Down-regulation of immune checkpoints by doxorubicin and carboplatin-containing neoadjuvant regimens in a murine breast cancer model

Document Type : Original Article


1 Department of Medical Oncology, Cancer Research Center, Cancer Institute of Iran, Tehran University of Medical Sciences, Tehran, Iran

2 Department of Hematology and Oncology, Vali-e-Asr Hospital, Tehran University of Medical Sciences, Tehran, Iran

3 Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran and Université Toulouse III Paul Sabatier, INSERM U1037, Cancer Research Centre of Toulouse (CRCT), Toulouse, France

4 Cancer Biology Research Center, Cancer Institute of Iran, Tehran University of Medical Sciences

5 Department of Medical Biotechnology, School of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran

6 Gene Therapy Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences, Tehran, Iran

7 Pediatric Cell Therapy Research Center, Tehran University of Medical Sciences, Tehran, Iran


Objective(s): Immune checkpoint expression on tumor-infiltrating lymphocytes (TILs) has a correlation with the outcome of neoadjuvant chemotherapy (NAC) in breast cancer. However, the reciprocal effect of these regimens on the quality and quantity of immune checkpoints has hitherto not been addressed. We aimed to evaluate the impact of three NAC regimens on TILs and immune checkpoints in a murine triple-negative breast cancer model.
Materials and Methods: Syngeneic model of locally-advanced breast cancer was established in immunocompetent mice using a 4T1 cell line. Tumor-bearing animals were treated with human-equivalent dosages of doxorubicin, paclitaxel, paclitaxel and carboplatin combination, and placebo. Infiltration of CD3+, CD8+, and FoxP3+ cells into the tumor was assessed by immunohistochemistry. Expression of immune checkpoints, including PD-1, CTLA-4, and TIM-3, was evaluated by real-time PCR.
Results: Doxorubicin led to a significant (p <0.01) increase in the percentage of the stromal infiltrating CD3+ and CD8+ lymphocytes. Doxorubicin also suppressed significantly (p <0.05) the relative expression of PD-1 compared with the placebo. PD-1 expression was significantly (p <0.05) lower in the group treated with paclitaxel and carboplatin combination as compared with the placebo. The relative expression of TIM-3 was significantly (p <0.05) suppressed in doxorubicin-treated mice in comparison with other interventions.
Conclusion: Our findings hypothesize that NAC with doxorubicin may potentiate antitumor immunity not merely by recruitment of TILs, but via down-regulation of PD-1 and TIM-3 checkpoints. Carboplatin-containing NAC may suppress PD-1 as well.


