Antibodies targeting the co-inhibitory receptor programmed cell death 1 (PDCD1, best known as PD-1) or its main ligand CD274 (best known as PD-L1) have shown some activity in patients with metastatic triple-negative breast cancer (TNBC), especially in a recent Phase III clinical trial combining PD-L1 blockade with taxane-based chemotherapy. Despite these encouraging findings, however, most patients with TNBC fail to derive significant benefits from PD-L1 blockade, calling for the identification of novel therapeutic approaches. Here, we used the 4T1 murine mammary cancer model of metastatic and immune-resistant TNBC to test whether focal radiation therapy (RT), a powerful inducer of immunogenic cell death, in combination with various immunotherapeutic strategies can overcome resistance to immune checkpoint blockade. Our results suggest that focal RT enhances the therapeutic effects of PD-1 blockade against primary 4T1 tumors and their metastases. Similarly, the efficacy of an antibody specific for V-set immunoregulatory receptor (VSIR, another co-inhibitory receptor best known as VISTA) was enhanced by focal RT. Administration of cyclophosphamide plus RT and dual PD-1/VISTA blockade had superior therapeutic effects, which were associated with activation of tumor-infiltrating CD8+ T cells and depletion of intratumoral granulocytic myeloid-derived suppressor cells (MDSCs). Overall, these results demonstrate that RT can sensitize immunorefractory tumors to VISTA or PD-1 blockade, that this effect is enhanced by the addition of cyclophosphamide and suggest that a multipronged immunotherapeutic approach may also be required to increase the incidence of durable responses in patients with TNBC.

Caryl and Isreal Englander Institute for Precision Medicine Weill Cornell Medical College New York NY USA

Department of Immunology Charles University 2nd Faculty of Medicine and University Hospital Motol Prague Czech Republic

Department of Pathology and Laboratory Medicine Weill Cornell Medical College New York NY USA

Department of Radiation Oncology Weill Cornell Medical College New York NY USA

Sandra and Edward Meyer Cancer Center New York NY USA

Sotio Prague Czech Republic

Zobrazit více v PubMed

Galluzzi L, Chan TA, Kroemer G, Wolchok JD, López-Soto A.. The hallmarks of successful anticancer immunotherapy. Sci Transl Med. 2018;10:1–14. PubMed

Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell. 2017;168:707–10. PubMed PMC

Emens LA. Breast cancer immunotherapy: facts and hopes. Clin Cancer Res. 2018;24:511–520. PubMed PMC

Schmid P, Adams S, Rugo HS, Schneeweiss A, Barrios CH, Iwata H, Diéras V, Hegg R, Im SA, Shaw Wright G, et al. Atezolizumab and nab-paclitaxel in advanced triple-negative breast cancer. N Engl J Med. 2018;379:2108–2121. PubMed

Ma Y, Kepp O, Ghiringhelli F, Apetoh L, Aymeric L, Locher C, Tesniere A, Martins I, Ly A, Haynes NM, et al. Chemotherapy and radiotherapy: cryptic anticancer vaccines. Semin Immunol. 2010;22:113–124. PubMed

Galluzzi L, Vitale I, Warren S, Adjemian S, Agostinis P, Martinez AB, Chan TA, Coukos G, Demaria S, Deutsch E, et al. Consensus guidelines for the definition, detection and interpretation of immunogenic cell death. J Immunother Cancer. 2020;8:e000337. PubMed PMC

Rodriguez-Ruiz ME, Vitale I, Harrington KJ, Melero I, Galluzzi L. Immunological impact of cell death signaling driven by radiation on the tumor microenvironment. Nat Immunol. 2020;21:120–134. PubMed

Sistigu A, Viaud S, Chaput N, Bracci L, Proietti E, Zitvogel L. Immunomodulatory effects of cyclophosphamide and implementations for vaccine design. Semin Immunopathol. 2011;33:369–383. PubMed

Demaria S, Golden EB, Formenti SC. Role of local radiation therapy in cancer immunotherapy. JAMA Oncol. 2015;1:1325–1332. PubMed

