a functional and resistant network. Nature 528, 93–98 (2015).

    Article  ADS  CAS  PubMed  Google Scholar 

  7. Venkatesh, H. S. et al. Neuronal activity promotes glioma growth through neuroligin-3 secretion. Cell 161, 803–816 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Venkatesh, H. S. et al. Electrical and synaptic integration of glioma into neural circuits. Nature 573, 539–545 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Zhao, C. M. et al. Denervation suppresses gastric tumorigenesis. Sci. Transl. Med. 6, 250ra115 (2014).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  10. Magnon, C. et al. Autonomic nerve development contributes to prostate cancer progression. Science 341, 1236361 (2013).

    Article  PubMed  Google Scholar 

  11. Balood, M. et al. Nociceptor neurons affect cancer immunosurveillance. Nature 611, 405–412 (2022).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Globig, A. M. et al. The β1-adrenergic receptor links sympathetic nerves to T cell exhaustion. Nature 622, 383–392 (2023).

    Article  ADS  CAS  PubMed  Google Scholar 

  13. Gerendai, I. et al. Transneuronal labelling of nerve cells in the CNS of female rat from the mammary gland by viral tracing technique. Neuroscience 108, 103–118 (2001).

    Article  CAS  PubMed  Google Scholar 

  14. Hebb, C. & Linzell, J. L. Innervation of the mammary gland. A histochemical study in the rabbit. Histochem. J. 2, 491–505 (1970).

    Article  CAS  PubMed  Google Scholar 

  15. Tavora, B. et al. Tumoural activation of TLR3–SLIT2 axis in endothelium drives metastasis. Nature 586, 299–304 (2020).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  16. Brose, K. et al. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795–806 (1999).

    Article  CAS  PubMed  Google Scholar 

  17. Nguyen-Ngoc, K. V. et al. ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc. Natl Acad. Sci. USA 109, E2595–E2604 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Aslakson, C. J. & Miller, F. R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 52, 1399–1405 (1992).

    CAS  PubMed  Google Scholar 

  19. Bujak, J. K., Kosmala, D., Szopa, I. M., Majchrzak, K. & Bednarczyk, P. Inflammation, cancer and immunity-implication of TRPV1 channel. Front. Oncol. 9, 1087 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Liebig, C., Ayala, G., Wilks, J. A., Berger, D. H. & Albo, D. Perineural invasion in cancer: a review of the literature. Cancer 115, 3379–3391 (2009).

    Article  CAS  PubMed  Google Scholar 

  21. Kastin, A. Handbook of Biologically Active Peptides (Academic, 2013).

  22. Otsuka, M. & Konishi, S. Release of substance P-like immunoreactivity from isolated spinal cord of newborn rat. Nature 264, 83–84 (1976).

    Article  ADS  CAS  PubMed  Google Scholar 

  23. Eshete, F. & Fields, R. D. Spike frequency decoding and autonomous activation of Ca2+-calmodulin-dependent protein kinase II in dorsal root ganglion neurons. J. Neurosci. 21, 6694–6705 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nishikawa, S. et al. Two histidine residues are essential for ribonuclease T1 activity as is the case for ribonuclease A. Biochemistry 26, 8620–8624 (1987).

    Article  CAS  PubMed  Google Scholar 

  25. Robertson, H. D., Webster, R. E. & Zinder, N. D. Purification and properties of ribonuclease III from Escherichia coli. J. Biol. Chem. 243, 82–91 (1968).

    Article  CAS  PubMed  Google Scholar 

  26. Bremnes, R. M., Sirera, R. & Camps, C. Circulating tumour-derived DNA and RNA markers in blood: a tool for early detection, diagnostics, and follow-up? Lung Cancer 49, 1–12 (2005).

    Article  PubMed  Google Scholar 

  27. Huang, W. et al. Site-specific RNase A activity was dramatically reduced in serum from multiple types of cancer patients. PLoS ONE 9, e96490 (2014).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  28. De Lamirande, G. Action of deoxyribonuclease and ribonuclease on the growth of Ehrlich ascites carcinoma in mice. Nature 192, 52–54 (1961).

