IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8+ T, and CD4+ T Cells

Abstract

Huan-Huan Shen,1,2,* Jie-Fei Peng,1,3,* Ran-Ran Wang,4,* Ping-Yu Wang,1,5 Jia-Xiang Zhang,1 Hong-Fang Sun,1 Yan Liang,1 Yan-Mei Li,6 Jiang-Nan Xue,7 You-Jie Li,1 Guang-Bin Sun,1,* Shu-Yang Xie1,2,* 1Department of Biochemistry and Molecular Biology, Binzhou Medical University, YanTai, Shandong, 264003, People’s Republic of China; 2Shandong Laboratory of Advanced Materials and Green Manufacturing, Yantai, Shandong, 264000, People’s Republic of China; 3Department of Clinical Laboratory, the Affiliated Taian City Central Hospital of Qingdao University, Taian, 271000, People’s Republic of China; 4Institute of Rehabilitation Medicine, School of Rehabilitation Medicine, Binzhou Medical University, Yantai, Shandong, 264003, People’s Republic of China; 5Department of Epidemiology, Binzhou Medical University, YanTai, ShanDong, 264003, People’s Republic of China; 6Department of Immune Rheumatism, Yantaishan Hospital, Yantai, Shandong, 264000, People’s Republic of China; 7Department of Immunology, Binzhou Medical University, Yantai, Shandong, 264003, People’s Republic of China*These authors contributed equally to this workCorrespondence: Shu-Yang Xie; Guang-Bin Sun, Department of Biochemistry and Molecular Biology, Binzhou Medical University, YanTai, Shandong, 264003, People’s Republic of China, Tel [phone], Fax [phone], Email [email protected]; [email protected]: The drug resistance and low response rates of immunotherapy limit its application. This study aimed to construct a new nanoparticle (CaCO3-polydopamine-polyethylenimine, CPP) to effectively deliver interleukin-12 (IL-12) and suppress cancer progress through immunotherapy.Methods: The size distribution of CPP and its zeta potential were measured using a Malvern Zetasizer Nano-ZS90. The morphology and electrophoresis tentative delay of CPP were analyzed using a JEM-1400 transmission electron microscope and an ultraviolet spectrophotometer, respectively. Cell proliferation was analyzed by MTT assay. Proteins were analyzed by Western blot. IL-12 and HMGB1 levels were estimated by ELISA kits. Live/dead staining assay was performed using a Calcein-AM/PI kit. ATP production was detected using an ATP assay kit. The xenografts in vivo were estimated in C57BL/6 mice. The levels of CD80+/CD86+, CD3+/CD4+ and CD3+/CD8+ were analyzed by flow cytometry.Results: CPP could effectively express EGFP or IL-12 and increase ROS levels. Laser treatment promoted CPP-IL-12 induced the number of dead or apoptotic cell. CPP-IL-12 and laser could further enhance CALR levels and extracellular HMGB1 levels and decrease intracellular HMGB1 and ATP levels, indicating that it may induce immunogenic cell death (ICD). The tumors and weights of xenografts in CPP-IL-12 or laser-treated mice were significantly reduced than in controls. The IL-12 expression, the CD80+/CD86+ expression of DC from lymph glands, and the number of CD3+/CD8+T or CD3+/CD4+T cells from the spleen increased in CPP-IL-12-treated or laser-treated xenografts compared with controls. The levels of granzyme B, IFN-γ, and TNF-α in the serum of CPP-IL-12-treated mice increased. Interestingly, CPP-IL-12 treatment in local xenografts in the back of mice could effectively inhibit the growth of the distant untreated tumor.Conclusion: The novel CPP-IL-12 could overexpress IL-12 in melanoma cells and achieve immunotherapy to melanoma through inducing ICD, activating CD4+ T cell, and enhancing the function of tumor-reactive CD8+ T cells.Keywords: cancer immunotherapy, CaCO3 nanoparticle, interleukin-12, immunogenic cell death, tumor therapy

Keywords

• cancer immunotherapy
• caco3 nanoparticle
• interleukin-12
• immunogenic cell death
• tumor therapy