Skip to main content

Advertisement

SpringerLink
Log in

Inhibition of the CCR6-CCL20 axis prevents regulatory T cell recruitment and sensitizes head and neck squamous cell carcinoma to radiation therapy

  • Research
  • Published:
Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

Background

 Radioresistance of HNSCCs remains a major challenge for effective tumor control. Combined radiotherapy (RT) and immunotherapy (IT) treatment improved survival for a subset of patients with inflamed tumors or tumors susceptible to RT-induced inflammation. To overcome radioresistance and improve treatment outcomes, an understanding of factors that suppress anti-tumor immunity is necessary. In this regard, regulatory T cells (Tregs) are critical mediators of immune suppression in HNSCCs. In this study, we investigated how radiation modulates Treg infiltration in tumors through the chemokine CCL20. We hypothesized that radiation induces CCL20 secretion resulting in Treg infiltration and suppression of anti-tumor immunity.

Methods

 Human and mouse HNSCC cell lines with different immune phenotypes were irradiated at doses of 2 or 10 Gy. Conditioned media, RNA and protein were collected for assessment of CCL20. qPCR was used to determine CCL20 gene expression. In vivo, MOC2 cells were implanted into the buccal cavity of mice and the effect of neutralizing CCL20 antibody was determined alone and in combination with RT. Blood samples were collected before and after RT for analysis of CCL20. Tumor samples were analyzed by flow cytometry to determine immune infiltrates, including CD8 T cells and Tregs. Mass-spectrometry was performed to analyze proteomic changes in the tumor microenvironment after anti-CCL20 treatment.

Results

 Cal27 and MOC2 HNSCCs had a gene signature associated with Treg infiltration, whereas SCC9 and MOC1 tumors displayed a gene signature associated with an inflamed TME. In vitro, tumor irradiation at 10 Gy significantly induced CCL20 in Cal27 and MOC2 cells relative to control. The increase in CCL20 was associated with increased Treg migration. Neutralization of CCL20 reversed radiation-induced migration of Treg cells in vitro and decreased intratumoral Tregs in vivo. Furthermore, inhibition of CCL20 resulted in a significant decrease in tumor growth compared to control in MOC2 tumors. This effect was further enhanced after combination with RT compared to either treatment alone.

Conclusion

 Our results suggest that radiation promotes CCL20 secretion by tumor cells which is responsible for the attraction of Tregs. Inhibition of the CCR6-CCL20 axis prevents infiltration of Tregs in tumors and suppresses tumor growth resulting in improved response to radiation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. McDonald MW et al (2011) ACR appropriateness criteria retreatment of recurrent head and neck cancer after prior definitive radiation expert panel on radiation oncology-head and neck cancer. Int J Radiat Oncol Biol Phys 80:1292–1298. https://doi.org/10.1016/j.ijrobp.2011.02.014

    Article  PubMed  Google Scholar 

  2. Bonner JA et al (2006) Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck. N Engl J Med 354:567–578. https://doi.org/10.1056/NEJMoa053422

    Article  CAS  PubMed  Google Scholar 

  3. Ang KK et al (2014) Randomized phase III trial of concurrent accelerated radiation plus cisplatin with or without cetuximab for stage III to IV head and neck carcinoma: RTOG 0522. J Clin Oncol 32:2940–2950. https://doi.org/10.1200/JCO.2013.53.5633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Gillison ML et al (2019) Radiotherapy plus cetuximab or cisplatin in human papillomavirus-positive oropharyngeal cancer (NRG Oncology RTOG 1016): a randomised, multicentre, non-inferiority trial. Lancet 393:40–50. https://doi.org/10.1016/S0140-6736(18)32779-X

    Article  CAS  PubMed  Google Scholar 

  5. Mehra R et al (2018) Efficacy and safety of pembrolizumab in recurrent/metastatic head and neck squamous cell carcinoma: pooled analyses after long-term follow-up in KEYNOTE-012. Br J Cancer 119:153–159. https://doi.org/10.1038/s41416-018-0131-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Qin Y, Zheng X, Gao W, Wang B, Wu Y (2021) Tumor microenvironment and immune-related therapies of head and neck squamous cell carcinoma. Mol Ther Oncolytics 20:342–351. https://doi.org/10.1016/j.omto.2021.01.011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Partlova S et al (2015) Distinct patterns of intratumoral immune cell infiltrates in patients with HPV-associated compared to non-virally induced head and neck squamous cell carcinoma. Oncoimmunology 4:e965570. https://doi.org/10.4161/21624011.2014.965570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Wang J et al (2019) HPV-positive status associated with inflamed immune microenvironment and improved response to anti-PD-1 therapy in head and neck squamous cell carcinoma. Sci Rep 9:13404. https://doi.org/10.1038/s41598-019-49771-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Oguejiofor K et al (2017) Distinct patterns of infiltrating CD8+ T cells in HPV+ and CD68 macrophages in HPV- oropharyngeal squamous cell carcinomas are associated with better clinical outcome but PD-L1 expression is not prognostic. Oncotarget. https://doi.org/10.18632/oncotarget.14796.

