Synergistic Induction of Immunogenic Cell Death by Biomineralized Manganese and Bisphosphonates Enhances Anti-PD-L1 Therapy in Triple-Negative Breast Cancer.

Background: Despite therapeutic benefits of anti-Programmed Death-Ligand 1 (PD-L1) therapy in triple-negative breast cancer (TNBC), low response rates and resistance limit its efficacy. Both manganese (Mn) and bisphosphonates (BPs) are known to induce immunogenic cell death (ICD). Strategies to synergistically enhance ICD induction and elucidate the underlying molecular mechanisms remain to be fully explored.

Methods: We analyzed the mode of apoptosis and immunogenicity of cancer cells post-treatment using Western blotting, flow cytometry, and confocal microscopy. RNA sequencing was employed to identify activated apoptotic pathways and elucidate the molecular mechanisms underlying ICD when Mn²⁺ and BPs act synergistically. In 4T1 tumor models, we evaluated the synergistic anti-tumor effect of Mn²⁺ and BPs with anti-PD-L1 antibodies.

Results: By leveraging the doping capacity of hydroxyapatite (HA) for Mn²⁺ and its high affinity for BPs, we developed MnHARis particles-a biocompatible slow-release system of biomineralized Mn²⁺ and risedronate (Ris). Compared to Mn2+ and Ris alone, MnHARis achieved a synergistic antitumor effect, manifesting as increased cytotoxicity (IC50 reduced by 17 times) and the emergence of more significant mitochondrial autophagic apoptosis (more pronounced nuclear fragmentation, increased ROS levels, significantly decreased ATP levels, depolarization of mitochondrial membrane potential, upregulation of autophagy markers (LC3B and Beclin), and obvious autophagosomes). MnHARis exerts its antitumor effects via the p38-MAPK pathway. Additionally, increased exposure of calreticulin and increased secretion of high mobility group box 1 indicated that MnHARis successfully induced ICD and promoted specific recognition and cross-presentation of damage-associated molecular patterns released by apoptotic tumor cells by activating dendritic cells and pattern recognition receptors, thereby altering TME of TNBC, increasing TILs, and sensitizing TNBC to anti-PD-L1 therapy.

Conclusion: MnHARis effectively synergizes Mn²⁺ and Ris to promote autophagic apoptosis and ICD, increasing TILs and sensitizing TNBC to anti-PD-L1 therapy, thus offering a new therapeutic strategy.