Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Hedgehog blockade remodels the gut microbiota and the intestinal effector CD8+ T cells in a mouse model of mammary carcinoma

Abstract

Given the gut microbiome’s rise as a potential frontier in cancer pathogenesis and therapy, leveraging microbial analyses in the study of breast tumor progression and treatment could unveil novel interactions between commensal bacteria and disease outcomes. In breast cancer, the Hedgehog (Hh) signaling pathway is a potential target for treatment due to its aberrant activation leading to poorer prognoses and drug resistance. There are limited studies that have investigated the influences of orally administered cancer therapeutics, such as Vismodegib (a pharmacological, clinically used Hh inhibitor) on the gut microbiota. Using a 4T1 mammary carcinoma mouse model and 16 S rRNA sequencing, we longitudinally mapped alterations in immunomodulating gut microbes during mammary tumor development. Next, we identified changes in the abundance of commensal microbiota in response to Vismodegib treatment of 4T1 mammary tumor-bearing mice. In addition to remodeling gut microbiota, Vismodegib treatment elicited an increase in proliferative CD8+ T cells in the colonic immune network, without any remarkable gastrointestinal-associated side effects. To our knowledge, this is the first study to assess longitudinal changes in the gut microbiome during mammary tumor development and progression. Our study also pioneers an investigation of the dynamic effects of an orally delivered Hh inhibitor on the gut microbiome and the gut-associated immune-regulatory adaptive effector CD8+ T cells. These findings inform future comprehensive studies on the consortium of altered microbes that can impact potential systemic immunomodulatory roles of Vismodegib.

Your institute does not have access to this article

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Progression of 4T1 mammary tumor development and progression induces changes within the fecal microbiome.
Fig. 2: Hh inhibition impacts the gut bacterial abundance in the 4T1 immunocompetent model of mouse mammary carcinoma.
Fig. 3: Hh inhibition alters the α and β diversity of the fecal and cecal microbiota.
Fig. 4: Hh blockade progressively modifies the gut microbiota in mammary tumor-bearing mice.
Fig. 5: Smo-i treatment augments the proliferation of CD8+ T cells in the colon and the mesenteric lymph nodes.
Fig. 6: The oral administration of Smo-i does not induce colitis in 4T1 tumor-bearing mice.

Data availability

The datasets used and/or analyzed in this study are available from the corresponding author upon reasonable request.

References

  1. Maynard C, Elson C, Hatton R, & Weaver C. Reciprocal interactions of the intestinal microbiota and immune system. Nature 489, 231-241 (2012).

  2. Lozupone C, Stombaugh J, Gordon J, Jansson J & Knight R. Diversity, stability and resilience of the human gut microbiota. Nature 489, 220-230 (2012).

  3. Schwabe R & Jobin C. The microbiome and cancer. Nature Reviews Cancer 13, 800-812 (2013).

  4. Hanahan D. Hallmarks of Cancer: New Dimensions. Cancer Discovery 12, 31-46 (2022).

  5. Cullin N, Azevedo Antunes C, Straussman R, Stein-Thoeringer C, & Elinav E. Microbiome and cancer. Cancer Cell 39, 1317-1341 (2021).

  6. Luu M, Riester Z, Baldrich A, Reichardt N, Yuille S, Busetti A et al. Microbial short-chain fatty acids modulate CD8+ T cell responses and improve adoptive immunotherapy for cancer. Nature Communications 12, 4077 (2021).

  7. Francescone R, Hou V & Grivennikov SI. Microbiome, Inflammation, and Cancer. The Cancer Journal 20 (2014).

  8. Grivennikov SI, Greten FR & Karin M. Immunity, Inflammation, and Cancer. Cell 140, 883-899 (2010).

  9. Banerjee S, Tian T, Wei Z, Shih N, Feldman MD, Peck KN et al. Distinct Microbial Signatures Associated With Different Breast Cancer Types. Frontiers in Microbiology 9, 951 (2018).