    Article  ADS  PubMed  Google Scholar 

  29. Ledoux, L. Action of ribonuclease on two solid tumours in vivo. Nature 176, 36–37 (1955).

    Article  ADS  CAS  PubMed  Google Scholar 

  30. Lund, J. M. et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl Acad. Sci. USA 101, 5598–5603 (2004).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kawasaki, T. & Kawai, T. Toll-like receptor signaling pathways. Front. Immunol. 5, 461 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Ojaniemi, M. et al. Phosphatidylinositol 3-kinase is involved in Toll-like receptor 4-mediated cytokine expression in mouse macrophages. Eur. J. Immunol. 33, 597–605 (2003).

    Article  CAS  PubMed  Google Scholar 

  33. Ha, T. et al. TLR2 ligands induce cardioprotection against ischaemia/reperfusion injury through a PI3K/Akt-dependent mechanism. Cardiovasc. Res. 87, 694–703 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hesketh, P. J. et al. The oral neurokinin-1 antagonist aprepitant for the prevention of chemotherapy-induced nausea and vomiting: a multinational, randomized, double-blind, placebo-controlled trial in patients receiving high-dose cisplatin-the Aprepitant Protocol 052 Study Group. J. Clin. Oncol. 21, 4112–4119 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Rosso, M., Robles-Frias, M. J., Covenas, R., Salinas-Martin, M. V. & Munoz, M. The NK-1 receptor is expressed in human primary gastric and colon adenocarcinomas and is involved in the antitumor action of L-733,060 and the mitogenic action of substance P on human gastrointestinal cancer cell lines. Tumour Biol. 29, 245–254 (2008).

    Article  CAS  PubMed  Google Scholar 

  36. Munoz, M. & Rosso, M. The NK-1 receptor antagonist aprepitant as a broad spectrum antitumor drug. Invest. N. Drugs 28, 187–193 (2010).

    Article  CAS  Google Scholar 

  37. Nagakawa, O. et al. Effect of prostatic neuropeptides on invasion and migration of PC-3 prostate cancer cells. Cancer Lett. 133, 27–33 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Nizam, E. & Erin, N. Differential consequences of neurokinin receptor 1 and 2 antagonists in metastatic breast carcinoma cells; effects independent of substance P. Biomed. Pharmacother. 108, 263–270 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Le, T. T. et al. Sensory nerves enhance triple-negative breast cancer invasion and metastasis via the axon guidance molecule PlexinB3. NPJ Breast Cancer 8, 116 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Austin, M., Elliott, L., Nicolaou, N., Grabowska, A. & Hulse, R. P. Breast cancer induced nociceptor aberrant growth and collateral sensory axonal branching. Oncotarget 8, 76606–76621 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Jurcak, N. R. et al. Axon guidance molecules promote perineural invasion and metastasis of orthotopic pancreatic tumors in mice. Gastroenterology 157, 838–850 (2019).

    Article  CAS  PubMed  Google Scholar 

  42. Kamiya, A. et al. Genetic manipulation of autonomic nerve fiber innervation and activity and its effect on breast cancer progression. Nat. Neurosci. 22, 1289–1305 (2019).

    Article  CAS  PubMed  Google Scholar 

  43. Zahalka, A. H. et al. Adrenergic nerves activate an angio-metabolic switch in prostate cancer. Science 358, 321–326 (2017).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  44. Partecke, L. I. et al. Subdiaphragmatic vagotomy promotes tumor growth and reduces survival via TNFα in a murine pancreatic cancer model. Oncotarget 8, 22501–22512 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Renz, B. W. et al. Cholinergic signaling via muscarinic receptors directly and indirectly suppresses pancreatic tumorigenesis and cancer stemness. Cancer Discov. 8, 1458–1473 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Boilly, B., Faulkner, S., Jobling, P. & Hondermarck, H. Nerve dependence: from regeneration to cancer. Cancer Cell 31, 342–354 (2017).

