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

Abstract

Purpose

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

Acknowledgments

We thank professional editing support from ShineWrite.com ([email protected]) for editing the English text of a draft of this manuscript. We created schematic illustration of in vivo study using the Figdraw software.

Disclosure

The authors declare no conflicts of interest in this work.

Additional information

Funding

The present study was supported by the National Natural Science Foundation of China (No.81772281, 31371321), the Shandong Science and Technology Committee (No. ZR2022LSW002, ZR2020KH015), the Education Department of Shandong Province (2019KJK014, 2021KJ101), and the Science Fund of Shandong Laboratory of Advanced Materials and Green Manufacturing (Yantai) (AMGM2023F16).