  10. Luu TH, Michel C, Bard J-M, Dravet F, Nazih H & Bobin-Dubigeon C. Intestinal Proportion of Blautia sp. is Associated with Clinical Stage and Histoprognostic Grade in Patients with Early-Stage Breast Cancer. Nutrition and Cancer 69, 267-275 (2017).

  11. Gopalakrishnan V, Spencer CN, Nezi L, Reuben A, Andrews MC, Karpinets TV et al. Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients. Science 359, 97-103 (2018).

  12. Armstrong H, Bording-Jorgensen M, Dijk S & Wine E. The Complex Interplay between Chronic Inflammation, the Microbiome, and Cancer: Understanding Disease Progression and What We Can Do to Prevent It. Cancers 10 (2018).

  13. Parida S, Wu S, Siddharth S, Wang G, Muniraj N, Nagalingam A et al. A Procarcinogenic Colon Microbe Promotes Breast Tumorigenesis and Metastatic Progression and Concomitantly Activates Notch and β-Catenin Axes. Cancer Discovery 11, 1138 (2021).

  14. Bhateja P, Cherian MM, Majumder S & Ramaswamy B. The Hedgehog Signaling Pathway: A Viable Target in Breast Cancer? Cancers 11 (2019).

  15. Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS et al. Metagenomic biomarker discovery and explanation. Genome Biology 12, R60 (2011).

  16. Amakye D, Jagani Z & Dorsch M. Unraveling the therapeutic potential of the Hedgehog pathway in cancer. Nature Medicine 19, 1410-1422 (2013).

  17. Hui M, Cazet A, Nair R, Watkins DN, O’Toole SA & Swarbrick A. The Hedgehog signalling pathway in breast development, carcinogenesis and cancer therapy. Breast Cancer Research 15, 203 (2013).

  18. Eggenschwiler JT & Anderson KV. Cilia and Developmental Signaling. Annual Review of Cell and Developmental Biology 23, 345-373 (2007).

  19. Rubin LL & de Sauvage FJ. Targeting the Hedgehog pathway in cancer. Nature Reviews Drug Discovery 5, 1026-1033 (2006).

  20. Hanna A, Metge BJ, Bailey SK, Chen D, Chandrashekar DS, Varambally S et al. Inhibition of Hedgehog signaling reprograms the dysfunctional immune microenvironment in breast cancer. Oncoimmunology 8, 1548241 (2019).

  21. National Library of Medicine (U.S.). Addition of Vismodegib to Neoadjuvant Chemotherapy in Triple Negative Breast Cancer Patients (SHH-CM). Identifier NCT02694224 (2016).

  22. Merchant JL & Ding L. Hedgehog Signaling Links Chronic Inflammation to Gastric Cancer Precursor Lesions. Cellular and Molecular Gastroenterology and Hepatology 3, 201-210 (2017).

  23. Woese CR & Gutell RR. Evidence for several higher order structural elements in ribosomal RNA. Proc Natl Acad Sci USA 86, 3119-3122 (1989).

  24. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proceedings of the National Academy of Sciences 108, 4516-4522 (2011).

  25. Kumar R, Eipers P, Little RB, Crowley M, Crossman DK, Lefkowitz EJ et al. Getting started with microbiome analysis: sample acquisition to bioinformatics. Curr Protoc Hum Genet 82, 18.18.11-29 (2014).

  26. Kozich JJ, Westcott SL, Baxter NT, Highlander SK & Schloss PD. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl Environ Microbiol 79, 5112-5120 (2013).

  27. Beller A, Kruglov A, Durek P, von Goetze V, Werner K, Heinz GA et al. Specific microbiota enhances intestinal IgA levels by inducing TGF-β in T follicular helper cells of Peyer’s patches in mice. European Journal of Immunology 50, 783-794 (2020).

  28. Fong W, Li Q & Yu J. Gut microbiota modulation: a novel strategy for prevention and treatment of colorectal cancer. Oncogene 39, 4925-4943 (2020).