  10. Mito I et al (2021) Comprehensive analysis of immune cell enrichment in the tumor microenvironment of head and neck squamous cell carcinoma. Sci Rep 11:16134. https://doi.org/10.1038/s41598-021-95718-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mandal R et al (2016) The head and neck cancer immune landscape and its immunotherapeutic implications. JCI Insight 1:e89829. https://doi.org/10.1172/jci.insight.89829

    Article  PubMed  PubMed Central  Google Scholar 

  12. Lassen P, Overgaard J, Eriksen JG (2013) Expression of EGFR and HPV-associated p16 in oropharyngeal carcinoma: correlation and influence on prognosis after radiotherapy in the randomized DAHANCA 5 and 7 trials. Radiother Oncol 108:489–494. https://doi.org/10.1016/j.radonc.2013.08.036

    Article  CAS  PubMed  Google Scholar 

  13. Lassen P et al (2014) Impact of HPV-associated p16-expression on radiotherapy outcome in advanced oropharynx and non-oropharynx cancer. Radiother Oncol 113:310–316. https://doi.org/10.1016/j.radonc.2014.11.032

    Article  PubMed  Google Scholar 

  14. Lindel K, Beer KT, Laissue J, Greiner RH, Aebersold DM (2001) Human papillomavirus positive squamous cell carcinoma of the oropharynx: a radiosensitive subgroup of head and neck carcinoma. Cancer 92:805–813. https://doi.org/10.1002/1097-0142(20010815)92:4%3c805::aid-cncr1386%3e3.0.co;2-9

    Article  CAS  PubMed  Google Scholar 

  15. Gottgens EL, Ostheimer C, Span PN, Bussink J, Hammond EM (2019) HPV, hypoxia and radiation response in head and neck cancer. Br J Radiol 92:20180047. https://doi.org/10.1259/bjr.20180047

    Article  PubMed  Google Scholar 

  16. Galvis MM et al (2020) Immunotherapy improves efficacy and safety of patients with HPV positive and negative head and neck cancer: a systematic review and meta-analysis. Crit Rev Oncol Hematol 150:102966. https://doi.org/10.1016/j.critrevonc.2020.102966

    Article  PubMed  Google Scholar 

  17. Bol V, Gregoire V (2014) Biological basis for increased sensitivity to radiation therapy in HPV-positive head and neck cancers. Biomed Res Int 2014:696028. https://doi.org/10.1155/2014/696028

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Duan Q, Zhang H, Zheng J, Zhang L (2020) Turning cold into hot: firing up the tumor microenvironment. Trends Cancer 6:605–618. https://doi.org/10.1016/j.trecan.2020.02.022

    Article  CAS  PubMed  Google Scholar 

  19. Oweida AJ et al (2019) STAT3 modulation of regulatory T cells in response to radiation therapy in head and neck cancer. J Natl Cancer Inst. https://doi.org/10.1093/jnci/djz036

    Article  PubMed  PubMed Central  Google Scholar 

  20. Huang B et al (2007) CCL2/CCR2 pathway mediates recruitment of myeloid suppressor cells to cancers. Cancer Lett 252:86–92. https://doi.org/10.1016/j.canlet.2006.12.012

    Article  CAS  PubMed  Google Scholar 

  21. Kalbasi A et al (2017) Tumor-derived CCL2 mediates resistance to radiotherapy in pancreatic ductal adenocarcinoma. Clin Cancer Res 23:137–148. https://doi.org/10.1158/1078-0432.CCR-16-0870

    Article  CAS  PubMed  Google Scholar 

  22. Wang D et al (2019) Colorectal cancer cell-derived CCL20 recruits regulatory T cells to promote chemoresistance via FOXO1/CEBPB/NF-kappaB signaling. J Immunother Cancer 7:215. https://doi.org/10.1186/s40425-019-0701-2

    Article  PubMed  PubMed Central  Google Scholar 

  23. Harlin H et al (2009) Chemokine expression in melanoma metastases associated with CD8+ T-cell recruitment. Cancer Res 69:3077–3085. https://doi.org/10.1158/0008-5472.CAN-08-2281

    Article  CAS  PubMed  Google Scholar 

  24. Bouchard G et al (2013) Pre-irradiation of mouse mammary gland stimulates cancer cell migration and development of lung metastases. Br J Cancer 109:1829–1838. https://doi.org/10.1038/bjc.2013.502

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Oweida AJ, Bhatia S, Van Court B, Darragh L, Serkova N, Karam SD (2019) Intramucosal inoculation of squamous cell carcinoma cells in mice for tumor immune profiling and treatment response assessment. J Vis Exp. Published online Jan. 2019.

