In situ vaccine (ISV) can activate the anti-tumor immune system by inducing immunogenic cell death (ICD) at the tumor site. However, the development of tumor ISV still faces challenges due to insufficient tumor antigens released by tumor cells and the existence of tumor immunosuppressive microenvironment (TIME). Targeting the STING pathway has been reported to enhance the adjuvant effects of in situ tumor vaccines by initiating innate immunity. Based on this, we developed a potent in situ cancer vaccine, MBMA-RGD ISV, which simultaneously induces ICD and activates the STING pathway to achieve sustained anti-tumor immunity. Specifically, a water-soluble prodrug Mit-ALA was synthesized from the chemotherapeutic agent mitoxantrone (Mit) and the photosensitizer precursor 5-aminolevulinic acid (5-ALA) by pH-responsive ester bonds, which was then loaded into pre-synthesized BSA-MnO2 nanoparticles and functionalized with the targeting Arg-Gly-Asp (RGD) peptide to obtain MBMA-RGD ISV. This ISV actively targets tumor cells by binding integrin receptors and then gradually releases antitumor components in response to tumor microenvironment (TME). The released 5-ALA is metabolized in mitochondria to produce photosensitizer PpIX. Under laser irradiation, the photodynamic property of PpIX coupled with the photothermal effect of Mit synergistically induced ICD, resulting in the release of tumor antigens and evoking adaptive immunity. Meanwhile, released Mn2+ and Mit synergistically activate the STING pathway by inducing DNA damage, further enhancing antitumor immunity. Moreover, large amounts of oxygen released by MnO2 relieved the hypoxia microenvironment, thus sensitizing photodynamic therapy and improving the immunosuppressive state of TME. Therefore, MBMA-RGD ISV efficiently activates systemic antitumor immunity in vitro and in vivo, providing new strategies and ideas for the development of tumor ISV. STATEMENT OF SIGNIFICANCE: Using a biocompatible BSA-MnO2 nanoplatform, we developed a dual-prodrug tumor in situ vaccine (ISV) with tumor microenvironment-responsive action for synergistic cancer immunotherapy. Once internalized by tumor cells, the MBMA-RGD ISV responded to intracellular H+, H2O2, and GSH, releasing its therapeutic "cargo." Under laser irradiation, the combined effects of photodynamic therapy (PDT) and photothermal therapy (PTT) induced immunogenic cell death (ICD), effectively recruiting and stimulating dendritic cells (DCs). Concurrently, STING pathway activation, triggered by DNA damage, enhanced DC maturation. Moreover, the MnO2 component alleviated hypoxia within the tumor microenvironment by releasing significant amounts of oxygen, which facilitated the repolarization of macrophages from the M2 phenotype to the M1 phenotype. Therefore, MBMA-RGD ISV demonstrated potent suppression of tumor metastasis and recurrence without notable side effects in mouse tumor models.Copyright © 2025. Published by Elsevier Inc.