Bystander effect-mediated suicide gene therapy of experimental brain tumor by genetically engineered tumor cells in rat

我们的前期研究表明，通过将转导有单纯疱疹病毒胸苷激酶（HSVtk）基因的9 L胶质瘤细胞（9 Ltk）注射到预先植入的野生型9 L胶质瘤附近，然后给予更昔洛韦（GCV）（TK细胞疗法），由于旁观者效应，可以有效地治疗颅内9 L胶质瘤。为了旁观者效应介导的细胞杀伤的可能临床应用，我们测试了HSVtk基因转染的同种异体C6胶质瘤（C6 tk）细胞而不是9 Ltk（同种异体TK细胞疗法）。颅内9 L胶质瘤也可以有效地治疗同种异体TK细胞治疗，虽然这种旁观者效应弱于同基因TK细胞治疗。由于同种异体肿瘤细胞最终会被排斥，因此同种异体TK细胞治疗是一种安全可行的治疗策略，本研究采用野生型细胞（9 Lwt和C6 wt）和各自的TK细胞（9 Ltk和C6 tk）混合群体，研究了不同肿瘤细胞系（9 L和C6细胞）之间的旁观者效应。在9 Lwt/9 Ltk和9 Lwt/C6 tk组合中观察到有效的体外旁观者效应，但在C6 wt/9 Ltk和C6 wt/C6 tk组合中未观察到。在无胸腺裸鼠皮下肿瘤模型中研究的体内旁观者效应在9 Lwt/9 Ltk和9 Lwt/C6 tk组合中也是有效的。由于连接蛋白家族基因产物中的主要蛋白连接蛋白43在9 L细胞中的表达远高于C6细胞中的表达，结果表明，靶野生型细胞中而不是效应TK细胞中的连接蛋白的量对于旁观者效应的产生是重要的。C6 wt/C6 tk组合中的体外旁观者效应通过将连接蛋白43基因转导至靶细胞而增强，这进一步证实了该假设

Our previous study demonstrated that intracranial 9L glioma could be efficiently treated due to the bystander effect by injecting the 9L glioma cells transduced with herpes simplex virus-thymidine kinase(HSVtk)gene(9Ltk)in the vicinity of the preimplanted wild-type 9L glioma and then administerin ganciclovir(GCV)(TK cell therapy). For a possible clinical application of the bystander effect-mediated cell killing, we tested HSVtk gene trransduced allogeneic C6 glioma(C6tk)cells instead of 9Ltk(allogeneic TK cell therapy). Intracranial 9L glioma could also be efficiently treated by allogeneic TK cell therapy, though this bystander effect was weaker than syngeneic TK cell therapy. Since allogeneic tumor cells are finally rejected, the allogeneic TK cell therapy is a safe and feasible strategy for glioma treatment.The bystander effect between different tumor cell lines(9L and C6 cells)was then studied using mixed populations of wild-type cells(9Lwt and C6wt)and respective TK cells(9Ltk and C6tk). A potent in vitro bystander effect was observed in 9Lwt/9Ltk and 9Lwt/C6tk combinations but not in C6wt/9Ltk and C6wt/C6tk combinations. In vivo bystander effect studied in a subcutaneous tumor model in athymic nude mice was also potent in 9Lwt/9Ltk and 9Lwt/C6tk combinations. Since the expression of connexin43, a major protein in the connexin family gene products, in 9L cells is much higher than that in C6 cells, the results suggest that the amount of connexin in target wild-type cells but not in effector TK cells is important for the generation of the bystander effect. This hypothesis was further confirmed by the observation that in vitro bystander effect in C6wt/C6tk combination was potentiated by transduction of the connexin43 gene to the target cells