Combined immunotherapy of metastatic carcinoma by resection of the primary tumor and subsequent reprogramming of macrophages and dendritic cells using a TLR4 agonist in laboratory mice

• Ekaterina I. Ushakova
• Ekaterina S. Lebedeva
• Alexander V. Bagaev
• Aleksey V. Pichugin
• Ravshan I. Ataullakhanov

Abstract

Introduction. The successes of the last decade in the development of new methods of treating cancer patients are associated with the use of inhibitory receptor blockers and their ligands (PD-1, PD-L1, CTLA4); agonists of activation receptors of immune cells; bi- and trispecific ligands that contribute to the antitumor effect of cells of the immune system; as well as CAR-T, CAR-NK, TIL cells grown ex vivo for cell therapy. Despite the fact that many of the aforementioned variants of immune therapy for cancer patients have already been approved for use or are in the final stages of clinical trials, the achieved treatment efficacy is still far from the desired level. Consequently, the development of new methods of immune therapy for cancer patients is urgent.

The aim of the investigation was to study the effectiveness of immunotherapy of a solid metastatic tumor using a TLR4 agonist for reprogramming myeloid cells in the tumor microenvironment from protumor state to antitumor one.

Material and methods. Experimental model of solid metastatic carcinoma 4T1 in BALB/c mice was used. 4T1 carcinoma cells were inoculated subcutaneously, after which solid tumors grew in 100 % of cases. All tumor-bearing animals died within 30-35 days from multiple metastases to the lymph nodes, spleen, lungs, liver and other organs. In this experimental model, we performed surgical removal of the primary tumor at its 2-3 mm size (11^th day after inoculation of 15 thousand 4T1 cells). The next day after resection, immunotherapy against metastatic disease was started. The TLR4-agonist i.e. the acidic peptidoglycan (hereinafter APG) from the plant source that is an active component of a pharmaceutical drug Immunomax was used for immunotherapy. The drug was injected intraperitoneally every 4-5 days, in total - 7 injections per course of treatment. Groups of mice treated with a combination of surgical resection and immunotherapy were compared with groups of mice treated using either surgical resection of the primary tumor alone or immunotherapy with TLR4-agonist alone. The rate of tumor growth, the survival time of the animals, the frequency of relapses after resection, and the number of metastatic CFUs in the lungs were investigated.

Results of our experiments showed that surgical resection of the primary tumor does not save animals from death, only postpones the moment of death by about 30 days. Monotherapy with TLR4-agonist (without surgical resection of the primary tumor) also proved to be ineffective. The growth rate of tumors and the lifespan of the animals treated with APG only did not change. In contrast, the combination of surgical treatment and immunotherapy has shown high therapeutic efficacy in 4T1 carcinoma. Resection of the primary tumor followed by a course of immunotherapy with injections of a TLR4-agonist allows a complete elimination of the malignant neoplasm in 20 % and a significant prolongation of the life span of the remaining 80 % animals.

Discussion contains an analysis of the possible mechanisms of the TLR4-agonist therapeutic action, based on the reprogramming of macrophages and dendritic cells to their anti-tumor state. Experimental data are presented that prove the feasibility and effectiveness of the use of a TLR4 agonist, capable of this reprogramming, in the complex treatment of animals with absolutely lethal metastatic carcinoma.

Conclusions. 1. The TLR4 agonist (APG) stand-alone use for the treatment of metastatic carcinoma does not significantly affect the growth rate of the primary tumor, the process of metastasis and the survival of tumor-bearing mice.

2. After surgical resection of the primary carcinoma, the TLR4 agonist (APG) use is highly effective for treatment of the lethal metastatic disease.

Keywords:4T1 breast carcinoma; TLR4-agonist; Immunomax; immunotherapy

References

1. Zhou J., Dudley M.E., Rosenberg S.A., Robbins P.F. Selective Growth. In vitro and in vivo, of individual T cell clones from tumor-infiltrating lymphocytes obtained from patients with melanoma. J Immunol. 2004; 173 (12): 7622–9. PMID: 15585890. DOI: http://dx.doi.org/10.4049/jimmunol.173.12.7622

2. Wang M., Shu X., Li M., Zhang Y., Yao Y., Huang X., Wei P., He Z., Lu J., Ying Y.A. Novel strategy conjugating PD-L1 polypeptide with doxorubicin alleviates chemotherapeutic resistance and enhances immune response in colon cancer. Res Sq. 2021; 1–19. DOI: http://dx.doi.org/10.21203/rs.3.rs-420385/v1