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. PubMed PMC

Rodriguez-Ruiz ME, Buque A, Hensler M, Chen J, Bloy N, Petroni G, Sato A, Yamazaki T, Fucikova J, Galluzzi L. Apoptotic caspases inhibit abscopal responses to radiation and identify a new prognostic biomarker for breast cancer patients. Oncoimmunology. 2019;8:e1655964. PubMed PMC

Demaria S, Kawashima N, Yang AM, Devitt ML, Babb JS, Allison JP, Formenti SC. Immune-mediated inhibition of metastases after treatment with local radiation and CTLA-4 blockade in a mouse model of breast cancer. Clin Cancer Res. 2005;11:728–734. PubMed

Deng L, Liang H, Burnette B, Beckett M, Darga T, Weichselbaum RR, Fu YX. Irradiation and anti-PD-L1 treatment synergistically promote antitumor immunity in mice. J Clin Invest. 2014;124:687–695. PubMed PMC

Dovedi SJ, Adlard AL, Lipowska-Bhalla G, McKenna C, Jones S, Cheadle EJ, Stratford IJ, Poon E, Morrow M, Stewart R, et al. Acquired resistance to fractionated radiotherapy can be overcome by concurrent PD-L1 blockade. Cancer Res. 2014;74:5458–5468. PubMed

Pilones KA, Vanpouille-Box C, Demaria S. Combination of radiotherapy and immune checkpoint inhibitors. Semin Radiat Oncol. 2015;25:28–33. PubMed

Shaverdian N, Lisberg AE, Bornazyan K, Veruttipong D, Goldman JW, Formenti SC, Garon EB, Lee P. Previous radiotherapy and the clinical activity and toxicity of pembrolizumab in the treatment of non-small-cell lung cancer: a secondary analysis of the KEYNOTE-001 phase 1 trial. Lancet Oncol. 2017;18:895–903. PubMed PMC

Antonia SJ, Villegas A, Daniel D, Vicente D, Murakami S, Hui R, Yokoi T, Chiappori A, Lee KH, de Wit M, et al. Durvalumab after chemoradiotherapy in stage III non-small-cell lung cancer. N Engl J Med. 2017;377:1919–1929. PubMed

Formenti SC, Rudqvist NP, Golden E, Cooper B, Wennerberg E, Lhuillier C, Vanpouille-Box C, Friedman K, Ferrari de Andrade L, Wucherpfennig KW, et al. Radiotherapy induces responses of lung cancer to CTLA-4 blockade. Nat Med. 2018;24:1845–1851. PubMed PMC

Sharma P, Allison JP. Dissecting the mechanisms of immune checkpoint therapy. Nat Rev Immunol. 2020;20:75–76. PubMed

Zarour HM. Reversing T-cell Dysfunction and Exhaustion in Cancer. Clin Cancer Res. 2016;22:1856–1864. PubMed PMC

Fucikova J, Rakova J, Hensler M, Kasikova L, Belicova L, Hladikova K, Truxova I, Skapa P, Laco J, Pecen L, et al. TIM-3 dictates functional orientation of the immune infiltrate in ovarian cancer. Clin Cancer Res. 2019;25:4820–4831. PubMed

Vanpouille-Box C, Formenti SC. Dual transforming growth factor-β and programmed death-1 blockade: a strategy for immune-excluded tumors? Trends Immunol. 2018;39:435–437. PubMed PMC

Tauriello DVF, Palomo-Ponce S, Stork D, Berenguer-Llergo A, Badia-Ramentol J, Iglesias M, Sevillano M, Ibiza S, Cañellas A, Hernando-Momblona X, et al. TGFβ drives immune evasion in genetically reconstituted colon cancer metastasis. Nature. 2018;554:538–543. PubMed

Mariathasan S, Turley SJ, Nickles D, Castiglioni A, Yuen K, Wang Y, Kadel EE III, Koeppen H, Astarita JL, Cubas R, et al. TGFβ attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells. Nature. 2018;554:544–548. PubMed PMC

Nowak EC, Lines JL, Varn FS, Deng J, Sarde A, Mabaera R, Kuta A, Le Mercier I, Cheng C, Noelle RJ. Immunoregulatory functions of VISTA. Immunol Rev. 2017;276:66–79. PubMed PMC

