Stem cell mediated targeting of tumor cells and associated vasculature in gliomas

描述（由申请人提供）：许多先前的临床前研究表明，抗血管生成剂产生“正常化窗口”，在此期间，同时施用的细胞毒性剂的递送和功效增强。然而，最近的研究表明，血管正常化导致血脑屏障的恢复，并因此导致细胞毒性药物向脑肿瘤的血管依赖性递送效率低下。这些结果提出了关于递送模式和抗血管生成和细胞毒性药物组合类型的基本问题，这些药物组合用于1）使肿瘤血管系统正常化;和2）增强脑肿瘤正常化后细胞毒性治疗的结果？这促使了新疗法的设计，其允许使用1）靶向脑中的原发性肿瘤和继发性微创沉积物的替代药物递送模式; 2）特异性靶向肿瘤细胞和相关脉管系统的工程化治疗性蛋白质;和3）允许跟踪药物递送载体、治疗性蛋白质和肿瘤在体内的命运的诊断性蛋白质。在这项提案中，我们将用可分泌的抗血管生成（aa）基因改造人类神经干细胞（NSC），血小板反应蛋白（TSP）-1和促凋亡分泌型肿瘤坏死因子凋亡诱导配体（S-TRAIL），其已知通过死亡受体（DR）4/5诱导凋亡，并初步比较NSC-aaTSP-1与已知抗-TNF-α的全身递送的“正常化”潜力。血管生成剂在已确定的恶性和原发性侵袭性脑肿瘤（神经胶质瘤）中的作用。根据我们的合作研究者Lawler博士最近的研究，TSP-1使血管正常化，还诱导肿瘤相关内皮细胞（EC）上的死亡受体（DR）4/5表达，使其启动TRAIL诱导的杀伤，将在恶性和侵袭性胶质瘤模型中测试血管正常化后S-TRAIL和aaTSP-1引发的EC的治疗效果。我们假设aaTSP-1将通过使血管正常化以及上调EC中的DR 4/5表达来增强S-TRAIL对肿瘤细胞的细胞毒性作用。这将最终导致增强的肿瘤细胞根除，并且还杀死原发性肿瘤块和逃离原发性肿瘤块的微侵袭性肿瘤细胞两者中的EC。将荧光和生物发光成像标记物紧密整合到胶质瘤细胞、EC和NSC中将使我们能够早期和快速地评估治疗效果，从而调整和微调所提出的治疗方法。为了模拟临床情况，治疗性NSC将被封装到生物相容性合成细胞外基质（sECM）中，并在我们最近开发的胶质瘤切除模型中进行测试。一旦得到验证，我们设想了一种治疗方式，在脑肿瘤手术时，将去除主要肿瘤块，并将基因工程治疗细胞引入sECM中，并允许靶向脑中剩余的肿瘤细胞和微侵袭性肿瘤沉积物。这将对挽救许多脑癌患者的生命产生重大影响。 公共卫生相关性：在这个提议中，我们将用抗血管生成、aaTSP-1和可调节的S-TRAIL工程化干细胞，并通过在小鼠胶质瘤模型中原位递送工程化干细胞来测试治疗效果。所开发的药物和策略将被设计为临床可翻译的，并且应该对理解当前的联合疗法和开发未来的脑肿瘤疗法产生重大影响。

DESCRIPTION (provided by applicant): A number of previous pre-clinical studies suggest that anti-angiogenic agents create "normalization window" during which delivery and efficacy of concurrently administered cytotoxic agents is enhanced. However, recent studies have shown that vessel normalization results in the restoration of blood brain barrier and consequential inefficient vessel dependent delivery of cytotoxic drugs to brain tumors. These results have raised fundamental questions about modes of delivery and the types of anti-angiogenic and cytotoxic drug combinations used to 1) normalize tumor vasculature; and 2) enhance the outcome of cytotoxic therapy post-normalization for brain tumors? This has prompted the design of novel therapies that allow use of 1) alternative modes of drug delivery that targets both the primary tumors and secondary micro-invasive deposits in the brain; 2) engineered therapeutic proteins that specifically target both tumor cells and associated vasculature; and 3) diagnostic proteins that allow tracking of drug delivery vehicles, therapeutic proteins and fate of tumors in vivo. In this proposal, we will engineer human neural stem cells (NSC) with secretable antiangiogenic (aa), thrombospondin (TSP)-1 and pro-apoptotic secretable tumor necrosis factor apoptosis inducing ligand (S-TRAIL) which is known to induce apoptosis via death receptor (DR)4/5 and initially compare the "normalization" potential of NSC-aaTSP-1 with systemic delivery of known anti-angiogenic agents in established malignant and primary invasive brain tumors (gliomas). Based on the recent studies by our Co-Investigator, Dr. Lawler, that TSP-1 normalizes vasculature and also induces death receptor (DR)4/5 expression on tumor associated endothelial cells (EC) priming them to TRAIL-induced killing, the therapeutic effect of S-TRAIL post vessel normalization and EC priming by aaTSP-1 will be tested in both malignant and invasive glioma models. We hypothesize that aaTSP-1 will enhance the cytotoxic effects of S-TRAIL on tumor cells by normalizing the vasculature and also upregulation of DR4/5 expression in EC. This will ultimately lead to enhanced eradication of tumor cells and also killing of EC in both the primary tumor mass and the micro-invasive tumor cells escaping the primary tumor mass. A close integration of fluorescent and bioluminescent imaging markers into glioma cells, EC and NSC will allow us to assess therapeutic efficacy early and quickly and thus to adjust and fine-tune the proposed therapeutic approaches. In an effort to simulate a clinical scenario, the therapeutic NSC will be encapsulated into biocompatible synthetic extracellular matrix (sECMs) and tested in our recently developed resection model of glioma. Once validated, we envision a therapeutic modality in which at the time of brain tumor surgery, the main tumor mass will be removed and genetically engineered therapeutic cells will be introduced in sECMs and allowed to target the remaining tumor cells and micro-invasive tumor deposits in the brain. This will have a major impact in saving the lives of many brain cancer patients. PUBLIC HEALTH RELEVANCE: In this proposal we will engineer stem cells with antiangiogenic, aaTSP-1 and regulatable S-TRAIL and test the therapeutic effect by on-site delivery of engineered stem cells in mouse glioma model. The developed agents and strategies will be designed to be clinically translatable and should have a major impact in understanding current combined therapies and also developing future therapies for brain tumors.