1. Denduluri N, Miller K, O’Regan RM. Using a neoadjuvant approach for evaluating novel therapies for patients with breast cancer. Am Soc Clin Oncol Educ B. 2018;38:47–55.
2. Rapoport BL, Demetriou GS, Moodley SD, Benn CA. When and how do i use neoadjuvant chemotherapy for breast cancer? Curr Treat Options Oncol. 2014;15:86–98.
3. Rivera Vargas T, Apetoh L. Danger signals: Chemotherapy enhancers? Immunol Rev 2017;280:175–193.
4. Disis ML, Stanton SE. Triple-negative breast cancer: Immune modulation as the new treatment paradigm. Am Soc Clin Oncol Educ B 2015;35:e25–30.
5. Gooden MJM, De Bock GH, Leffers N, Daemen T, Nijman HW. The prognostic influence of tumour-infiltrating lymphocytes in cancer: A systematic review with meta-analysis. Br J Cancer. 2011;105:93–103.
6. Ansell SM, Vonderheide RH. Cellular composition of the tumor microenvironment. Am Soc Clin Oncol Educ B 2013;33:e91–7.
7. DeLeeuw RJ, Kost SE, Kakal JA, Nelson BH. The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: A critical review of the literature. Clin Cancer Res 2012;18:3022–3029.
8. Velcheti V, Schalper K. Basic overview of current immunotherapy approaches in cancer. Am Soc Clin Oncol Educ B 2016;35:298–308.
9. Silverman GJ, Azzouz DF, Mor A. Immune checkpoint inhibitors and the union of bugs against cancer. Kidney Int. 2018;93:1030–1032.
10. Ibrahim EM, Al-Foheidi ME, Al-Mansour MM, Kazkaz GA. The prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancer: a meta-analysis. Breast Cancer Res Treat. 2014;148:467–476.
11. Melichar B. The biology of tumor-infiltrating Leukocytes in breast cancer. Anticancer Res 2014;1126:1115–1125.
12. Lee SY, Ju MK, Jeon HM, Jeong EK, Lee YJ, Kim CH, et al. Review article regulation of tumor progression by programmed necrosis. 2018;2018????.
13. Lee H, Lee M, Seo J, Gong G, Lee HEEJIN. Changes in tumor-infiltrating lymphocytes after neoadjuvant chemotherapy and clinical. Anticancer Res 2020;1890:1883–1890.
14. Pelekanou V, Carvajal-Hausdorf DE, Altan M, Wasserman B, Carvajal-Hausdorf C, Wimberly H, et al. Erratum: Effect of neoadjuvant chemotherapy on tumor-infiltrating lymphocytes and PD-L1 expression in breast cancer and its clinical significance. Breast Cancer Res 2017;19:91-101.
15. Denkert C, Von Minckwitz G, Brase JC, Sinn BV, Gade S, Kronenwett R, et al. Tumor-infiltrating lymphocytes and response to neoadjuvant chemotherapy with or without carboplatin in human epidermal growth factor receptor 2-positive and triple-negative primary breast cancers. J Clin Oncol. 2015;33:983–991.
16. Steenbrugge J, Breyne K, Demeyere K, Wever O De, Sanders NN, Broeck W Van Den, et al. Anti-inflammatory signaling by mammary tumor cells mediates prometastatic macrophage polarization in an innovative intraductal mouse model for triple-negative breast cancer. J Exp Clin Cancer Res 2018;37:191-208.
17. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24:148–154.
18. Arifin WN, Zahiruddin WM. Sample size calculation in animal studies using resource equation approach. Malays J Med Sci 2017;24:101–105.
19. Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited. FASEB J 2008;33:659–661.
20. Vittoria M, Radosevic-robin N, Fineberg S, Eynden G Van Den, Ternes N, Penault-llorca F, et al. Seminars in cancer biology update on tumor-in fi ltrating lymphocytes ( TILs ) in breast cancer , including recommendations to assess TILs in residual disease after neoadjuvant therapy and in carcinoma in situ : A report of the International Immuno- Oncol. Semin Cancer Biol 2018;52:16–25.
21. Jia L, Liu Y, Wang L, Zhu J, Huang Y. Effects of topical sphingosine-1-phosphate 1 receptor agonist on corneal allograft in mice. Cornea 2014;33:398–404.
22. Lee JS, Yost SE, Yuan Y. Neoadjuvant treatment for triple negative breast cancer: Recent progresses and challenges. Cancers (Basel) 2020;12: 1404-1436.
23. Von Minckwitz G, Schneeweiss A, Loibl S, Salat C, Denkert C, Rezai M, et al. Neoadjuvant carboplatin in patients with triple-negative and HER2-positive early breast cancer (GeparSixto; GBG 66): A randomised phase 2 trial. Lancet Oncol 2014;15:747–756.
24. Mao Y, Qu Q, Zhang Y, Liu J, Chen X, Shen K. The value of tumor infiltrating lymphocytes (TILs) for predicting response to neoadjuvant chemotherapy in breast cancer: A systematic review and meta-analysis. PLoS One 2014;9:1–21.
25. Dieci M V., Criscitiello C, Goubar A, Viale G, Conte P, Guarneri V, et al. Prognostic value of tumor-infiltrating lymphocytes on residual disease after primary chemotherapy for triple-negative breast cancer: A retrospective multicenter study. Ann Oncol 2014;25:611–618.
26. Mattarollo SR, Loi S, Duret H, Ma Y, Zitvogel L, Smyth MJ. Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of established tumors. Cancer Res 2011; 71:4809–4820.
27. Sistigu A, Yamazaki T, Vacchelli E, Chaba K, Enot DP, Adam J, et al. Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy. Nat Med 2014;20:1301–1309.
28. Liu M, Guo S, Stiles JK. The emerging role of CXCL10 in cancer (Review). Oncol Lett 2011;2:583–589.
