Localized co-delivery of hyaluronidase enhances CSPG4 CAR-T cells cytotoxicity against chordoma

Background: Chordoma is a rare malignant bone tumor characterized by a dense extracellular matrix (ECM) and an immunologically “cold” microenvironment that limits immune cell infiltration and therapeutic efficacy. Chondroitin sulfate proteoglycan 4 (CSPG4) has emerged as a promising tumor-associated antigen in chordoma. However, the hyaluronan-rich ECM forms a physical and biochemical barrier that hampers CAR-T cell trafficking and function. We hypothesized that localized co-delivery of hyaluronidase (PH20) could remodel the ECM and enhance the cytotoxic activity of CSPG4-directed CAR-T cells. Methods: CSPG4 CAR-T cells were generated using a third-generation lentiviral construct containing CD28-Ox40-CD3ζ signaling domains. Human chordoma cell lines and patient-derived 3D organoids were used to evaluate cytotoxicity and infiltration. Electrical impedance assays were applied to monitor real-time changes in surface charge and cell-matrix interactions following PH20 treatment. Transwell migration assays and 3D organoid co-cultures were used to assess CAR-T infiltration, and cytokine secretion was quantified by ELISA. In vivo efficacy and safety were examined in xenograft models following localized co-delivery of PH20 and CAR-T cells. Results: CSPG4 CAR-T cells exhibited antigen-specific cytolytic activity against chordoma cells, but their infiltration was markedly restricted in hyaluronan-rich environments. Electrical impedance analysis revealed that PH20-mediated ECM degradation significantly altered the charge distribution and impedance profile, indicating reduced matrix density and improved accessibility for CAR-T cells. In Transwell assays, PH20 enhanced CSPG4 CAR-T transmigration across hyaluronan-containing barriers. Similarly, in 3D chordoma organoids, PH20 co-treatment promoted deeper CAR-T infiltration and led to greater cytotoxicity with elevated IFN-gamma and Granzyme B production. Consistent findings were observed across multiple patient-derived organoid models. In xenograft studies, localized co-delivery of PH20 with CSPG4 CAR-T cells resulted in stronger tumor regression and prolonged survival compared with CAR-T monotherapy (p < 0.01), without observable systemic toxicity. Conclusion: ECM degradation through localized hyaluronidase co-delivery effectively enhances CSPG4 CAR-T cell infiltration and cytotoxicity against chordoma. This combinatorial approach overcomes stromal barriers, reshapes the tumor microenvironment, and represents a feasible strategy to improve CAR-T efficacy in solid tumors with dense ECM architecture.