Enhancing antitumor immunity through the combination of cholesterolized TLR7 agonist liposomes and radiotherapy: a role for IL-1β and the inflammasome pathway

Abstract

Background: Radiotherapy (RT) is a key treatment modality in cancer therapy, utilizing high-energy radiation to directly kill tumor cells. Recent research has increasingly highlighted RT's potential to indirectly enhance antitumor immunity. However, this immune activation alone often fails to generate sustained systemic antitumor responses. In this study, we aimed to investigate the antitumor effects of combining cholesterolized toll-like receptor 7 (TLR7) agonist liposomes, specifically 1V209-Cho-Lip, with RT.

Methods: Mouse tumor models were used to assess the impact of combining 1V209-Cho-Lip with RT on tumor progression and modification of the tumor microenvironment. In vitro, primary mouse bone marrow-derived dendritic cells (BMDCs) were utilized to investigate changes in function and the activated pathways through RNA sequencing. Additionally, we explored the role of oxidized mitochondrial DNA (ox-mtDNA) released from irradiated tumor cells as a damage-associated molecular pattern in modulating immune responses. The involvement of interleukin-1β (IL-1β) and the inflammasome pathway in the antitumor efficacy of the combined treatment was evaluated using Il-1β^−/− and cysteinyl aspartate specific proteinase 1 knockout (Casp1^−/−) mouse models.

Results: The combination of 1V209-Cho-Lip and RT significantly inhibited tumor growth and induced antitumor immunity in tumor models. This combination therapy enhanced maturation, antigen presentation and IL-1β secretion of dendritic cells (DCs) in vitro. Ox-mtDNA released from irradiated tumor cells synergized with 1V209-Cho-Lip to activate the inflammasome pathway in DCs. The antitumor effect of the combined therapy was significantly reduced in Il-1β^−/− and Casp1^−/− mice.

Conclusions: This study suggests that the combination of 1V209-Cho-Lip with RT might be a promising antitumor strategy and further studies are warranted to explore the clinical relevance of this combination therapy.

Keywords

cholesterolized liposome / combination therapy / IL-1β / immunotherapy / inflammasome / oxidized mitochondrial DNA / radiotherapy / TLR7 agonist

References

[1] Thariat J, Hannoun-Levi JM, Sun Myint A, Vuong T, Gérard JP. Past, present, and future of radiotherapy for the benefit of patients. Nat Rev Clin Oncol. 2013; 10(1): 52-60.

[2] Kono H, Rock KL. How dying cells alert the immune system to danger. Nat Rev Immunol. 2008; 8(4): 279-89.

[3] Golden EB, Apetoh L. Radiotherapy and immunogenic cell death. Semin Radiat Oncol. 2015; 25(1): 11-7.

[4] Wu Y, Yi M, Niu M, Zhou B, Mei Q, Wu K. Beyond success: unveiling the hidden potential of radiotherapy and immunotherapy in solid tumors. Cancer Commun (Lond). 2024; 44(7): 739-60.

[5] Demaria S, Ng B, Devitt ML, Babb JS, Kawashima N, Liebes L, et al. Ionizing radiation inhibition of distant untreated tumors (abscopal effect) is immune mediated. Int J Radiat Oncol Biol Phys. 2004; 58(3): 862-70.

[6] Dovedi SJ, Melis MH, Wilkinson RW, Adlard AL, Stratford IJ, Honeychurch J, et al. Systemic delivery of a TLR7 agonist in combination with radiation primes durable antitumor immune responses in mouse models of lymphoma. Blood. 2013; 121(2): 251-9.

[7] Castro F, Pinto ML, Pereira CL, Serre K, Barbosa MA, Vermaelen K, et al. Chitosan/γ-PGA nanoparticles-based immunotherapy as adjuvant to radiotherapy in breast cancer. Biomaterials. 2020; 257: 120218.

[8] Formenti SC, Rudqvist NP, Golden E, Cooper B, Wennerberg E, Lhuillier C, et al. Radiotherapy induces responses of lung cancer to CTLA-4 blockade. Nat Med. 2018; 24(12): 1845-51.