29. Hu J, Zhu S, Xia X, Zhang L, Kleinerman ES, Li S. CD8+T cell-specific induction of NKG2D receptor by doxorubicin plus interleukin-12 and its contribution to CD8+T cell accumulation in tumors. Mol Cancer 2014;13:1–13.
30. Wang YJ, Fletcher R, Yu J, Zhang L. Immunogenic effects of chemotherapy-induced tumor cell death. Genes Dis 2018;5:194–203.
31. Zhang Z, Yu X, Wang Z, Wu P, Huang J. Anthracyclines potentiate antitumor immunity: A new opportunity for chemoimmunotherapy. Cancer Lett 2015;369:331–335.
32. Galluzzi L, Senovilla L, Zitvogel L, Kroemer G. The secret ally: Immunostimulation by anticancer drugs. Nat Rev Drug Discov 2012;11:215–233.
33. Miyashita M, Sasano H, Tamaki K, Hirakawa H, Takahashi Y, Nakagawa S, et al. Prognostic significance of tumor-infiltrating CD8+ and FOXP3+ lymphocytes in residual tumors and alterations in these parameters after neoadjuvant chemotherapy in triple-negative breast cancer: A retrospective multicenter study. Breast Cancer Res 2015;17:1–13.
34. Alizadeh D, Trad M, Hanke NT, Larmonier CB, Janikashvili N, Bernard B, et al. Doxorubicin eliminates myeloid-derived suppressor cells and enhances the efficacy of adoptive T cell transfer in breast cancer. Cancer Res 2014;74:104–118.
35. De Sousa Linhares A, Leitner J, Grabmeier-Pfistershammer K, Steinberger P. Not all immune checkpoints are created equal. Front Immunol 2018;9:1–15.
36. Sakuishi K, Apetoh L, Sullivan JM, Blazar BR, Kuchroo VK, Anderson AC. Targeting tim-3 and PD-1 pathways to reverse T cell exhaustion and restore antitumor immunity. J Exp Med 2010;207:2187–2194.
37. Demaria S, Volm MD, Shapiro RL, Yee HT, Oratz R, Formenti SC, et al. Development of tumor-infiltrating lymphocytes in breast cancer after neoadjuvant paclitaxel chemotherapy. Clin Cancer Res. 2001;7:3025–3030.
38. Kotsakis A, Sarra E, Peraki M, Koukourakis M, Apostolaki S, Souglakos J, et al. Docetaxel-induced lymphopenia in patients with solid tumors: A prospective phenotypic analysis. Cancer 2000;89:1380–1386.
39. Zhang L, Dermawan K, Jin M, Liu R, Zheng H, Xu L, et al. Differential impairment of regulatory T cells rather than effector T cells by paclitaxel-based chemotherapy. Clin Immunol 2008;129:219–229.
40. Zhou L, Xu Q, Huang L, Jin J, Zuo X, Zhang Q, et al. Low-dose carboplatin reprograms tumor immune microenvironment through STING signaling pathway and synergizes with PD-1 inhibitors in lung cancer. Cancer Lett 2021;500:163–171.
41. Lesterhuis WJ, Punt CJ a, Hato S V, Eleveld-trancikova D, Jansen BJH, Nierkens S, et al. Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice. J Clin Invest 2011;121:3100-3108.
42. Rojkó L, Reiniger L, Téglási V, Fábián K, Pipek O, Vágvölgyi A, et al. Chemotherapy treatment is associated with altered PD-L1 expression in lung cancer patients. J Cancer Res Clin Oncol 2018;144:1219–1226.
43. Heinhuis KM, Ros W, Kok M, Steeghs N, Beijnen JH, Schellens JHM. Enhancing antitumor response by combining immune checkpoint inhibitors with chemotherapy in solid tumors. Ann Oncol [Internet] 2019;30:219–35.
44. Wang X, Teng F, Kong L, Yu J. PD-L1 expression in human cancers and its association with clinical outcomes. Onco Targets The 2016;9:5023-5039.
45. Ock C-Y, Kim S, Keam B, Kim S, Ahn Y-O, Chung E-J, et al. Changes in programmed death-ligand 1 expression during cisplatin treatment in patients with head and neck squamous cell carcinoma. Oncotarget 2017; 8:97920-97927.
46. Homma Y, Taniguchi K, Nakazawa M, Matsuyama R, Mori R, Takeda K, et al. Changes in the immune cell population and cell proliferation in peripheral blood after gemcitabine-based chemotherapy for pancreatic cancer. Clin Transl Oncol 2014;16:330–335.
47. Vincent J, Mignot G, Chalmin F, Ladoire S, Bruchard M, Chevriaux A, et al. 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced t cell-dependent antitumor immunity. Cancer Res 2010; 70:3052-3061.
48. Wang W, Wu L, Zhang J, Wu H, Han E, Guo Q. Chemoimmunotherapy by combining oxaliplatin with immune checkpoint blockades reduced tumor burden in colorectal cancer animal model. Biochem Biophys Res Commun 2017; 487:1-7.
49. Shujin A, Corresponding C, Cui S. Immunogenic chemotherapy sensitizes renal cancer to immune checkpoint blockade therapy in preclinical models. Med Sci Monit 2017;23:3360–3366.
50. Bespalov A, Michel MC, Steckler T. Good research practice in non-clinical pharmacology and biomedicine. Springer Nature; 2020.
51. Xing K, Gu B, Zhang P, Wu X. Dexamethasone enhances programmed cell death 1 (PD-1) expression during T cell activation: An insight into the optimum application of glucocorticoids in anti-cancer therapy. BMC Immunol 2015;16:1–9.
52. Geng H, Zhang G-M, Li D, Zhang H, Yuan Y, Zhu H-G, et al. Soluble form of T cell Ig mucin 3 is an inhibitory molecule in T cell-mediated immune response. J Immunol 2006;176:1411–1420.
53. Stojanovic A, Fiegler N, Brunner-Weinzierl M, Cerwenka A. CTLA-4 Is expressed by activated mouse NK cells and inhibits NK cell IFN-  production in response to mature dendritic cells. J Immunol 2014;192:4184–4191.