PO.TB10.16 · 肿瘤生物学
局部共递送透明质酸酶增强CSPG4 CAR-T细胞对脊索瘤的细胞毒性
Localized co-delivery of hyaluronidase enhances CSPG4 CAR-T cells cytotoxicity against chordoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景：脊索瘤是一种罕见的恶性骨肿瘤，其特征为致密的细胞外基质（ECM）和免疫学“冷”微环境，限制了免疫细胞浸润和治疗疗效。硫酸软骨素蛋白聚糖4（CSPG4）已成为脊索瘤中一个前景广阔的肿瘤相关抗原。然而，富含透明质酸的ECM形成物理和生化屏障，妨碍CAR-T细胞的运输和功能。我们假设局部共递送透明质酸酶（PH20）可重塑ECM并增强CSPG4靶向CAR-T细胞的细胞毒活性。
方法：使用含CD28-Ox40-CD3ζ信号结构域的第三代慢病毒构建体生成CSPG4 CAR-T细胞。使用人脊索瘤细胞系和患者来源的3D类器官评估细胞毒性和浸润。应用电阻抗实验监测PH20处理后表面电荷和细胞-基质相互作用的实时变化。使用Transwell迁移实验和3D类器官共培养评估CAR-T浸润，并通过ELISA定量细胞因子分泌。在局部共递送PH20和CAR-T细胞后，于异种移植模型中检测体内疗效和安全性。
结果：CSPG4 CAR-T细胞对脊索瘤细胞表现出抗原特异性的细胞裂解活性，但其浸润在富含透明质酸的环境中明显受限。电阻抗分析显示，PH20介导的ECM降解显著改变了电荷分布和阻抗特征，表明基质密度降低、CAR-T细胞可及性改善。在Transwell实验中，PH20增强了CSPG4 CAR-T穿越含透明质酸屏障的迁移。同样，在3D脊索瘤类器官中，PH20共处理促进了CAR-T更深的浸润，并带来更强的细胞毒性以及IFN-gamma和Granzyme B产生的升高。在多个患者来源的类器官模型中观察到一致的结果。在异种移植研究中，局部共递送PH20与CSPG4 CAR-T细胞相比CAR-T单药治疗带来更强的肿瘤消退和更长的生存（p < 0.01），且无可观察到的全身毒性。
结论：通过局部共递送透明质酸酶降解ECM可有效增强CSPG4 CAR-T细胞对脊索瘤的浸润和细胞毒性。这种联合方法克服了基质屏障，重塑了肿瘤微环境，代表了一种在具有致密ECM结构的实体瘤中改善CAR-T疗效的可行策略。
查看英文原文 English abstract
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.
利益披露 Disclosure
M. Qi, None..
G. Cattaneo, None..
Z. Chen, None..
C. Ferrone, None..
J. Schwab, None.