    Article  CAS  PubMed  Google Scholar 

  47. Amit, M. et al. Loss of p53 drives neuron reprogramming in head and neck cancer. Nature 578, 449–454 (2020).

  48. Kalinichenko, V. V., Mokyr, M. B., Graf, L. H. Jr, Cohen, R. L. & Chambers, D. A. Norepinephrine-mediated inhibition of antitumor cytotoxic T lymphocyte generation involves a β-adrenergic receptor mechanism and decreased TNF-α gene expression. J. Immunol. 163, 2492–2499 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Mohammadpour, H. et al. β2 adrenergic receptor-mediated signaling regulates the immunosuppressive potential of myeloid-derived suppressor cells. J. Clin. Invest. 129, 5537–5552 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Benci, J. L. et al. Opposing functions of interferon coordinate adaptive and innate immune responses to cancer immune checkpoint blockade. Cell 178, 933–948 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Cao, Y. Q. et al. Primary afferent tachykinins are required to experience moderate to intense pain. Nature 392, 390–394 (1998).

    Article  ADS  CAS  PubMed  Google Scholar 

  52. Wang, Y. et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483–486 (2010).

    Article  ADS  CAS  PubMed  Google Scholar 

  53. Guy, C. T., Cardiff, R. D. & Muller, W. J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol. Cell. Biol. 12, 954–961 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Maroulakou, I. G., Anver, M., Garrett, L. & Green, J. E. Prostate and mammary adenocarcinoma in transgenic mice carrying a rat C3(1) simian virus 40 large tumor antigen fusion gene. Proc. Natl Acad. Sci. USA 91, 11236–11240 (1994).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hale, J. J. et al. Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent, orally active, long-acting morpholine acetal human NK-1 receptor antagonist. J. Med. Chem. 41, 4607–4614 (1998).

    Article  CAS  PubMed  Google Scholar 

  56. Padmanaban, V. et al. Organotypic culture assays for murine and human primary and metastatic-site tumors. Nat. Protoc. 15, 2413–2442 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Young, L., Sung, J., Stacey, G. & Masters, J. R. Detection of Mycoplasma in cell cultures. Nat. Protoc. 5, 929–934 (2010).

    Article  CAS  PubMed  Google Scholar 

  58. Foty, R. A simple hanging drop cell culture protocol for generation of 3D spheroids. J. Vis. Exp. https://doi.org/10.3791/2720 (2011).

  59. Heil, F. et al. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 303, 1526–1529 (2004).

    Article  ADS  CAS  PubMed  Google Scholar 

  60. Alexopoulou, L., Holt, A. C., Medzhitov, R. & Flavell, R. A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413, 732–738 (2001).

    Article  ADS  CAS  PubMed  Google Scholar 

  61. Hemmi, H. et al. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat. Immunol. 3, 196–200 (2002).

    Article  CAS  PubMed  Google Scholar 

  62. Jurk, M. et al. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat. Immunol. 3, 499 (2002).

    Article  CAS  PubMed  Google Scholar 

  63. Maira, S. M. et al. Identification and characterization of NVP-BKM120, an orally available pan-class I PI3-kinase inhibitor. Mol. Cancer Ther. 11, 317–328 (2012).

    Article  CAS  PubMed  Google Scholar 

  64. Davies, B. R. et al. Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background. Mol. Cancer Ther. 11, 873–887 (2012).

    Article  CAS  PubMed  Google Scholar 

  65. Folkes, A. J. et al. The identification of 2-(1H-indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as a potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer. J. Med. Chem. 51, 5522–5532 (2008).

    Article  CAS  PubMed  Google Scholar 

  66. Padmanaban, V. et al. E-cadherin is required for metastasis in multiple models of breast cancer. Nature 573, 439–444 (2019).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  67. Krause, S., Brock, A. & Ingber, D. E. Intraductal injection for localized drug delivery to the mouse mammary gland. J. Vis. Exp. https://doi.org/10.3791/50692 (2013).

  68. Jancso, G., Kiraly, E., Such, G., Joo, F. & Nagy, A. Neurotoxic effect of capsaicin in mammals. Acta Physiol. Hung. 69, 295–313 (1987).

    CAS  PubMed  Google Scholar 

  69. Chi, J. et al. Three-dimensional adipose tissue imaging reveals regional variation in beige fat biogenesis and PRDM16-dependent sympathetic neurite density. Cell Metab. 27, 226–236 (2018).

    Article  CAS  PubMed  Google Scholar 

  70. Luppi, P. H., Fort, P. & Jouvet, M. Iontophoretic application of unconjugated cholera toxin B subunit (CTb) combined with immunohistochemistry of neurochemical substances: a method for transmitter identification of retrogradely labeled neurons. Brain Res. 534, 209–224 (1990).