  29. Miyake S, Kim S, Suda W, Oshima K, Nakamura M, Matsuoka T et al. Dysbiosis in the Gut Microbiota of Patients with Multiple Sclerosis, with a Striking Depletion of Species Belonging to Clostridia XIVa and IV Clusters. PLOS ONE 10, e0137429 (2015).

  30. Zito PM, Nassereddin A, & Scharf R. Vismodegib. In: StatPearls [Internet]. Treasure Island (FL): Vismodegib. StatPearls Publishing (2022).

  31. Han L, Jin H, Zhou L, Zhang X, Fan Z, Dai M et al. Intestinal Microbiota at Engraftment Influence Acute Graft-Versus-Host Disease via the Treg/Th17 Balance in Allo-HSCT Recipients. Front Immunol 9 (2018).

  32. He B, Hoang TK, Wang T, Ferris M, Taylor CM, Tian X et al. Resetting microbiota by Lactobacillus reuteri inhibits T reg deficiency-induced autoimmunity via adenosine A2A receptors. J Exp Med 214, 107-123 (2017).

  33. Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT et al. Up-Regulation of PD-L1, IDO, and Tregs in the Melanoma Tumor Microenvironment Is Driven by CD8+ T Cells. Science Translational Medicine 5, 200ra116-200ra116 (2013).

  34. Sivan A, Corrales L, Hubert N, Williams JB, Aquino-Michaels K, Earley ZM et al. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti–PD-L1 efficacy. Science 350, 1084-1089 (2015).

  35. Escalante J, McQuade RM, Stojanovska V & Nurgali K. Impact of chemotherapy on gastrointestinal functions and the enteric nervous system. Maturitas 105, 23-29 (2017).

  36. Song JL, Choi JH, Seo JH, Lim YI & Park KY. Anti-Colitic Effects of Kanjangs (Fermented Soy Sauce and Sesame Sauce) in Dextran Sulfate Sodium-Induced Colitis in Mice. Journal of Medicinal Food 17, 1027-1035 (2014).

  37. Magne F, Gotteland M, Gauthier L, Zazueta A, Pesoa S, Navarrete P et al. The Firmicutes/Bacteroidetes Ratio: A Relevant Marker of Gut Dysbiosis in Obese Patients? Nutrients 12, 1474 (2020).

  38. Aguirre M & Venema K. Does the Gut Microbiota Contribute to Obesity? Going beyond the Gut Feeling. Microorganisms 3, 213-235 (2015).

  39. Mell B, Jala VR, Mathew AV, Byun J, Waghulde H, Zhang Y et al. Evidence for a link between gut microbiota and hypertension in the Dahl rat. Physiol Genomics 47, 187-197 (2015).

  40. Sears CL. The toxins of Bacteroides fragilis. Toxicon 39, 1737-1746 (2001).

  41. Sears CL & Pardoll DM. Perspective: Alpha-Bugs, Their Microbial Partners, and the Link to Colon Cancer. The Journal of Infectious Diseases 203, 306-311 (2011).

  42. Lu Y, Chen J, Zheng J, Hu G, Wang J, Huang C et al. Mucosal adherent bacterial dysbiosis in patients with colorectal adenomas. Sci Rep 6, 26337-26337 (2016).

  43. Wells JM. Immunomodulatory mechanisms of lactobacilli. Microbial Cell Factories 10, S17 (2011).

  44. Liu Y, Fatheree NY, Dingle BM, Tran DQ & Rhoads JM. Lactobacillus reuteri DSM 17938 Changes the Frequency of Foxp3+ Regulatory T Cells in the Intestine and Mesenteric Lymph Node in Experimental Necrotizing Enterocolitis. PLOS ONE 8, e56547 (2013).

  45. Oh NS, Lee JY, Kim Y-T, Kim SH & Lee J-H. Cancer-protective effect of a synbiotic combination between Lactobacillus gasseri 505 and a Cudrania tricuspidata leaf extract on colitis-associated colorectal cancer. Gut Microbes 12, 1785803 (2020).

  46. Pan F, Zhang L, Li M, Hu Y, Zeng B, Yuan H et al. Predominant gut Lactobacillus murinus strain mediates anti-inflammaging effects in calorie-restricted mice. Microbiome 6, 54-54 (2018).

