Enhancing the response rate of immunotherapy will aid in the success of cancer treatment. Here, we aimed to explore the combined effect of immunogenic radiotherapy with anti-PD-L1 treatment in immunotherapy-resistant HNSCC mouse models.
The SCC7 and 4MOSC2 cell lines were irradiated in vitro. SCC7-bearing mice were treated with hypofractionated or single-dose radiotherapy followed by anti-PD-L1 therapy. The myeloid-derived suppressive cells (MDSCs) were depleted using an anti-Gr-1 antibody. Human samples were collected to evaluate the immune cell populations and ICD markers.
Irradiation increased the release of immunogenic cell death (ICD) markers (calreticulin, HMGB1 and ATP) in SCC7 and 4MOSC2 in a dose-dependent manner. The supernatant from irradiated cells upregulated the expression of PD-L1 in MDSCs. Mice treated with hypofractionated but not single-dose radiotherapy were resistant to tumour rechallenge by triggering ICD, when combined with anti-PD-L1 treatment. The therapeutic efficacy of combination treatment partially relies on MDSCs. The high expression of ICD markers was associated with activation of adaptive immune responses and a positive prognosis in HNSCC patients.
These results present a translatable method to substantially improve the antitumor immune response by combining PD-L1 blockade with immunogenic hypofractionated radiotherapy in HNSCC.
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The data that support the findings of this study are available from the corresponding author upon reasonable request.
Galon J, Bruni D. Tumor immunology and tumor evolution: intertwined histories. Immunity. 2020;52:55–81.
Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med. 2012;366:2443–54.
Garon EB, Rizvi NA, Hui R, Leighl N, Balmanoukian AS, Eder JP, et al. Pembrolizumab for the treatment of non-small-cell lung cancer. N Engl J Med. 2015;372:2018–28.
Burtness B, Harrington KJ, Greil R, Soulieres D, Tahara M, de Castro G Jr. et al. Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study. Lancet. 2019;394:1915–28.
Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. 2017;168:707–23.
Whiteside TL. Head and neck carcinoma immunotherapy: facts and hopes. Clin Cancer Res. 2018;24:6–13.
Mandal R, Senbabaoglu Y, Desrichard A, Havel JJ, Dalin MG, Riaz N, et al. The head and neck cancer immune landscape and its immunotherapeutic implications. JCI Insight. 2016;1:e89829.
Ferris RL, Blumenschein G Jr, Fayette J, Guigay J, Colevas AD, Licitra L, et al. Nivolumab vs investigator’s choice in recurrent or metastatic squamous cell carcinoma of the head and neck: 2-year long-term survival update of CheckMate 141 with analyses by tumor PD-L1 expression. Oral Oncol. 2018;81:45–51.
Ferris RL, Blumenschein G Jr, Fayette J, Guigay J, Colevas AD, Licitra L, et al. Nivolumab for recurrent squamous-cell carcinoma of the head and neck. N Engl J Med. 2016;375:1856–67.
Chow LQM, Haddad R, Gupta S, Mahipal A, Mehra R, Tahara M, et al. Antitumor activity of pembrolizumab in biomarker-unselected patients with recurrent and/or metastatic head and neck squamous cell carcinoma: results from the phase Ib KEYNOTE-012 expansion cohort. J Clin Oncol. 2016;34:3838–45.
Cohen EEW, Soulieres D, Le Tourneau C, Dinis J, Licitra L, Ahn MJ, et al. Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study. Lancet. 2019;393:156–67.
Powell SF, Gold KA, Gitau MM, Sumey CJ, Lohr MM, McGraw SC, et al. Safety and efficacy of pembrolizumab with chemoradiotherapy in locally advanced head and neck squamous cell carcinoma: a phase IB study. J Clin Oncol. 2020;38:2427–37.
Weiss J, Sheth S, Deal AM, Grilley Olson JE, Patel S, Hackman TG, et al. Concurrent definitive immunoradiotherapy for patients with stage III-IV head and neck cancer and cisplatin contraindication. Clin Cancer Res. 2020;26:4260–7.
Lee NY, Ferris RL, Psyrri A, Haddad RI, Tahara M, Bourhis J, et al. Avelumab plus standard-of-care chemoradiotherapy versus chemoradiotherapy alone in patients with locally advanced squamous cell carcinoma of the head and neck: a randomised, double-blind, placebo-controlled, multicentre, phase 3 trial. Lancet Oncol. 2021;22:450–62.
Demaria S, Coleman CN, Formenti SC. Radiotherapy: changing the game in immunotherapy. Trends Cancer. 2016;2:286–94.
Karam SD, Raben D. Radioimmunotherapy for the treatment of head and neck cancer. Lancet Oncol. 2019;20:e404–16.
Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer G. Immunogenic cell death in cancer and infectious disease. Nat Rev Immunol. 2017;17:97–111.
Deng L, Liang H, Xu M, Yang X, Burnette B, Arina A, et al. STING-dependent cytosolic DNA sensing promotes radiation-induced type I interferon-dependent antitumor immunity in immunogenic tumors. Immunity. 2014;41:843–52.
