Intracellular and extracellular stiffness regulation in osteosarcoma: Overcoming mechano-immune checkpoints to enhance anti-osteosarcoma immunity

• Dual Mechano-modulation Strategy: Z@CMDH is engineered to simultaneously target intracellular and extracellular biomechanical properties. • Synergistic Immunotherapy: Z@CMDH synergizes with αPDL1 to suppres tumor growth and immune evasion. • Clinical Translation Potential: Patient-derived organoid co-culture models confirm personalized therapeutic efficacy of Z@CMDH + αPDL1. Immune checkpoint blockade therapy shows great potential in cancer treatment, but its low response rate (∼20 %) limits its effectiveness. This study introduces a novel approach for treating osteosarcoma (OS) that targets intracellular and extracellular biomechanical signals to enhance immunotherapy. Z@CMDH, a core–shell nanoparticle system combining ZnO nanoparticles and an engineered cell membrane, was developed. Intracellularly, cholesterol depletion from tumor cell membranes increases cell stiffness, weakening the resistance to T cell cytotoxicity. This depletion destabilizes lipid rafts, reduces PD-L1 membrane stability, inhibits PI3K-Akt signaling, and impairs cellular resistance to oxidative stress. ZnO nanoparticles induce oxidative stress, promoting immunogenic cell death (ICD). Extracellularly, hyaluronidase-mediated extracellular matrix degradation reduces the stiffness of tumor tissue to counteract Piezo1-mediated toxicity suppression in CTLs. This dual modulation synergistically enhances immune responses, promoting tumor suppression and immune cell infiltration. In vitro and in vivo, Z@CMDH + αPDL1 significantly suppressed tumor growth, reduced metastasis, and induced robust ICD. Transcriptomic and steroid metabolic profiling revealed key alterations in tumor-associated pathways, including upregulation of immunogenic pathways and modulation of steroid metabolism, supporting the synergistic effects of biomechanical regulation and immune checkpoint inhibition. These findings suggest that Z@CMDH + αPDL1 is a promising strategy for improving OS immunotherapy outcomes.