  47. Saresella M, Mendozzi L, Rossi V, Mazzali F, Piancone F, LaRosa F et al. Immunological and Clinical Effect of Diet Modulation of the Gut Microbiome in Multiple Sclerosis Patients: A Pilot Study. Front Immunol 8, 1391-1391 (2017).

  48. Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y et al. Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species. Science 331, 337-341 (2011).

  49. Jones-Hall YL, Kozik A & Nakatsu C. Ablation of Tumor Necrosis Factor Is Associated with Decreased Inflammation and Alterations of the Microbiota in a Mouse Model of Inflammatory Bowel Disease. PLOS ONE 10, e0119441 (2015).

  50. Konjar Š, Ferreira C, Blankenhaus B & Veldhoen M. Intestinal Barrier Interactions with Specialized CD8 T Cells. Front Immunol 8 (2017).

  51. Dzutsev A, Badger JH, Perez-Chanona E, Roy S, Salcedo R, Smith CK et al. Microbes and Cancer. Annu Rev Immunol 35, 199-228 (2017).

  52. Luu M, Pautz S, Kohl V, Singh R, Romero R, Lucas S et al. The short-chain fatty acid pentanoate suppresses autoimmunity by modulating the metabolic-epigenetic crosstalk in lymphocytes. Nature Communications 10, 760 (2019).

  53. Tanoue T, Morita S, Plichta DR, Skelly AN, Suda W, Sugiura Y et al. A defined commensal consortium elicits CD8 T cells and anti-cancer immunity. Nature 565, 600-605 (2019).

  54. Heshiki Y, Vazquez-Uribe R, Li J, Ni Y, Quainoo S, Imamovic L et al. Predictable modulation of cancer treatment outcomes by the gut microbiota. Microbiome 8, 28 (2020).

  55. de Oliveira GLV. Chapter 33 - The Gut Microbiome in Autoimmune Diseases. In: Joel Faintuch & Salomao Faintuch (eds). Microbiome and Metabolome in Diagnosis, Therapy, and other Strategic Applications. Academic Press 332 (2019).

Download references

Acknowledgements

The authors acknowledge funding from the following sources: Department of Defense (W81XWH-14-1-0516, W81XWH-18-1-0036, W81XWH-10-1-0755) and O’Neal Invests awarded to L.A.S. The work is supported in part by BXAl3374 (VA) and CA194048 (NCI/NIH) to R.S.S. The authors acknowledge funding provided by T32AI007051-42 (awarded to D.C.H.) and 2T32GM8361-29 (awarded to C.A.S.). The authors thank Sarah Kammerud and Brandon Metge for editorial suggestions. The authors would like to thank the UAB Comprehensive Flow Cytometry Core supported by NIH Grants P30 AR048311 and P30 AI027667 and the UAB Comprehensive Cancer Center’s Preclinical Imaging Shared Facility supported by NIH Grants P30 CA013148 and 1S1 0OD021697.

Author information

Authors and Affiliations

Authors

Contributions

All authors have read and approved the manuscript. D.C.H., A.H., C.A.S., C.L.M., P.A.M., and L.A.S. contributed to experimental design. D.C.H., A.H., C.A.S., C.L.M., P.A.M. were responsible for acquisition of data. D.C.H., C.A.S., D.C., C.L.M., P.A.M., G.L., B.C.M., R.S.S., and L.A.S. performed analysis and interpretation of data. C.A.S., D.C.H., A.H., R.S.S, D.C., C.L.M., P.A.S., G.L., B.C.M., and L.A.S. were involved in writing and revision of the manuscript.

Corresponding author

Correspondence to Lalita A. Shevde.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hinshaw, D.C., Swain, C.A., Chen, D. et al. Hedgehog blockade remodels the gut microbiota and the intestinal effector CD8+ T cells in a mouse model of mammary carcinoma. Lab Invest (2022). https://doi.org/10.1038/s41374-022-00828-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41374-022-00828-1

Search

Quick links