    Article  CAS  PubMed  Google Scholar 

  71. Gee, K. R. et al. Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes. Cell Calcium 27, 97–106 (2000).

    Article  CAS  PubMed  Google Scholar 

  72. Curtis, C. et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 486, 346–352 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the members of our laboratory for discussions and feedback on the manuscript text; P. Rajasethupathy for advice on calcium imaging analysis; M. Klatt for technical help with several animal experiments; the members of the various resource centres at Rockefeller University, including A. North, C. Pyrgaki, Banerjee P., and other staff of the Bio Imaging Resource Center, including C. Zhao, the staff of the Genomics Resource Center, S. Mazel and the staff of the Flow Cytometry Resource Center. The results published here are in part based on data generated by the TCGA Research Network. This work was supported by U54CA261701, R35CA274446, the Black Family Metastasis Center, the Breast Cancer Research Foundation and the Reem Kayden award. V.P. was supported by the Hope Funds for Cancer Research postdoctoral fellowship. I.K. is member of the German Academic Scholarship Foundation (Studienstiftung des deutschen Volkes) and was awarded a fellowship from Boehringer Ingelheim Fonds (BIF). B.N.O. was supported by a Max Eder grant of the German Cancer Aid (70114327) and is a fellow of the digital clinician scientist program at BIH-Charité.

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Authors and Affiliations

  1. Laboratory of Systems Cancer Biology, The Rockefeller University, New York, NY, USA

    Veena Padmanaban, Isabel Keller, Ethan S. Seltzer, Benjamin N. Ostendorf & Sohail F. Tavazoie

  2. Department of Hematology, Oncology, and Tumor Immunology and Berlin Institute of Health, Charité-Universitätsmedizin Berlin, Berlin, Germany

    Benjamin N. Ostendorf

  3. Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany

    Benjamin N. Ostendorf

  4. Laboratory of Mucosal Immunology, The Rockefeller University, New York, NY, USA

    Zachary Kerner

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  1. Veena Padmanaban
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Contributions

V.P. and S.F.T. conceptualized the study, designed experiments, supervised research and wrote the manuscript with input from all of the authors. V.P. performed most of the experiments with technical assistance from I.K., E.S.S. and Z.K.; B.N.O. analysed mRNA-sequencing data. S.F.T. obtained funding and supervised scientists.

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Correspondence to Sohail F. Tavazoie.

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Extended data figures and tables

Extended Data Fig. 1 Breast tumours are frequently innervated by sensory nerves.

(a) We previously uncovered a requirement for endothelial-derived SLIT2 (ecSLIT2) in metastasis15. (b) Quantification of ecSLIT2 expression in poorly vs highly metastatic breast tumour models. ****p < 0.0001, Mann-Whitney test. Mean ± SD. (c-d) Quantification of total (c) and CGRP+ sensory (d) innervation in 4T1 tumours grown in ecSLIT2 knockout (endothelial-specific depletion of SLIT2) mice. **** p < 0.0001, *p = 0.0489, t-test. Mean ± SD. (e) Quantification of innervation in SLIT2-knockout mammary tumours in Slit2fl/fl; MMTV-PyMT; MMTV-Cre mice. nsp = 0.6414, Mann-Whitney test. Mean ± SD. (f) Kaplan-Meier plots showing distant metastasis-free survival (DMFS) of breast cancer patients sorted by the median expression level of βIII-tubulin (mRNA, protein) or PGP9.5 (mRNA) of their tumours. The x-axes were set to have >10 surviving patients in each arm. (g) Nerve bundle abundance in highly metastatic primary tumours relative to corresponding isogenic poorly metastatic tumours. Between 4 and 8 tumours were analysed per group. **p (67NR vs 4T1) = 0.001, **p (4T07 vs 4T1) = 0.0019, nsp (67NR vs 4T07) = 0.9095, ANOVA; **p (EO771 Par vs LM2) = 0.0065, *p (HCC1806 Par vs LM2) = 0.0159, Mann-Whitney test. Mean ± SD. (h) Expression of pan-neuronal βIII-tubulin in 67NR vs 4T1 primary tumours, stroma-depleted primary tumour organoids, or cell lines. **p = 0.0048, t-test. Mean ± SD. MW: βIII-tubulin (50 kDa). (i) Liver metastasis from a 4T1 tumour-bearing mouse immuno-stained for nerve fibres. r = 3/group. (j) Retrograde tracing of DRG neurons innervating abdominal mammary glands of wild-type mice. Mean ± SEM. (k-l) Sensory, CGRP+ innervation observed within the normal murine mammary gland (k), murine models of breast cancer (l), and primary human tumours (l). r = 3 tumours/ group.

