Synergistic Effects of Nanoscale CaO2 Combined with PD-1 Inhibitors in the Treatment of Hepatocellular Carcinoma: A Promising Combination

Purpose: To explore the effect of calcium peroxide nanoparticles (CaO 2 NPs) combined with programmed cell death protein 1 (PD-1) inhibitors in the treatment of liver cancer and its related mechanism. Methods: Hepa1-6 cells were cultured to construct the Hepa1-6 mouse liver cancer model. In vivo mechanism study, a unilateral tumor model was established. Eighteen tumor-bearing mice were randomly divided into the control group (intra-tumoral injection of PBS solution) and the experimental group (intra-tumoral injection of CaO 2 NPs). A hypoxic probe, pH probe, and micro-CT were used to evaluate the effect of CaO 2 NPs on improving hypoxia, neutralizing acidity, and inducing calcium overload within the tumor. To study the effect of CaO 2 NPs combined with PD-1 inhibitors on proximal and distal tumors, the bilateral tumor model was established. Forty tumor-bearing mice were randomly divided into the control group (intra-tumoral/intra-peritoneal injection of PBS solution), CaO 2 NPs group (intra-tumoral injection of CaO 2 NPs), PD-1 group (intra-peritoneal injection of PD-1 inhibitor), and the combination group (intra-tumoral injection of CaO 2 NPs and intra-peritoneal injection of PD-1 inhibitors). The administered side was recorded as the proximal tumor. Tumor volume and body weight were measured every 2 days after treatment. On day 8, serum and tumor samples were collected. The immune factors in serum (Interferon-γ (IFN-γ), Tumour necrosis factor-α (TNF-α), Interleukin-2 (IL-2), and Interleukin-10 (IL-10)) and tumor tissue (IFN-γ and TNF-α) were detected by ELISA. H&E staining was used to detect tumor necrosis. Immunohistochemical staining was used to detect the amount of CD4 + and CD8 + T cells within the tumor. By analyzing the tumor volume, pathological indexes, and immune-related indexes, the effects of CaO 2 NPs combined with PD-1 inhibitors on proximal and distal tumors were evaluated, and they mediated immunomodulatory effects (including local and systemic immunity), and their effects on tumor burden were studied. In addition, a unilateral tumor model was established to study the effect of CaO 2 NPs combined with PD-1 inhibitors on survival time. Results: The results of in vivo mechanism study showed that CaO 2 NPs can improve hypoxia, neutralize acidity, and induce calcium overload within tumors. The results of the study on the effect of CaO 2 NPs combined with PD-1 inhibitor on proximal and distal tumors showed that, compared with the other three groups, the bilateral tumor burden of the combination group was significantly reduced, the intra-tumoral infiltration of CD8 + and CD4 + T cells were significantly increased, the secretion of anti-tumor immune factors in tumor and serum was increased, and the secretion of pro-tumor immune factors was decreased. Mice in the combination group showed the longest survival compared with the other groups. Conclusion: CaO 2 NPs can improve hypoxia, neutralize acidity, and induce calcium overload within tumors, so as to reduce tumor burden and realize an immunosuppressive tumor transformation to a hot tumor, and play a synergistic role with PD-1 inhibitors in anti-liver cancer. Keywords: calcium peroxide nanoparticles, programmed cell death protein 1 inhibitor, Hepa1-6 model, tumor microenvironment, liver cancer