Multifunctional Magnetic Nanoclusters Induce Immunogenic Cell Death and Suppress Tumor Recurrence and Metastasis
Introduction
Immune checkpoint blockade (ICB) has been approved for treating late-stage cancers. However, the overall response rate of solid tumors is relatively low. A large proportion of patients cannot benefit from ICB due to various reasons such as low immunogenicity of the tumors. It has been shown that ICB can be improved by combining it with other cancer treatments that trigger immunogenic cell death (ICD). Among these methods, local magnetic heating provides a promising approach due to minimal invasiveness and low side effect. In this work, we developed a combination therapeutic approach, magneto-immunotherapy, combining magnetic heating, free radical generation, and ICB for cancer treatment. We demonstrated that the simultaneous generation of heat and free radicals induced immunogenic cell death and efficiently eradicated the primary tumors. When it is further combined with ICB, the resulting magneto-immunotherapy led to efficient abscopal effect and immune memory, thus demonstrating the potential in improving ICB and suppressing tumor recurrence and metastasis.
Methods
The iron oxide nanocrystal clusters (IONCs) were synthesized through the hydrolysis of iron salts in a solvothermal reaction. The IONCs were loaded with an azo compound 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) through a polymer-mediated chemical reaction. The formation of the nanostructure, IONC-AAPH, was confirmed using Fourier transform infrared (FTIR) spectroscopy. The generation of intracellular free radicals by IONC-AAPH was determined in MC-38 cells using H2DCFDA probe. The cell killing activity of IONC-AAPH was examined using fluorescence imaging and flow cytometry. The damage-associated molecular patterns (DAMPs) were analyzed at different time points after treating cells with IONC-AAPH. The anti-tumor activities, including debulking of primary tumor, abscopal effect, and immune memory effect, were determined in vivo in C57BL/6 mice with subcutaneous MC-38 tumors.
Results
The IONC was ~40 nm in diameter and composed of ~300 primary magnetic iron oxide nanocrystals (MIONs) with a diameter of ~5 nm. The heating efficiency of IONCs was 40 folds higher than the primary MIONs. The FTIR spectra confirmed the successful loading of AAPH to IONCs. Treating MC-38 cells with IONC-AAPH under an alternating magnetic field (AMF) led to a large amount of intracellular free radicals. The cells were efficiently killed by IONC-AAPH under AMF under both normoxia and hypoxia. High levels of DAMPs, including calreticulin, heat shock protein 70, and adenosine triphosphate were detected after the treatment, indicating IONC-AAPH caused immunogenic cell death. Co-incubation of the treated cells with dendritic cells (DCs) induced DC maturation. Treating the subcutaneous MC-38 tumors with IONC-AAPH under AMF reduced the tumor volume by 88.2% compared with the saline control. Using a bilateral subcutaneous tumor model, we further found combining IONC-AAPH under AMF with anti-PD-1 significantly inhibited the growth of untreated distant tumors by inducing tumor-specific T cell response and increasing tumor-infiltrating CD8+ T cells. In addition, the cured mice completely rejected tumor rechallenge, suggesting the development of anti-tumor immune memory.
Conclusion
In this study, we developed a magneto-immunotherapy for solid tumors by combining local heat and free radical generation with ICB therapy. The simultaneous generation of heat and free radicals from IONC-AAPH effectively killed tumor cells and caused the exposure and release of DAMPs. Treating primary tumors with IONC-AAPH led to removal of the tumors. Further combining IONC-AAPH with anti-PD-1 ICB dramatically inhibited the growth of untreated distant tumors. In addition, this magneto-immunotherapy also induced long-term immune memory against tumor rechallenge. Hence, the IONC-AAPH-based magneto-immunotherapy has the potential to effectively control cancer recurrence and combat cancer metastasis.