Source Data

Extended Data Fig. 2 Sensory neurons promote cancer invasion and proliferation across multiple ex vivo murine and human models of breast cancer.

(a) Frequency of apoptosis (cleaved caspase 3, CC3 +) in DRG neurons cultured alone or in the presence of 67NR cancer cells. (b) Micrographs of mCherry+ 67NR spheroids co-cultured with βIII-tubulin+ DRG neurons. Neuron and spheroid boundaries are manually traced to illustrate little physical contact. n = 10 ROIs/ group, r = 4. (c) 2D invasion assay of 67NR cancer cells that were cultured alone or in the presence of primary DRG neurons. *p = 0.0432, t-test. Mean ± SD. (d) Quantification of the number of mitotically active cells within regions of 4T1 primary tumours adjacent to or far away from a nerve bundle. **p = 0.0043, Mann-Whitney test. Mean ± SD. (e-f) 3D colony formation assay. 67NR cancer cells were cultured alone or in the presence of DRG neurons in 3D Matrigel. (e) Schematic. (f) Quantification. **p = 0.0067, Kruskal-Wallis test. Mean ± SD. (g-i) 3D co-culture assays of MMTV-PyMT mammary tumour organoids and primary DRG neurons. (g) Schematic. (h) Invasion assay. ****p < 0.0001, Mann-Whitney test. (i) Colony formation assay. **p = 0.0033, t-test. Mean ± SD. (j-m) 3D co-culture assays of 4 independent primary human breast tumours and DRG neurons. (j) Schematic of organoid isolation. (k) Invasion quantification. ****p < 0.0001, *p = 0.0266, Mann-Whitney test. Mean ± SD. (l) Proliferation quantification. ****p < 0.0001, *p = 0.0401 (#2), 0.0267 (#4), t-test. Mean ± SD. (m) Basic de-identified clinical information for human tumour samples cultured.

Source Data

Extended Data Fig. 3 Co-transplantation of breast cancer cells with DRG neurons drives metastasis.

(a-h) Co-transplantation of mCherry+ (mCh) 67NR cancer cells and DRG neurons. (a) Schematic. (b) Optically cleared 67NR tumours transplanted with or without DRG neurons and immunostained for CGRP+ sensory nerves. r = 2 tumours/ group. (c) Mean fluorescence intensity (MFI) for SP in 67NR primary tumours transplanted with or without DRG neurons. **p = 0.0046, t-test. Mean ± SD. (d) Tumour growth. **p = 0.0017, t-test. Mean ± SEM. (e) Percentage of the tumour-stroma boundary with a pushing vs invasive morphology. ****p < 0.0001, Chi-square test. (f) Association of mCh+ 67NR cancer cells with the lung endothelium in mice transplanted with orthotopic 67NR tumours. r = 3 lungs/ group. (g) CTC enumeration in mice transplanted with mCh+ 67NR cancer cells with or without DRG neurons. *p = 0.0287, t-test. Mean ± SD. (h) mCh+ micro-metastases. **p = 0.0079, Mann-Whitney test. Mean ± SD. (i) Co-transplantation of 67NR cancer cells with increasing numbers of DRG neurons. nsp = 0.4124, **p = 0.0026, ****p < 0.0001, ANOVA. Mean ± SEM.

Source Data

Extended Data Fig. 4 Sensory neurons drive metastatic colonization in multiple breast cancer models.

