Xiuli Zhang,1,2,* Nan Liu,1,2,* Mingjing Wei,1,2 Chuanlai Yang,1,2 Yanhua Lin,1,2 Yarong Zeng,1,2 Yufang Li,1,2 Lizhi Zhou,1,2 Tingting Li,1,2 Qingbing Zheng,1,2 Hai Yu,1,2 Jun Zhang,1,2 Ying Gu,1,2 Ningshao Xia,1,2 Shaowei Li1,2 

1State Key Laboratory of Vaccines for Infectious Diseases, XiangAn Biomedicine Laboratory, School of Public Health, Xiamen University, Xiamen, People’s Republic of China; 2National Institute of Diagnostics and Vaccine Development in Infectious Diseases, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Collaborative Innovation Center of Biologic Products, National Innovation Platform for Industry-Education Integration in Vaccine Research, The Research Unit of Frontier Technology of Structural Vaccinology of Chinese Academy of Medical Sciences, Xiamen, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Shaowei Li, Email shaowei@xmu.edu.cn Ying Gu, Email guying@xmu.edu.cn

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.

Keywords: breast cancer, tumor infiltrating lymphocyte, tumor microenvironment, immunotherapy, immune checkpoint inhibitor