Wang L, Rubinstein R, Lines JL, Wasiuk A, Ahonen C, Guo Y, Lu LF, Gondek D, Wang Y, Fava RA, et al. VISTA, a novel mouse Ig superfamily ligand that negatively regulates T cell responses. J Exp Med. 2011;208:577–592. PubMed PMC

Flies DB, Han X, Higuchi T, Zheng L, Sun J, Ye JJ, Chen L. Coinhibitory receptor PD-1H preferentially suppresses CD4⁺ T cell-mediated immunity. J Clin Invest. 2014;124:1966–1975. PubMed PMC

Liu J, Yuan Y, Chen W, Putra J, Suriawinata AA, Schenk AD, Miller HE, Guleria I, Barth RJ, Huang YH, et al. Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses. Proc Natl Acad Sci U S A. 2015;112:6682–6687. PubMed PMC

Mosely SI, Prime JE, Sainson RC, Koopmann JO, Wang DY, Greenawalt DM, Ahdesmaki MJ, Leyland R, Mullins S, Pacelli L, et al. Rational selection of syngeneic preclinical tumor models for immunotherapeutic drug discovery. Cancer Immunol Res. 2017;5:29–41. PubMed

Markowitz J, Wesolowski R, Papenfuss T, Brooks TR, Carson WE 3rd.. Myeloid-derived suppressor cells in breast cancer. Breast Cancer Res Treat. 2013;140:13–21. PubMed PMC

Pilones KA, Charpentier M, Garcia-Martinez E, Daviaud C, Kraynak J, Aryankalayil J, Formenti SC, Demaria S. Radiotherapy cooperates with IL15 to induce antitumor immune responses. Cancer Immunol Res. 2020;8:1054–1063. PubMed PMC

Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol. 1989;24:148–154. PubMed

Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, Smyth GK. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43:e47. PubMed PMC

Gu Z, Eils R, Schlesner M. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics. 2016;32:2847–2849. PubMed

Becht E, Giraldo NA, Lacroix L, Buttard B, Elarouci N, Petitprez F, Selves J, Laurent-Puig P, Sautès-Fridman C, Fridman WH, et al. Estimating the population abundance of tissue-infiltrating immune and stromal cell populations using gene expression. Genome Biol. 2016;17:218. PubMed PMC

Angelova M, Charoentong P, Hackl H, Fischer ML, Snajder R, Krogsdam AM, Waldner MJ, Bindea G, Mlecnik B, Galon J, et al. Characterization of the immunophenotypes and antigenomes of colorectal cancers reveals distinct tumor escape mechanisms and novel targets for immunotherapy. Genome Biol. 2015;16:64. PubMed PMC

Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS. 2012;16:284–287. PubMed PMC

Korotkevich G, Sukhov V, Sergushichev A. Fast gene set enrichment analysis. bioRxiv. 2019. 060012.

Pulaski BA, Ostrand-Rosenberg S. Mouse 4T1 breast tumor model. Curr Protoc Immunol. Chapter 20: Unit20.2. 2001. PubMed

Aslakson CJ, Miller FR. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res. 1992;52:1399–1405. PubMed

Savas P, Salgado R, Denkert C, Sotiriou C, Darcy PK, Smyth MJ, Loi S. Clinical relevance of host immunity in breast cancer: from TILs to the clinic. Nat Rev Clin Oncol. 2016;13:228–241. PubMed

Vanpouille-Box C, Diamond JM, Pilones KA, Zavadil J, Babb JS, Formenti SC, Barcellos-Hoff MH, Demaria S. TGFβ is a master regulator of radiation therapy-induced antitumor immunity. Cancer Res. 2015;75:2232–2242. PubMed PMC

Sinha P, Clements VK, Bunt SK, Albelda SM, Ostrand-Rosenberg S. Cross-talk between myeloid-derived suppressor cells and macrophages subverts tumor immunity toward a type 2 response. J Immunol. 2007;179:977–983. PubMed

Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 2009;9:162–174. PubMed PMC

Liu Y, Wei G, Cheng WA, Dong Z, Sun H, Lee VY, Cha SC, Smith DL, Kwak LW, Qin H. Targeting myeloid-derived suppressor cells for cancer immunotherapy. Cancer Immunol Immunother. 2018;67:1181–1195. PubMed PMC