(a-c) Tail vein injections of mCh+ 67NR cancer cells with or without DRG neurons. (a) Schematic. (b) CGRP + / βIII-tubulin+ neuronal cell bodies in mice co-injected with DRG neurons. r = 3 lungs/ group. (c) mCh+ metastases. ****p < 0.0001, Mann-Whitney test. Mean ± SD. (d-f) Co-transplantation of EO771 cancer cells and DRG neurons. (d) Schematic. (e) Tumour growth. *p = 0.0345, t-test. Mean ± SEM. (f) Number of micro-metastases counted by H&E. *p = 0.0249, t-test. Mean ± SD.

Source Data

Extended Data Fig. 5 Capsaicin does not alter growth or invasion of cancer cells in vitro and reduces tumour growth in immune-compromised mice in vivo.

(a) Sensory (CGRP +) and sympathetic (TH +) innervation in ipsilateral or contralateral mammary glands 7 days after capsaicin administration. (b) Quantification of CGRP+ nerve bundles post-capsaicin administration. *p = 0.0403, t-test. Mean ± SD. (c-d) 3D culture of 4T1 spheroids in the presence of capsaicin (1–100 μM). (c) Invasion quantification. nsp > 0.9999, Kruskal-Wallis test. (d) Spheroid surface area. nsp = 0.7108, Kruskal-Wallis test. Mean ± SD. (e-f) Sensory-specific denervation of 4T1 tumours grown in NSG mice. (e) Schematic. (f) Tumour growth. ****p < 0.0001, t-test. Mean ± SEM.

Source Data

Extended Data Fig. 6 Neuronal substance-P drives breast cancer metastasis via the activation of the tumoral TACR1 receptor.

(a) Invasion assay of 67NR cancer cells in response to SP. *p = 0.0254, t-test. Mean ± SD. (b-c) 67NR cancer cell spheroids cultured with neuropeptides galanin or CGRP, with or without RNase A. (b) Invasion quantification. **p = 0.0011, ****p < 0.0001, nsp (CGRP) = 0.1066, nsp (galanin) > 0.9999, Kruskal-Wallis test. Vehicle-treated 67NR spheroids (−/+RNase A) are also plotted in Fig. 2d and Fig. 3d respectively since the experiments were conducted together. (c) Proliferation quantification. ***p = 0.0006, Mann-Whitney test. Mean ± SD. (d) ELISA-based quantification for SP levels in conditioned medium. p values are based on comparisons with base medium (NGM). nsp = 0.7426 (vs DRG only), 0.8724 (vs 67NR only); *p = 0.0213; **p = 0.0024, ANOVA. Mean ± SD. (e-g) In vivo measurements of SP expression. (e) Mean fluorescence intensity (MFI) of SP in 4T1 vs 67NR primary tumours. ****p < 0.0001, t-test. (f) Plasma SP levels in 67NR vs 4T1 tumour-bearing mice. *p = 0.0359, t-test. (g) MFI of SP in 4T1 vehicle vs capsaicin-treated primary tumours. ****p < 0.0001, t-test. Mean ± SD. (h-k) Effects of an SP-blocking antibody on 4T1 tumour growth and metastasis. (h) Schematic. (i) MFI of SP in IgG vs anti-SP treated 4T1 tumours. ****p < 0.0001, Mann-Whitney test. Mean ± SD. (j) Tumour growth. ****p < 0.0001, Mann-Whitney test. Mean ± SEM. (k) Metastatic area quantified by H&E. **p = 0.0012, t-test. Mean ± SD. (l-o) Orthotopic transplantation of EO771 LM2 cells into the abdominal mammary glands of Tac1-WT and Tac1-null host mice. (l) Schematic. (m) MFI of SP in primary tumours. ***p = 0.0003, t-test. Mean ± SD. (n) Tumour growth. ***p = 0.0003, t-test. Mean ± SEM. (o) Number of macro-metastases quantified by H&E. ***p = 0.0006, t-test. Mean ± SD. (p-r) Depletion of SP’s receptor, TACR1 in 4T1 cancer cell spheroids. (p) Validation of knockdown. ****p < 0.0001, ANOVA. MW: TACR1 (46 kDa). Quantification of spheroid invasion (q) and proliferation (r). ****p < 0.0001, Mann-Whitney test. Mean ± SD. (s-u) Orthotopic transplantation of 4T1 cancer cells depleted for TACR1. (s) Schematic. (t) Tumour growth. ****p < 0.0001, ANOVA. Mean ± SEM. (u) Metastatic area quantified by H&E. ***p = 0.0003, ANOVA. Mean ± SD.