  26. Judd NP, Allen CT, Winkler AE, Uppaluri R (2012) Comparative analysis of tumor-infiltrating lymphocytes in a syngeneic mouse model of oral cancer. Otolaryngol Head Neck Surg 147:493–500. https://doi.org/10.1177/0194599812442037

    Article  PubMed  PubMed Central  Google Scholar 

  27. Lu E, Su J, Zhou Y, Zhang C, Wang Y (2017) CCL20/CCR6 promotes cell proliferation and metastasis in laryngeal cancer by activating p38 pathway. Biomed Pharmacother 85:486–492. https://doi.org/10.1016/j.biopha.2016.11.055

    Article  CAS  PubMed  Google Scholar 

  28. Gopal YN et al (2014) Inhibition of mTORC1/2 overcomes resistance to MAPK pathway inhibitors mediated by PGC1alpha and oxidative phosphorylation in melanoma. Cancer Res 74:7037–7047. https://doi.org/10.1158/0008-5472.CAN-14-1392

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Inoue Y et al (2000) Induction of human leukocyte antigen-A26-restricted and tumor-specific cytotoxic T lymphocytes by a single peptide of the SART1 antigen in patients with cancer with different A26 subtypes. J Immunother 23:296–303. https://doi.org/10.1097/00002371-200005000-00002

    Article  CAS  PubMed  Google Scholar 

  30. Narita M et al (2015) Immune responses in patients with esophageal cancer treated with SART1 peptide-pulsed dendritic cell vaccine. Int J Oncol 46:1699–1709. https://doi.org/10.3892/ijo.2015.2846

    Article  CAS  PubMed  Google Scholar 

  31. Oweida A, Harrah MK, Phan, A, Binder D, Bhatia S, Lennon S, Bukkapatnam S, Vancourt B, Uyanga N, Darragh L, Kim HM, Raben D, Tan AC, Heasley L, Clambey E, Nemenoff R, Karam SD (2018) Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation and regulatory T-cell infiltration. Clin Cancer Res

  32. Gasparoto TH et al (2010) Patients with oral squamous cell carcinoma are characterized by increased frequency of suppressive regulatory T cells in the blood and tumor microenvironment. Cancer Immunol Immunother 59:819–828. https://doi.org/10.1007/s00262-009-0803-7

    Article  CAS  PubMed  Google Scholar 

  33. Wing K, Sakaguchi S (2010) Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat Immunol 11:7–13. https://doi.org/10.1038/ni.1818

    Article  CAS  PubMed  Google Scholar 

  34. Shevach EM (2009) Mechanisms of foxp3+ T regulatory cell-mediated suppression. Immunity 30:636–645. https://doi.org/10.1016/j.immuni.2009.04.010

    Article  CAS  PubMed  Google Scholar 

  35. Moore EC et al (2016) Enhanced tumor control with combination mTOR and PD-L1 inhibition in syngeneic oral cavity cancers. Cancer Immunol Res 4:611–620. https://doi.org/10.1158/2326-6066.CIR-15-0252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Zhang CY et al (2015) The role of CCL20/CCR6 axis in recruiting Treg cells to tumor sites of NSCLC patients. Biomed Pharmacother 69:242–248. https://doi.org/10.1016/j.biopha.2014.12.008

    Article  CAS  PubMed  Google Scholar 

  37. Bromley SK, Mempel TR, Luster AD (2008) Orchestrating the orchestrators: chemokines in control of T cell traffic. Nat Immunol 9:970–980. https://doi.org/10.1038/ni.f.213

    Article  CAS  PubMed  Google Scholar 

  38. Nagarsheth N, Wicha MS, Zou W (2017) Chemokines in the cancer microenvironment and their relevance in cancer immunotherapy. Nat Rev Immunol 17:559–572. https://doi.org/10.1038/nri.2017.49

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gajewski TF, Schreiber H, Fu YX (2013) Innate and adaptive immune cells in the tumor microenvironment. Nat Immunol 14:1014–1022. https://doi.org/10.1038/ni.2703

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Xu L, Xu W, Qiu S, Xiong S (2010) Enrichment of CCR6+Foxp3+ regulatory T cells in the tumor mass correlates with impaired CD8+ T cell function and poor prognosis of breast cancer. Clin Immunol 135:466–475. https://doi.org/10.1016/j.clim.2010.01.014

    Article  CAS  PubMed  Google Scholar 

  41. Lee JJ et al (2011) Increased prevalence of interleukin-17-producing CD4(+) tumor infiltrating lymphocytes in human oral squamous cell carcinoma. Head Neck 33:1301–1308. https://doi.org/10.1002/hed.21607