Taieb J, Chaput N, Schartz N, Roux S, Novault S, Ménard C, Ghiringhelli F, Terme M, Carpentier AF, Darrasse-Jèze G, et al. Chemoimmunotherapy of tumors: cyclophosphamide synergizes with exosome based vaccines. J Immunol. 2006;176:2722–2729. PubMed

Dewan MZ, Vanpouille-Box C, Kawashima N, DiNapoli S, Babb JS, Formenti SC, Adams S, Demaria S. Synergy of topical toll-like receptor 7 agonist with radiation and low-dose cyclophosphamide in a mouse model of cutaneous breast cancer. Clin Cancer Res. 2012;18:6668–6678. PubMed PMC

Machiels JP, Reilly RT, Emens LA, Ercolini AM, Lei RY, Weintraub D, Okoye FI, Jaffee EM. Cyclophosphamide, doxorubicin, and paclitaxel enhance the antitumor immune response of granulocyte/macrophage-colony stimulating factor-secreting whole-cell vaccines in HER-2/neu tolerized mice. Cancer Res. 2001;61:3689–3697. PubMed

Zachariae C, Overgaard J. Interactions of radiation, cyclophosphamide and nimorazole in a C3H mammary carcinoma in vivo. Int J Radiat Oncol Biol Phys. 1986;12:1445–1448. PubMed

Thommen DS, Schumacher TN. T cell dysfunction in cancer. Cancer Cell. 2018;33:547–562. PubMed PMC

Scrimieri F, Askew D, Corn DJ, Eid S, Bobanga ID, Bjelac JA, Tsao ML, Allen F, Othman YS, Wang SC, et al. Murine leukemia virus envelope gp70 is a shared biomarker for the high-sensitivity quantification of murine tumor burden. Oncoimmunology. 2013;2:e26889. PubMed PMC

Pilones KA, Aryankalayil J, Babb JS, Demaria S. Invariant natural killer T cells regulate anti-tumor immunity by controlling the population of dendritic cells in tumor and draining lymph nodes. J Immunother Cancer. 2014;2:37. PubMed PMC

Gabrilovich DI. Myeloid-derived suppressor cells. Cancer Immunol Res. 2017;5:3–8. PubMed PMC

Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid-derived suppressor cells in tumor-bearing mice. J Immunol. 2008;181:5791–5802. PubMed PMC

Allard B, Allard D, Buisseret L, Stagg J. The adenosine pathway in immuno-oncology. Nat Rev Clin Oncol. 2020;17:611–629. PubMed

Wennerberg E, Spada S, Rudqvist NP, Lhuillier C, Gruber S, Chen Q, Zhang F, Zhou XK, Gross SS, Formenti SC, et al. CD73 blockade promotes dendritic cell infiltration of irradiated tumors and tumor rejection. Cancer Immunol Res. 2020;8:465–478. PubMed PMC

Martinek J, Wu TC, Cadena D, Banchereau J, Palucka K. Interplay between dendritic cells and cancer cells. Int Rev Cell Mol Biol. 2019;348:179–215. PubMed

Lee YS, Radford KJ. The role of dendritic cells in cancer. Int Rev Cell Mol Biol. 2019;348:123–178. PubMed

Kennedy BE, Sadek M, Elnenaei MO, Reiman A, Gujar SA. Targeting NAD(+) synthesis to potentiate CD38-based immunotherapy of multiple myeloma. Trends Cancer. 2020;6:9–12. PubMed

Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science. 2018;359:1350–1355. PubMed PMC

Boutros C, Tarhini A, Routier E, Lambotte O, Ladurie FL, Carbonnel F, Izzeddine H, Marabelle A, Champiat S, Berdelou A, et al. Safety profiles of anti-CTLA-4 and anti-PD-1 antibodies alone and in combination. Nat Rev Clin Oncol. 2016;13:473–486. PubMed

Hellmann MD, Ciuleanu TE, Pluzanski A, Lee JS, Otterson GA, Audigier-Valette C, Minenza E, Linardou H, Burgers S, Salman P, et al. Nivolumab plus ipilimumab in lung cancer with a high tumor mutational burden. N Engl J Med. 2018;378:2093–2104. PubMed PMC

Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM, Segal NH, Ariyan CE, Gordon RA, Reed K, et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med. 2013;369:122–133. PubMed PMC