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Extended Data Fig. 7 Neuronal SP drives metastasis in a ssRNA-dependent manner.

(a-b) Schematic for isolation of conditioned medium from tumour only, DRG only, or tumour-DRG cultures (a). Invasion quantification, nsp = 0.1463, ****p < 0.0001, Kruskal-Wallis test (b). Mean ± SD. (c) Calcium fluorescence traces (ΔF/F0) of DRG neurons cultured alone or in the presence of 67NR cancer cells. F = measured fluorescence, F0 = baseline fluorescence. (d) Invasion quantification of 67NR spheroids co-cultured with DRG neurons in the presence of a sodium channel blocker, tetrodotoxin (TTX). nsp > 0.9999, ****p < 0.0001, Kruskal-Wallis test. Mean ± SD. (e) Invasion quantification of 67NR spheroids cultured with DNase-treated or heat inactivated tumour-CM or DRG-CM. ***p = 0.001, ****p < 0.0001, Kruskal-Wallis test. Mean ± SD. (f) Invasion quantification of 67NR spheroids cultured with solely RNase A, RNase T1 or RNase III. nsp > 0.9999, Kruskal-Wallis test. Mean ± SD. (g) Proliferation quantification of 67NR spheroids cultured with or without ssRNA40. ****p < 0.0001, Mann-Whitney test. Mean ± SD. (h) Invasion quantification of 67NR spheroids cultured with a dsRNA mimetic, Poly (I:C). **p = 0.0072, Kruskal-Wallis test. Mean ± SD. (i-k) Effect of RNase A treatment on 4T1 tumour growth and metastasis. (i) Schematic. (j) Tumour growth. ****p < 0.0001, ANOVA. Mean ± SEM. (k) Metastatic area quantified by H&E. *p = 0.0392 (vehicle vs RNase A #1), *p = 0.0347 (vehicle vs RNase A #2), ANOVA. Mean ± SD. (l) MFI of dsRNA (measured using an anti-dsRNA antibody, J2) in vehicle vs RNase A treated 4T1 primary tumours. nsp = 0.22 (RNase A #1), 0.0625 (RNase A #2), ANOVA. Mean ± SD. (m-o) Intra-cardiac injections of mCherry+ 67NR cancer cells pre-treated with tumour-CM or DRG-CM. (m) Schematic. (n) Representative mCherry+ metastases. (o) Quantification of mCherry+ metastases. **p = 0.0028, *p = 0.0366 (liver), *p = 0.0313 (brain), t-test. Mean ± SD.

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Extended Data Fig. 8 SP-driven activation of TACR1 and ssRNA-driven activation of TLR7 promote breast cancer invasiveness across multiple models of breast cancer.

(a) Invasion quantification of Py8119, 4T1, and MDA-MB-231 breast cancer spheroids in the presence of SP. **p = 0.0013, ****p < 0.0001, Kruskal-Wallis test. Mean ± SD. (b) Invasion quantification of Py8119, 4T1, and MDA-MB-231 breast cancer spheroids in the presence of ssRNA40. *p = 0.0235, ****p < 0.0001, Kruskal-Wallis test. Mean ± SD.

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Extended Data Fig. 9 Neuronal SP signals via tumoral TLR7 receptors to drive metastasis.