3. Ostrand-Rosenberg S., Horn L.A., Alvarez J.A. Novel strategies for inhibiting PD-1 pathway-mediated immune suppression while simultaneously delivering activating signals to tumor-reactive T cells. Cancer Immunol Immunother. 2016; 176: 139–48. PMID: 25792524. DOI: http://dx.doi.org/10.1007/s00262-015-1677-5

4. O’Day S.J., Hamid O., Urba W.J. Targeting cytotoxic T-lymphocyte antigen-4 (CTLA-4): a novel strategy for the treatment of melanoma and other malignancies. Cancer. 2007; 110: 2614–27. PMID: 18000991. DOI: http://dx.doi.org/10.1002/cncr.23086

5. Mullins S.R., Vasilakos J.P., Deschler K., Grigsby I., Gillis P., John J., Elder M.J., Swales J., Timosenko E., Cooper Z., Dovedi S.J., Leishman A.J., Luheshi N., Elvecrog J., Tilahun A., Goodwin R., Herbst R., Tomai M.A., Wilkinson R.W. Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies. J Immunother Cancer. 2018; 8: 711. DOI: http://dx.doi.org/10.1158/1538-7445.am2018-711

6. Miliotou A.N., Papadopoulou L.C. CAR T-cell therapy: a new era in cancer immunotherapy. Curr Pharm Biotechnol. 2018; 19: 5–18. PMID: 29667553. DOI: http://dx.doi.org/10.2174/1389201019666180418095526

7. Gellrich F., Schmitz M., Beissert S., Meier F. Anti-PD-1 and Novel Combinations in the Treatment of Melanoma – An Update. J Clin Med. 2020; 9 (1): 223. PMID: 31947592. DOI: http://dx.doi.org/10.3390/jcm9010223

8. Bhatia S., Miller N.J., Lu H., Longino N.V., Ibrani D., Shinohara M.M., Byrd D.R., Parvathaneni U., Kulikauskas R., Ter Meulen J., Hsu F.J., Koelle D.M., Nghiem P. Intratumoral G100, a TLR4 agonist, induces antitumor immune responses and tumor regression in patients with merkel cell carcinoma. Clin Cancer Res. 2020; 25 (4): 1185–95. DOI: http://dx.doi.org/10.1158/1078-0432.CCR-18-0469.Intratumoral

9. Wei R., Liu S., Zhang S., Min L., Zhu S. Cellular and extracellular components in tumor microenvironment and their application in early diagnosis of cancers. Anal Cell Pathol. 2020; 2020: 6283796. DOI: http://dx.doi.org/]10.1155/2020/6283796

10. Kaufman H.L., Wolchok J.D. General Principles of Tumor Immunotherapy. Basic and Clinical Applications of Tumor Immunology. Springer Netherlands. 2008, 503 p. ISBN 978-1-4020-6086-1. DOI: http://dx.doi.org/10.1007/978-1-4020-6087-8

11. Ataullakhanov R.I., Pichugin A.V., Mel’nikova T.M., Khaitov R.M. A method of producing a substance with immunostimulating, antiviral and, antimicrobial activity, a substance produced by this method and its pharmaceutical compositions. Russian Patent Application RU 2195308, priority date: November 11, 2001. (in Russian)

12. Ghochikyan A., Pichugin A., Bagaev A., Davtyan A., Hovakimyan A., Tukhvatulin A., Davtyan H., Shcheblyakov D., Logunov D., Chulkina M., Savilova A., Trofimov D., Nelson E.L., Agadjanyan M.G., Ataullakhanov R.I. Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax, as a therapeutic strategy for metastatic breast cancer. J Translational Medicine. 2014; 12 (1): 322. PMID: 25432242. DOI: http://dx.doi.org/10.1186/s12967-014-0322-y

13. Ataullakhanov R.I., Pichugin A.V., Shishkova N.M., Masternak T.B., Malkina E.Yu., Ulyanova L.I., Stetsenko O.N. Cellular mechanisms of immunomodulating action of «Immunomax». Immunologiya. 2005; 2: 111–20. (in Russian)

14. Bagaev A., Pichugin A., Nelson E.L., Agadjanyan M.G., Ghochikyan A., Ataullakhanov R.I. Anti-cancer mechanisms in two murine bone marrow-derived DC subsets activated with Toll-like receptor 4 agonists. J Immunol. 2019; 200 (8): 2656–69. PMID: 29500244. DOI: http://dx.doi.org/10.4049/jimmunol.1701126.Anti-cancer