Oweida AJ, Darragh L, Phan A, Binder D, Bhatia S, Mueller A, et al. STAT3 modulation of regulatory T cells in response to radiation therapy in head and neck cancer. J Natl Cancer Inst. 2019;111:1339–49.
Jayaraman P, Parikh F, Newton JM, Hanoteau A, Rivas C, Krupar R, et al. TGF-beta1 programmed myeloid-derived suppressor cells (MDSC) acquire immune-stimulating and tumor killing activity capable of rejecting established tumors in combination with radiotherapy. Oncoimmunology. 2018;7:e1490853.
Lin L, Kane N, Kobayashi N, Kono EA, Yamashiro JM, Nickols NG, et al. High-dose per fraction radiotherapy induces both antitumor immunity and immunosuppressive responses in prostate tumors. Clin Cancer Res. 2021;27:1505–15.
Oweida A, Hararah MK, Phan A, Binder D, Bhatia S, Lennon S, et al. Resistance to radiotherapy and PD-L1 blockade is mediated by TIM-3 upregulation and regulatory T-cell infiltration. Clin Cancer Res. 2018;24:5368–80.
Deng L, Liang H, Burnette B, Beckett M, Darga T, Weichselbaum RR, et al. Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J Clin Invest. 2014;124:687–95.
Vanpouille-Box C, Diamond JM, Pilones KA, Zavadil J, Babb JS, Formenti SC, et al. TGFbeta is a master regulator of radiation therapy-induced antitumor immunity. Cancer Res. 2015;75:2232–42.
Dovedi SJ, Adlard AL, Lipowska-Bhalla G, McKenna C, Jones S, Cheadle EJ, et al. Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res. 2014;74:5458–68.
Oweida A, Lennon S, Calame D, Korpela S, Bhatia S, Sharma J, et al. Ionizing radiation sensitizes tumors to PD-L1 immune checkpoint blockade in orthotopic murine head and neck squamous cell carcinoma. Oncoimmunology. 2017;6:e1356153.
Newton JM, Hanoteau A, Liu HC, Gaspero A, Parikh F, Gartrell-Corrado RD, et al. Immune microenvironment modulation unmasks therapeutic benefit of radiotherapy and checkpoint inhibition. J Immunother Cancer. 2019;7:216.
Wennerberg E, Spada S, Rudqvist NP, Lhuillier C, Gruber S, Chen Q, et al. CD73 blockade promotes dendritic cell infiltration of irradiated tumors and tumor rejection. Cancer Immunol Res. 2020;8:465–78.
Wang Z, Wu VH, Allevato MM, Gilardi M, He Y, Luis Callejas-Valera J, et al. Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4. Nat Commun. 2019;10:5546.
El-Naggar AK, Chan JKC, Grandis JR, Takata T, Slootweg PJ. WHO classification of head and neck tumours. Lyon, Fracnce: International Agency for Research on Cancer (IARC); 2017.
Huang CF, Chen L, Li YC, Wu L, Yu GT, Zhang WF, et al. NLRP3 inflammasome activation promotes inflammation-induced carcinogenesis in head and neck squamous cell carcinoma. J Exp Clin Cancer Res. 2017;36:116.
Wu L, Mao L, Liu JF, Chen L, Yu GT, Yang LL, et al. Blockade of TIGIT/CD155 signaling reverses T-cell exhaustion and enhances antitumor capability in head and neck squamous cell carcinoma. Cancer Immunol Res. 2019;7:1700–13.
Roberts EW, Broz ML, Binnewies M, Headley MB, Nelson AE, Wolf DM, et al. Critical role for CD103(+)/CD141(+) dendritic cells bearing CCR7 for tumor antigen trafficking and priming of T cell immunity in melanoma. Cancer Cell. 2016;30:324–36.
Golden EB, Frances D, Pellicciotta I, Demaria S, Helen Barcellos-Hoff M, Formenti SC. Radiation fosters dose-dependent and chemotherapy-induced immunogenic cell death. Oncoimmunology. 2014;3:e28518.
Kondo Y, Ohno T, Nishii N, Harada K, Yagita H, Azuma M. Differential contribution of three immune checkpoint (VISTA, CTLA-4, PD-1) pathways to antitumor responses against squamous cell carcinoma. Oral Oncol. 2016;57:54–60.
Veglia F, Perego M, Gabrilovich D. Myeloid-derived suppressor cells coming of age. Nat Immunol. 2018;19:108–19.
Xu J, Escamilla J, Mok S, David J, Priceman S, West B, et al. CSF1R signaling blockade stanches tumor-infiltrating myeloid cells and improves the efficacy of radiotherapy in prostate cancer. Cancer Res. 2013;73:2782–94.
Carmeliet P, Jain RK. Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases. Nat Rev Drug Discov. 2011;10:417–27.
Sharabi AB, Lim M, DeWeese TL, Drake CG. Radiation and checkpoint blockade immunotherapy: radiosensitisation and potential mechanisms of synergy. Lancet Oncol. 2015;16:e498–509.
Pfister DG, Spencer S, Adelstein D, Adkins D, Anzai Y, Brizel DM, et al. Head and neck cancers, version 2.2020, NCCN clinical practice guidelines in oncology. J Natl Compr Cancer Netw. 2020;18:873–98.