(a) Percent apoptotic area within 67NR spheroids cultured with galanin. nsp = 0.0807, Mann-Whitney test. Mean ± SD. (b) 67NR spheroids cultured with or without DRG neurons and immunostained for TACR1. n = 10 spheroids/ group. (c) Flow cytometry analysis of TACR1 expression in 67NR cancer cells. **p = 0.0089, t-test. Mean ± SD. (d-f) 67NR spheroids depleted for TACR1 and cultured with SP. (e) Percent apoptotic area. ***p = 0.0003, Mann-Whitney test, ****p < 0.0001, Kruskal-Wallis test. (f) Invasion quantification. ****p < 0.0001, Kruskal-Wallis test. Mean ± SD. (g) Validation of Tlr7 knockdown in 67NR cancer cells. ****p < 0.0001, ANOVA. Mean ± SD. MW: TLR7 (135 kDa). Validation of Tlr3 knockdown in 67NR cancer cells. ****p < 0.0001, ANOVA. Mean ± SD. Invasion quantification of 67NR spheroids depleted for TLR3 and cultured with or without DRG neurons. ****p < 0.0001, Kruskal-Wallis test. Mean ± SD. Invasion quantification of 67NR spheroids cultured in the presence of TLR7 agonists, R837 or R848. ****p < 0.0001, **p = 0.0011, Kruskal-Wallis test. Mean ± SD. Validation of Tlr7 knockdown in 4T1 cancer cells. ***p = 0.0006, ANOVA. Mean ± SD. MW: TLR7 (135 kDa). Invasion quantification of 4T1 spheroids depleted of TLR7. ****p < 0.0001, Kruskal-Wallis test. Mean ± SD. Immunostaining for CD45 (all immune), CD3 (T cells), or NK1.1 (NK cells) on 4T1 primary tumours depleted of TLR7. nsp > 0.9999 (all conditions), Kruskal-Wallis test. Mean ± SD. (n-p) Orthotopic transplantations of 4T1 cancer cells depleted for TLR7 in NSG mice. (n) Schematic. (o) Tumour growth. ****p < 0.0001, ANOVA. Mean ± SEM. (p) Metastatic area quantified by H&E. ****p < 0.0001, ANOVA. Mean ± SD. (q-s) Orthotopic transplantation of 4T1 cancer cells depleted for TLR7, followed by periodic injections of RNase A. (q,r) Schematics. (s) Tumour growth. **p = 0.0033, nsp = 0.9923, ANOVA. Mean ± SD.

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Extended Data Fig. 10 Sensory neurons activate a non-canonical TLR7 signalling axis in cancer cells which correlates with poor patient outcome.

(a) mRNA sequencing of control vs Tlr7 depleted 4T1 spheroids. Top five downregulated pathways in 4T1 spheroids depleted for Tlr7 as assessed by gene set enrichment analysis (p values according to permutation testing). (b) Validation of MyD88 knockdown in 67NR cancer cells. MW: MyD88 (33 kDa). r = 3. (c) Invasion quantification of 67NR spheroids depleted for MyD88 and cultured with or without DRG neurons. ****p < 0.0001, Mann-Whitney test. nsp > 0.9999 (si#1), nsp = 0.0783 (si#2), nsp = 0.4140 (si#3), Kruskal-Wallis test. Mean ± SD. (d) Invasion quantification of 67NR spheroids cultured with or without SP and a PI3K inhibitor (left to right: buparlisib, capivasertib, or pictilisib). nsp (Veh vs buparlisib) = 0.1273, nsp (Veh vs capivasertib) = 0.6967, nsp (Veh vs pictilisib) > 0.9999, ****p < 0.0001, Kruskal-Wallis test. Mean ± SD. (e-f) Multivariate analysis of the association of age, tumour stage and a Tlr7-dependent gene signature with survival in breast cancer patients from the METABRIC (e) and TCGA (f) datasets (p values according to multivariate Cox proportional hazard models, error bars indicate 95% confidence intervals).

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Extended Data Fig. 11 Aprepitant impairs tumour growth and metastasis of Py8119 and MMTV-PyMT models of breast cancer.

(a) Invasion quantification of Py8119, 4T1, and MDA-MB-231 breast cancer spheroids cultured in the presence of aprepitant. ****p < 0.0001, nsp = 0.9621, **p = 0.0013, Kruskal-Wallis test. Mean ± SD. (b-d) Aprepitant was evaluated for its potential in inhibiting breast cancer progression and metastasis. (b) Schematic. (c) Py8119 tumour growth and metastasis count. **p = 0.0012, *p = 0.0117, Mann-Whitney test. (d) MMTV-PyMT tumour growth. **p = 0.0047, Mann-Whitney test. Mean ± SEM.

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Padmanaban, V., Keller, I., Seltzer, E.S. et al. Neuronal substance P drives metastasis through an extracellular RNA–TLR7 axis. Nature 633, 207–215 (2024). https://doi.org/10.1038/s41586-024-07767-5

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