15. Lebedeva E.S., Bagaev A.V., Garaeva A.Y., Chulkina M.M., Pichugin A.V., Ataullakhanov R.I. The cooperative interaction of TLR4-, TLR9- and NOD2-signaling pathways in mouse macrophages. Immunologiya. 2018; 39 (1): 4–11. DOI: http://dx.doi.org/10.18821/0206-4952-2018-39-1-4-11 (in Russian)

16. Lebedeva E., Bagaev A., Pichugin A., Chulkina M., Lysenko A., Tutykhina I., Shmarov M., Logunov D., Naroditsky B., Ataullakhanov R. The differences in immunoadjuvant mechanisms of TLR3 and TLR4 agonists on the level of antigen-presenting cells during immunization with recombinant adenovirus vector. BMC immunology. 2018; 19 (1): 26. PMID: 30055563. DOI: http://dx.doi.org/10.1186/s12865-018-0264-x

17. Nikonova A., Pichugin A., Chulkina M., Lebedeva E., Gaisina A., Shilovskiy I., Ataullakhanov R., Khaitov M., Khaitov R. The TLR4 Agonist Immunomax Affects the Phenotype of Mouse Lung Macrophages during Respiratory Syncytial Virus Infection. Acta Naturae. 2018; 10 (4): 95–9. PMID: 30713767. (in Russian)

18. Lebedeva E.S., Bagaev A.V., Chulkina M.M., Pichugin A.V., Ataullakhanov R.I. Synergistic activation of gene transcription encoding type I interferons and cytokines in macrophages and dendritic cells by the combinations of two PRR-agonists. Immunologiya. 2017; 38 (1): 64–71. DOI: http://dx.doi.org/10.18821/0206-4952-2017-38-1-64-71 (in Russian)

19. Pichugin A.V., Bagaev A.V., Lebedeva E.S., Chulkina M.M., Ataullakhanov R.I. Synergistic cytokine production by murine dendritic cells in response to their simultaneous activation with pairs of agonists of different innate immune receptors. Immunologiya. 2017; 38 (1): 118–23. DOI: http://dx.doi.org/10.18821/0206-4952-2017-38-2-118-123 (in Russian)

20. Lebedeva E.S., Bagaev A.V., Chulkina M.M., Pichugin A.V., Ataullakhanov R.I. NF-kB-, but not MAPK-signaling pathway determines synergistic response of macrophages to the simultaneous activation of two types receptors TLR4 + NOD2 or TLR9 + NOD2. Immunologiya. 2017; 38 (2): 76–82. DOI: http://dx.doi.org/10.18821/0206-4952-2017-38-2-76-82 (in Russian)

21. Lebedeva E.S., Bagaev A.V., Chulkina M.M., Pichugin A.V., Lysenko A.A., Shmarov M.M., Logunov D.Yu., Naroditsky B.S., Ataullakhanov R.I. Mechanisms of TLR4 agonists enhance the expression of target protein in the composition of the adenoviral vector into antigen presenting cells. Immunologiya. 2017; 38 (6): 295–306. DOI: http://dx.doi.org/10.18821/0206-4952-2017-38-6-295-306 (in Russian)

22. Bagaev A.V., Pichugin A.V., Lebedeva E.S., Lysenko A.A., Shmarov M.M., Logunov D.Yu., Naroditsky B.S., Ataullakhanov R.I., Khaitov R.M., Gintsburg A.L. Influence of TLR-agonists on expression by antigen-presenting cells of the target protein antigen encoded in adenoviral vector. Immunologiya. 2015; 36 (4): 188–95. (in Russian)

23. Demicheli R., Abbattista A., Miceli R., Valagussa P., Bonadonna G. Time distribution of the recurrence risk for breast cancer patients undergoing mastectomy: Further support about the concept of tumor dormancy. Breast Cancer Res Treat. 1996; 41 (2): 177–85. PMID: 8944336. DOI: http://dx.doi.org/10.1007/BF01807163

24. Saphner T., Tormey D.C., Gray R. Annual hazard rates of recurrence for breast cancer after primary therapy. J Clin Oncol. 1996; 14 (10): 2738–46. PMID: 8874335. DOI: http://dx.doi.org/10.1200/JCO.1996.14.10.2738

25. Karrison T.G., Ferguson D.J., Meier P. Dormancy of mammary carcinoma after mastectomy. J Natl Cancer Inst. 1999; 91 (1): 80–5. PMID: 9890174. DOI: http://dx.doi.org/10.1093/jnci/91.1.80

26. Pichugin A.V., Bagaev A.V., Chulkina M.M., Berzhitskaya D.A., Shishkova N.M., Ataullakhanov R.I. Immunomodulator «Immunomax» activates dendritic cells. Immunologiya. 2015; 36 (4): 200–5. (in Russian)

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