Thorsson V, Gibbs DL, Brown SD, Wolf D, Bortone DS, Ou Yang TH, et al. The immune landscape of cancer. Immunity. 2018;48:812–30.e814.
Dudzinski SO, Cameron BD, Wang J, Rathmell JC, Giorgio TD, Kirschner AN. Combination immunotherapy and radiotherapy causes an abscopal treatment response in a mouse model of castration resistant prostate cancer. J Immunother Cancer. 2019;7:218.
Poty S, Carter LM, Mandleywala K, Membreno R, Abdel-Atti D, Ragupathi A, et al. Leveraging bioorthogonal click chemistry to improve (225)Ac-radioimmunotherapy of pancreatic ductal adenocarcinoma. Clin Cancer Res. 2019;25:868–80.
Dewan MZ, Galloway AE, Kawashima N, Dewyngaert JK, Babb JS, Formenti SC, et al. Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody. Clin Cancer Res. 2009;15:5379–88.
Galluzzi L, Kepp O, Kroemer G. Immunogenic cell death in radiation therapy. Oncoimmunology. 2013;2:e26536.
Weichselbaum RR, Liang H, Deng L, Fu YX. Radiotherapy and immunotherapy: a beneficial liaison? Nat Rev Clin Oncol. 2017;14:365–79.
Obeid M, Panaretakis T, Joza N, Tufi R, Tesniere A, van Endert P, et al. Calreticulin exposure is required for the immunogenicity of gamma-irradiation and UVC light-induced apoptosis. Cell Death Differ. 2007;14:1848–50.
Werthmoller N, Frey B, Wunderlich R, Fietkau R, Gaipl US. Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner. Cell Death Dis. 2015;6:e1761.
Ko A, Kanehisa A, Martins I, Senovilla L, Chargari C, Dugue D, et al. Autophagy inhibition radiosensitizes in vitro, yet reduces radioresponses in vivo due to deficient immunogenic signalling. Cell Death Differ. 2014;21:92–9.
Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, et al. Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med. 2012;4:127ra137.
Campian JL, Sarai G, Ye X, Marur S, Grossman SA. Association between severe treatment-related lymphopenia and progression-free survival in patients with newly diagnosed squamous cell head and neck cancer. Head Neck. 2014;36:1747–53.
Bernstein MB, Krishnan S, Hodge JW, Chang JY. Immunotherapy and stereotactic ablative radiotherapy (ISABR): a curative approach? Nat Rev Clin Oncol. 2016;13:516–24.
Yang X, Lu Y, Hang J, Zhang J, Zhang T, Huo Y, et al. Lactate-modulated immunosuppression of myeloid-derived suppressor cells contributes to the radioresistance of pancreatic cancer. Cancer Immunol Res. 2020;8:1440–51.
Oweida AJ, Mueller AC, Piper M, Milner D, Van Court B, Bhatia S, et al. Response to radiotherapy in pancreatic ductal adenocarcinoma is enhanced by inhibition of myeloid-derived suppressor cells using STAT3 anti-sense oligonucleotide. Cancer Immunol Immunother. 2021;70:989–1000.
Morisada M, Clavijo PE, Moore E, Sun L, Chamberlin M, Van Waes C, et al. PD-1 blockade reverses adaptive immune resistance induced by high-dose hypofractionated but not low-dose daily fractionated radiation. Oncoimmunology. 2018;7:e1395996.
Barker HE, Paget JT, Khan AA, Harrington KJ. The tumour microenvironment after radiotherapy: mechanisms of resistance and recurrence. Nat Rev Cancer. 2015;15:409–25.
We want to thank Shuyan Liang and Zhixin Qiu from Wuhan Biobank Co. Ltd., Hubei, China for their excellent technical assistance on flow cytometry and cells isolation. And we also want to thank Ji Chen, Yan-Peng Ding and Yan Qi from Zhongnan Hospital of Wuhan University and Ying Liu and Jin Jing from the Core Facility of Medical Research Institute at Wuhan University for their kind guidance on radiotherapy experiments.
This work was supported by the National Natural Science Foundation of China (NFSC): 82103333, 82072996, 81874131 and the Fundamental Research Funds for the Central Universities (2042021kf0174, 2042021kf0216).
The authors declare no competing interests.
Ethics approval and consent to participate
All animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Wuhan University (2019038, 2019108, 2019161, 20220283). All experiments performed using the patients’ samples were reviewed and approved by the Institutional Medical Ethics Committee of School and Hospital of Stomatology, Wuhan University (2012LUNSHENZI46, 2018LUNSHENZIA28), and informed consent was obtained from all patients. All studies were conducted in accordance with the Declaration of Helsinki.
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Mao, L., Zhou, JJ., Xiao, Y. et al. Immunogenic hypofractionated radiotherapy sensitising head and neck squamous cell carcinoma to anti-PD-L1 therapy in MDSC-dependent manner. Br J Cancer 128, 2126–2139 (2023). https://doi.org/10.1038/s41416-023-02230-0