Gray JE, Villegas A, Daniel D, Vicente D, Murakami S, Hui R, Kurata T, Chiappori A, Lee KH, Cho BC, et al. Three-year overall survival with durvalumab after chemoradiotherapy in stage III NSCLC-update from PACIFIC. J Thorac Oncol. 2020;15:288–293. PubMed PMC

Le Mercier I, Chen W, Lines JL, Day M, Li J, Sergent P, Noelle RJ, Wang L. VISTA regulates the development of protective antitumor immunity. Cancer Res. 2014;74:1933–1944. PubMed PMC

Ghiringhelli F, Larmonier N, Schmitt E, Parcellier A, Cathelin D, Garrido C, Chauffert B, Solary E, Bonnotte B, Martin F. CD4+CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol. 2004;34:336–344. PubMed

Viaud S, Saccheri F, Mignot G, Yamazaki T, Daillère R, Hannani D, Enot DP, Pfirschke C, Engblom C, Pittet MJ, et al. The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide. Science. 2013;342:971–976. PubMed PMC

Vanpouille-Box C, Alard A, Aryankalayil MJ, Sarfraz Y, Diamond JM, Schneider RJ, Inghirami G, Coleman CN, Formenti SC, Demaria S. DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity. Nat Commun. 2017;8:15618. PubMed PMC

Gao J, Ward JF, Pettaway CA, Shi LZ, Subudhi SK, Vence LM, Zhao H, Chen J, Chen H, Efstathiou E, et al. VISTA is an inhibitory immune checkpoint that is increased after ipilimumab therapy in patients with prostate cancer. Nat Med. 2017;23:551–555. PubMed PMC

Blando J, Sharma A, Higa MG, Zhao H, Vence L, Yadav SS, Kim J, Sepulveda AM, Sharp M, Maitra A, et al. Comparison of immune infiltrates in melanoma and pancreatic cancer highlights VISTA as a potential target in pancreatic cancer. Proc Natl Acad Sci U S A. 2019;116:1692–1697. PubMed PMC

Nickols NG, Ganapathy E, Nguyen C, Kane N, Lin L, Diaz-Perez S, Nazarian R, Mathis C, Felix C, Basehart V, et al. The intraprostatic immune environment after stereotactic body radiotherapy is dominated by myeloid cells. Prostate Cancer Prostatic Dis. 2020. PubMed PMC

Vatner RE, Formenti SC. Myeloid-derived cells in tumors: effects of radiation. Semin Radiat Oncol. 2015;25:18–27. PubMed

Ahn GO, Tseng D, Liao CH, Dorie MJ, Czechowicz A, Brown JM. Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment. Proc Natl Acad Sci U S A. 2010;107:8363–8368. PubMed PMC

Xu J, Escamilla J, Mok S, David J, Priceman S, West B, Bollag G, McBride W, Wu L. CSF1R signaling blockade stanches tumor-infiltrating myeloid cells and improves the efficacy of radiotherapy in prostate cancer. Cancer Res. 2013;73:2782–2794. PubMed PMC

Liang H, Deng L, Hou Y, Meng X, Huang X, Rao E, Zheng W, Mauceri H, Mack M, Xu M, et al. Host STING-dependent MDSC mobilization drives extrinsic radiation resistance. Nat Commun. 2017;8:1736. PubMed PMC

Loyher PL, Rochefort J, Baudesson de Chanville C, Hamon P, Lescaille G, Bertolus C, Guillot-Delost M, Krummel MF, Lemoine FM, Combadiere C, et al. CCR2 influences T regulatory cell migration to tumors and serves as a biomarker of cyclophosphamide sensitivity. Cancer Res. 2016;76:6483–6494. PubMed

Mondini M, Loyher PL, Hamon P, Gerbe de Thore M, Laviron M, Berthelot K, Clemenson C, BL S, Combadiere C, Deutsch E, et al. CCR2-dependent recruitment of tregs and monocytes following radiotherapy is associated with tnfalpha-mediated resistance. Cancer Immunol Res. 2019;7:376–387. PubMed

LTX-315-enabled, radiotherapy-boosted immunotherapeutic control of breast cancer by NK cells