    Article  CAS  PubMed  Google Scholar 

  42. Lee JJ et al (2017) Enrichment of Human CCR6(+) Regulatory T Cells with Superior Suppressive Activity in Oral Cancer. J Immunol 199:467–476. https://doi.org/10.4049/jimmunol.1601815

    Article  CAS  PubMed  Google Scholar 

  43. Xuan W, Qu Q, Zheng B, Xiong S, Fan GH (2015) The chemotaxis of M1 and M2 macrophages is regulated by different chemokines. J Leukoc Biol 97:61–69. https://doi.org/10.1189/jlb.1A0314-170R

    Article  CAS  PubMed  Google Scholar 

  44. Sierra-Filardi E et al (2014) CCL2 shapes macrophage polarization by GM-CSF and M-CSF: identification of CCL2/CCR2-dependent gene expression profile. J Immunol 192:3858–3867. https://doi.org/10.4049/jimmunol.1302821

    Article  CAS  PubMed  Google Scholar 

  45. Zorn E et al (2006) IL-2 regulates FOXP3 expression in human CD4+CD25+ regulatory T cells through a STAT-dependent mechanism and induces the expansion of these cells in vivo. Blood 108:1571–1579. https://doi.org/10.1182/blood-2006-02-004747

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Balermpas P et al (2014) Head and neck cancer relapse after chemoradiotherapy correlates with CD163+ macrophages in primary tumour and CD11b+ myeloid cells in recurrences. Br J Cancer 111:1509–1518. https://doi.org/10.1038/bjc.2014.446

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Nandi B et al (2016) Stromal CCR6 drives tumor growth in a murine transplantable colon cancer through recruitment of tumor-promoting macrophages. Oncoimmunology 5:e1189052. https://doi.org/10.1080/2162402X.2016.1189052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Boyle ST, Faulkner JW, McColl SR, Kochetkova M (2015) The chemokine receptor CCR6 facilitates the onset of mammary neoplasia in the MMTV-PyMT mouse model via recruitment of tumor-promoting macrophages. Mol Cancer 14:115. https://doi.org/10.1186/s12943-015-0394-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. He H et al (2017) CCR6(+) B lymphocytes responding to tumor cell-derived CCL20 support hepatocellular carcinoma progression via enhancing angiogenesis. Am J Cancer Res 7:1151–1163

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This reseach was funded by the Cancer Research Society and the CIHR-Institute of Musculoskeletal Health and Arthritis (Grant # 837109).

Author information

Authors and Affiliations

  1. Department of Nuclear Medicine and Radiobiology, Faculté de Médecine et des Sciences de La Santé, Université de Sherbrooke, Sherbrooke, QC, Canada

    Cleopatra Rutihinda, Ryma Haroun, Nour Elhouda Saidi, Juan Pablo Ordoñez, Sahar Naasri, Vincent Hubert-Tremblay, Guy Anne Turgeon, Chang Shu Wang, Patrick Delage, Étienne Rousseau, Benoît Paquette & Ayman J. Oweida

  2. Department of Immunology and Cell Biology, Faculté de Médecine Et Des Sciences de La Santé, Université de Sherbrooke, Sherbrooke, QC, Canada

    Dominique Lévesque & François-Michel Boisvert

  3. Department of Surgery, Faculté de Médecine Et Des Sciences de La Santé, Université de Sherbrooke, Sherbrooke, QC, Canada

    Pierre-Hugues Fortier, Mathieu Belzile & Laurent Fradet

Authors
  1. Cleopatra Rutihinda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ryma Haroun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nour Elhouda Saidi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Juan Pablo Ordoñez
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sahar Naasri
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dominique Lévesque
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. François-Michel Boisvert
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Pierre-Hugues Fortier
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Mathieu Belzile
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Laurent Fradet
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Vincent Hubert-Tremblay
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Guy Anne Turgeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Chang Shu Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Patrick Delage
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Étienne Rousseau
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Benoît Paquette
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Ayman J. Oweida
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

CR and AO designed the studies, performed the experiments and wrote the manuscript. RH, NS and JPO performed experiments. DL and FMB contributed to Table 1 and performed mass-spectrometry experiments. PHF, MB and LF assisted in experimental design and manuscript preparation. GAT, CSW, PD, ER, VHT and BP assisted with design of radiation-based experiments and associated data analysis.

Corresponding author

Correspondence to Ayman J. Oweida.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

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 (e.g. a society or other partner) 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

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rutihinda, C., Haroun, R., Saidi, N.E. et al. Inhibition of the CCR6-CCL20 axis prevents regulatory T cell recruitment and sensitizes head and neck squamous cell carcinoma to radiation therapy. Cancer Immunol Immunother 72, 1089–1102 (2023). https://doi.org/10.1007/s00262-022-03313-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00262-022-03313-2

Keywords

Search

Navigation