Synergistic Chemoimmunotherapy Augmentation via Sequential Nanocomposite Hydrogel‐Mediated Reprogramming of Cancer‐Associated Fibroblasts in Osteosarcoma

Abstract In osteosarcoma, immunotherapy often faces hurdles posed by cancer‐associated fibroblasts (CAFs) that secrete dense extracellular matrix components and cytokines. Directly removing CAFs may prove ineffective and even promote tumor metastasis. To address this challenge, a sequential nanocomposite hydrogel that reshapes CAF behavior is developed, enhancing tumor‐infiltrating T‐cells in osteosarcoma. The approach utilizes an injectable blend of carboxymethyl chitosan and tetrabasic polyethylene glycol, forming a hydrogel for controlled release of a potent CAF suppressor (Nox4 inhibitor, Nox4i) and liposomal Doxorubicin (L‐Dox) to induce immunogenic cell death (ICD) upon in situ administration. Nox4i effectively counters CAF activation, overcoming T‐cell exclusion mechanisms, followed by programmed L‐Dox release for ICD induction in stroma‐rich osteosarcoma models. Combining the co‐delivery gel with αPD‐1 checkpoint inhibitor further enhances its effectiveness in an orthotopic osteosarcoma model. Immunophenotyping data underscore a significant boost in tumor T‐cell infiltration and favorable anti‐tumor immunity at the whole‐animal level.