[9] Voorwerk L, Slagter M, Horlings HM, Sikorska K, van de Vijver KK, de Maaker M, et al. Immune induction strategies in metastatic triple-negative breast cancer to enhance the sensitivity to PD-1 blockade: the TONIC trial. Nat Med. 2019; 25(6): 920-8.

[10] Shao C, Zhang Y, Li H, Chen J, Huang T, Li J, et al. Radiotherapy-resistant prostate cancer cells escape immune checkpoint blockade through the senescence-related ataxia telangiectasia and Rad3-related protein. Cancer Commun (Lond). 2024.

[11] Kawai T, Ikegawa M, Ori D, Akira S. Decoding Toll-like receptors: Recent insights and perspectives in innate immunity. Immunity. 2024; 57(4): 649-73.

[12] Bekeredjian-Ding I, Jego G. Toll-like receptors–sentries in the B-cell response. Immunology. 2009; 128(3): 311-23.

[13] Girart MV, Fuertes MB, Domaica CI, Rossi LE, Zwirner NW. Engagement of TLR3, TLR7, and NKG2D regulate IFN-gamma secretion but not NKG2D-mediated cytotoxicity by human NK cells stimulated with suboptimal doses of IL-12. J Immunol. 2007; 179(6): 3472-9.

[14] Walshaw RC, Honeychurch J, Choudhury A, Illidge TM. Toll-Like Receptor Agonists and Radiation Therapy Combinations: An Untapped Opportunity to Induce Anticancer Immunity and Improve Tumor control. Int J Radiat Oncol Biol Phys. 2020; 108(1): 27-37.

[15] Latz E, Verma A, Visintin A, Gong M, Sirois CM, Klein DC, et al. Ligand-induced conformational changes allosterically activate Toll-like receptor 9. Nat Immunol. 2007; 8(7): 772-9.

[16] Frank MJ, Reagan PM, Bartlett NL, Gordon LI, Friedberg JW, Czerwinski DK, et al. In Situ Vaccination with a TLR9 Agonist and Local Low-Dose Radiation Induces Systemic Responses in Untreated Indolent Lymphoma. Cancer Discov. 2018; 8(10): 1258-69.

[17] Lee J, Chuang TH, Redecke V, She L, Pitha PM, Carson DA, et al. Molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of Toll-like receptor 7. Proc Natl Acad Sci U S A. 2003; 100(11): 6646-51.

[18] Nishiya T, DeFranco AL. Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling properties of the Toll-like receptors. J Biol Chem. 2004; 279(18): 19008-17.

[19] Baenziger S, Heikenwalder M, Johansen P, Schlaepfer E, Hofer U, Miller RC, et al. Triggering TLR7 in mice induces immune activation and lymphoid system disruption, resembling HIV-mediated pathology. Blood. 2009; 113(2): 377-88.

[20] Schön MP, Schön M. TLR7 and TLR8 as targets in cancer therapy. Oncogene. 2008; 27(2): 190-9.

[21] Wan D, Que H, Chen L, Lan T, Hong W, He C, et al. Lymph-Node-Targeted Cholesterolized TLR7 Agonist Liposomes Provoke a Safe and Durable Antitumor Response. Nano Lett. 2021; 21(19): 7960-9.

[22] Zhang Z, Yuan B, Bao M, Lu N, Kim T, Liu YJ. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat Immunol. 2011; 12(10): 959-65.

[23] Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol. 2011; 12(3): 222-30.

[24] Wu X, Xiong E, Wang W, Scali M, Cresti M. Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis. Nat Protoc. 2014; 9(2): 362-74.

[25] Vanpouille-Box C, Alard A, Aryankalayil MJ, Sarfraz Y, Diamond JM, Schneider RJ, et al. DNA exonuclease Trex1 regulates radiotherapy-induced tumour immunogenicity. Nat Commun. 2017; 8: 15618.

[26] Zhang Z, Li X, Wang Y, Wei Y, Wei X. Involvement of inflammasomes in tumor microenvironment and tumor therapies. J Hematol Oncol. 2023; 16(1): 24.

