不可逆电穿孔联合免疫调节基因的原位导入——一种骨肉瘤综合治疗新策略的基础研究

Currently, in the comprehensive treatment of osteosarcoma, the main approach is prosthesis replacement surgery, supplemented with chemotherapy. However, the patients’ prognosis and postoperative function are unsatisfied. Our group have a lot of innovative experiment about the limb salvage treatment of osteosarcoma with microwave ablation technology in-situ. To further expanding the advantage, we explore the possibility of application with irreversible electroporation(IRE) in the treatment of osteosarcoma. Our previous study indicated that IRE could not only locally destroy the osteosarcoma cells, with faster bone regeneration and larger scope of applications, but also change the status of immunity. Nonetheless, the activation of immune response induced by IRE cannot prevent recurrence and metastasis effectively, this may be related to the intensity of immune activation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene therapy, an important immunoregulatory factor, has been proved to enhance the anti-tumor immune effects in a variety of malignancies treatment, but the complexity and inconveniences to get the patients’ cancer/immune cell is the mainly problem in the gene transfer in vitro. Our preliminary experiments show, when IRE is applied the electric field decreases as it radiates out and enables reversible electroporation and the uptake of gene plasmids at the periphery of the area of ablation. Further, necrotic cells caused by IRE is a kind of whole-cell vaccine. Thus, we propose that facilitating gene transfer of GM-CSF plasmid during IRE can enhance the activation of immune response induced by IRE, and the recurrence and metastasis of osteosarcoma after limb salvage surgery may be prevented, that means patient's prognosis will be improved.

目前骨肉瘤综合治疗以假体置换为主、化疗为辅，但预后和术后功能均不理想。本组在利用微波原位消融技术对骨肉瘤进行原位灭活保肢方面有创新性的积累，并进一步扩大优势，尝试将不可逆电穿孔（IRE）用于骨肉瘤治疗，初步结果表明IRE能有效灭活骨肉瘤的同时骨段再生更快应用范围更广，且能引起机体免疫反应，但其免疫激活尚不能有效的预防肿瘤的复发转移，这可能与免疫激活的强度有关。粒细胞-巨噬细胞集落刺激因子（GM-CSF）是免疫治疗中重要的免疫调节因子，多种肿瘤的临床实践证实其基因治疗能够解决这一问题，但行体外基因导入存在操作复杂、患者自体细胞获取不便等问题。我们预实验显示，IRE能够使核心灭活区域周围低电场区的细胞产生瞬时微孔，是外源基因导入的天然基础，再加上IRE原位灭活的肿瘤细胞本身就是全细胞疫苗，因此联合GM-CSF原位基因修饰有望高效调动机体免疫反应，进而改善骨肉瘤患者预后。

对于骨肉瘤综合治疗以假体置换为主、化疗为辅，但预后和术后功能均不理想。本组在利用微波原位消融技术对骨肉瘤进行原位灭活保肢方面有创新性的积累，并进一步扩大优势，尝试将不可逆电穿孔（IRE）用于骨肉瘤治疗，初步结果表明IRE能有效灭活骨肉瘤的同时骨段再生更快应用范围更广，且能引起机体免疫反应，但其免疫激活尚不能有效的预防肿瘤的复发转移，这可能与免疫激活的强度有关。粒细胞-巨噬细胞集落刺激因子（GM-CSF）是免疫治疗中重要的免疫调节因子，多种肿瘤的临床实践证实其基因治疗能够解决这一问题，但行体外基因导入存在操作复杂、患者自体细胞获取不便等问题。我们预实验显示，IRE能够使核心灭活区域周围低电场区的细胞产生瞬时微孔，是外源基因导入的天然基础，再加上IRE原位灭活的肿瘤细胞本身就是全细胞疫苗，因此联合GM-CSF原位基因修饰有望高效调动机体免疫反应，进而改善骨肉瘤患者预后。.为进行上述假设的验证，本项目首先利用有限元模型分析辅助基因转导最优参数确定，确定 IRE 实施过程中周边可逆电穿孔区域基因转导效率最佳区域；在非荷瘤鼠动物模型体内利用IRE核心灭活区域周边可逆性电穿孔效应进行GM-CSF载体质粒基因转导可行性在体验证；最后，利用荷瘤鼠动物模型，验证IRE联合GM-CSF基因治疗增强特异性免疫反应增强及免疫保护效应。.本项目研究结果提示：利用IRE核心灭活区域周围低电场区的细胞产生瞬时微孔将外源基因导入效应，实现免疫刺激性细胞因子 GM-CSF 的基因转导。联合免疫治疗一定程度上能够进一步提高 IRE 治疗后残留的肿瘤全细胞疫苗所致的免疫激活，可能在一定程度上影响骨肉瘤复发及转移，改善预后。