Novel, biologically relevant, in vitro model of the blood-brain tumour barrier (BBTB)

胶质母细胞瘤（GBM）是脑癌最常见的形式。尽管如此，在过去20年里，治疗并没有明显改善。目前的治疗包括手术、放疗和化疗，但结果仍然很差，新诊断患者的平均预期寿命为12至14个月。治疗方案缺乏进展是多方面的，但靶向治疗方案尚未克服跨越血脑屏障（BBB）/血脑肿瘤屏障（BBTB）的挑战。目前的BBTB模型未能提供该疾病的准确代表。体外2D细胞培养不能反映BBTB的复杂性，并且与人体生理学的相关性有限。体内模型可能无法模拟临床疾病;对GBM异种移植模型起作用的新药在临床上尚未成功。本项目的目的是通过将GBM细胞与当前的BBB模型结合，建立并生物学验证BBTB的新型体外模型。这将使用水凝胶transwell来实现，所述水凝胶transwell由人源GBM细胞组成，与源自诱导多能干细胞（iPSC）的脑样内皮细胞共培养。该项目的工作假设是，该方法将在BBB处生成GBM的生物学相关模型，然后可以对其进行验证，并用作新型靶向治疗的筛选。NC 3R资助了这个项目，因为这个模型可以用来取代一些啮齿动物异种移植研究。我们在开放大学的合作者最近建立了一种新的BBB模型，该模型基于从iPSC中获得脑样内皮细胞并与星形胶质细胞在水凝胶上共培养。该模型已得到充分验证，并形成了表达关键BBB蛋白的紧密单层。水凝胶transwell培养物顶部的BEC单层提供了一个理想的模型，其中可以直接研究屏障性质和功能。为了在BBB模型中包含GBM细胞，我们在爱丁堡大学的合作者提供了神经干细胞（NSC）和患者来源的GBM干细胞样细胞（GSC）。这些GBM细胞与星形胶质细胞一起将在模型的水凝胶组分中培养。该技术使得能够建立体外BBTB和研究GBM细胞的作用的方法。将使用几个标准确定该模型的有效性：将评估屏障紧密性、星形胶质细胞末端足、基因表达、药物交叉和紧密连接蛋白表达。最后，使用转录组学，将新的体外BBTB模型与BBTB的GBM患者数据集进行比较，以确定体外模型与人类疾病的生物学相关性。该模型的影响将是显著减少动物使用，并且在长期内具有替代GBM异种移植模型的潜在模型。我们估计，在英国，每年至少有4,500只啮齿动物用于GBM异种移植实验。对于GBM异种移植啮齿动物的全球使用，检索显示去年发表的近800篇使用GBM异种移植模型的主要出版物。由此，我们估计全球每年至少有60，000只啮齿动物用于GBM异种移植研究。使用体外模型作为新型药物治疗的初步筛选将导致动物工作减少约20%，因此每年约有12，000只啮齿动物不会用于GBM研究。总之，该项目旨在生成一个经过验证的BBTB模型，可以推广到GBM实验室，从而减少GBM异种移植模型的使用，并改善对人类疾病的转化。

Glioblastoma (GBM) is the most common form of brain cancer. Despite this, treatment has not improved significantly in the past 20 years. Current therapy involves surgery, radiotherapy and chemotherapy, but outcomes remain very poor, with average life expectancy for newly diagnosed patients ranging from 12 to 14 months. The lack of progress in treatment options is multifaceted, but targeted therapy options have yet to overcome the challenge of crossing the blood-brain barrier (BBB) / blood-brain tumour barrier (BBTB). Current models of the BBTB fail to provide an accurate representation of the disease. In vitro 2D cell cultures do not reflect the complexity of the BBTB, and have limited relevance to human physiology. In vivo models may not mimic the clinical disease; novel drugs that work on GBM xenograft models have not been successful in the clinic. The objective of this project is to establish and biologically validate a novel in vitro model of the BBTB by inclusion of GBM cells with the current BBB model. This will be achieved using a hydrogel transwell, consisting of human derived GBM cells, co cultured with brain like endothelial cells derived from induced pluripotent stem cells (iPSCs). The working hypothesis of this project is that this method will generate a biologically relevant model of GBM at the BBB which can then be validated, and used as a screen for novel, targeted therapies. The NC3Rs have funded this project as this model could be used to replace some rodent xenograft studies. Our collaborators at the Open University have recently established a novel BBB model based on deriving brain-like endothelial cells from iPSCs and co-culturing with astrocytes on a hydrogel. This model has been fully validated and forms a tight monolayer with expression of key BBB proteins. The monolayer of BECs on top of the hydrogel transwell culture provides an ideal model where the barrier properties and function can be investigated directly. To achieve the inclusion of GBM cells within the BBB model, our collaborator at University of Edinburgh has provided neural stem cells (NSCs) and patient derived GBM stem-like cells (GSCs). These GBM cells, alongside astrocytes, will be cultured in the hydrogel component of the model. This technique enables the establishment of an in vitro BBTB and a way to study the effects of the GBM cells. The validity of this model will be determined using several criteria: barrier tightness; astrocyte end feet; gene expression; drug crossing and tight junction protein expression will be assessed. Finally, using transcriptomics, the novel in vitro BBTB model will be compared to GBM patient datasets of the BBTB to define the biological relevance of the in vitro model to the human disease. The impact of this model will be to reduce animal usage significantly and in the longer-term have the potential model to replace GBM xenograft models. We estimate an approximate use of at least 4,500 rodents a year for GBM xenograft experiments in the UK. For the worldwide usage of GBM xenograft rodents, a search shows nearly 800 primary publications published in the last year that used GBM xenograft models. From this we estimate at least 60,000 rodents are used per year worldwide for GBM xenograft studies. The use of an in vitro model as a primary screen for novel drug therapies would lead to an estimated 20% reduction in animal work, and therefore approximately 12,000 rodents a year that would not be used in GBM research. In conclusion, this project aims to generate a validated BBTB model that can be rolled out to GBM labs, thus leading to the reduction in the use of GBM xenograft models and improved translation to the human disease.