[27] Fucikova J, Kepp O, Kasikova L, Petroni G, Yamazaki T, Liu P, et al. Detection of immunogenic cell death and its relevance for cancer therapy. Cell Death Dis. 2020; 11(11): 1013.

[28] Wei X, Shao B, He Z, Ye T, Luo M, Sang Y, et al. Cationic nanocarriers induce cell necrosis through impairment of Na(+)/K(+)-ATPase and cause subsequent inflammatory response. Cell Res. 2015; 25(2): 237-53.

[29] Srinivas US, Tan BWQ, Vellayappan BA, Jeyasekharan AD. ROS and the DNA damage response in cancer. Redox Biol. 2019; 25: 101084.

[30] Sabharwal SS, Schumacker PT. Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel? Nat Rev Cancer. 2014; 14(11): 709-21.

[31] Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J. 1996; 313(Pt 1): 17-29.

[32] Shimada K, Crother TR, Karlin J, Dagvadorj J, Chiba N, Chen S, et al. Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis. Immunity. 2012; 36(3): 401-14.

[33] Xian H, Liu Y, Rundberg Nilsson A, Gatchalian R, Crother TR, Tourtellotte WG, et al. Metformin inhibition of mitochondrial ATP and DNA synthesis abrogates NLRP3 inflammasome activation and pulmonary inflammation. Immunity. 2021; 54(7): 1463-77.e11.

[34] Martinon F, Mayor A, Tschopp J. The inflammasomes: guardians of the body. Annu Rev Immunol. 2009; 27: 229-65.

[35] Guo H, Callaway JB, Ting JP. Inflammasomes: mechanism of action, role in disease, and therapeutics. Nat Med. 2015; 21(7): 677-87.

[36] Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell. 2010; 140(6): 805-20.

[37] Ye J, Mills BN, Qin SS, Garrett-Larsen J, Murphy JD, Uccello TP, et al. Toll-like receptor 7/8 agonist R848 alters the immune tumor microenvironment and enhances SBRT-induced antitumor efficacy in murine models of pancreatic cancer. J Immunother Cancer. 2022; 10(7): e004784.

[38] Panaretakis T, Kepp O, Brockmeier U, Tesniere A, Bjorklund AC, Chapman DC, et al. Mechanisms of pre-apoptotic calreticulin exposure in immunogenic cell death. Embo j. 2009; 28(5): 578-90.

[39] Lotze MT, Tracey KJ. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol. 2005; 5(4): 331-42.

[40] 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.

[41] Leach JK, Van Tuyle G, Lin PS, Schmidt-Ullrich R, Mikkelsen RB. Ionizing radiation-induced, mitochondria-dependent generation of reactive oxygen/nitrogen. Cancer Res. 2001; 61(10): 3894-901.

[42] Tulard A, Hoffschir F, de Boisferon FH, Luccioni C, Bravard A. Persistent oxidative stress after ionizing radiation is involved in inherited radiosensitivity. Free Radic Biol Med. 2003; 35(1): 68-77.

[43] Fang C, Mo F, Liu L, Du J, Luo M, Men K, et al. Oxidized mitochondrial DNA sensing by STING signaling promotes the antitumor effect of an irradiated immunogenic cancer cell vaccine. Cell Mol Immunol. 2021; 18(9): 2211-23.

[44] Ghiringhelli F, Apetoh L, Tesniere A, Aymeric L, Ma Y, Ortiz C, et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. Nat Med. 2009; 15(10): 1170-8.

[45] Zhong Z, Liang S, Sanchez-Lopez E, He F, Shalapour S, Lin XJ, et al. New mitochondrial DNA synthesis enables NLRP3 inflammasome activation. Nature. 2018; 560(7717): 198-203.

[46] Han C, Godfrey V, Liu Z, Han Y, Liu L, Peng H, et al. The AIM2 and NLRP3 inflammasomes trigger IL-1-mediated antitumor effects during radiation. Sci Immunol. 2021; 6(59): eabc6998.

RIGHTS & PERMISSIONS

2025 The Author(s). Cancer Communications published by John Wiley & Sons Australia, Ltd. on behalf of Sun Yat-sen University Cancer Center.