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

新型、生物学相关的血脑肿瘤屏障（BBTB）体外模型

• 批准号: 2887667
• 金额: --
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2887667
• 关键词: Novel; biologically; relevant; vitro; model

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.

胶质母细胞瘤（GBM）是最常见的脑癌形式。尽管如此，过去20年来治疗并没有显着改善。目前的治疗方法包括手术、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 模型无法准确描述该疾病。体外 2D 细胞培养不能反映 BBTB 的复杂性，并且与人类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尚未在临床上取得成功。该项目的目标是通过将 GBM 细胞纳入当前的 BBB 模型中，建立一种新型的 BBTB 体外模型并进行生物学验证。这将通过水凝胶 Transwell 来实现，该水凝胶 Transwell 由人源 GBM 细胞组成，与诱导多能干细胞 (iPSC) 衍生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。 NC3R 资助了该项目，因为该模型可用于替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型 BBB 模型，该模型基于从 iPSC 中衍生出类脑内皮细胞并与水凝胶上的星形胶质细胞共培养。该模型已得到充分验证，并形成紧密的单层，表达关键 BBB 蛋白。水凝胶 Transwell 培养物顶部的单层 BEC 提供了一个理想的模型，可以直接研究其屏障特性和功能。为了实现将 GBM 细胞纳入 BBB 模型，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在模型的水凝胶组件中培养。该技术能够建立体外 BBTB 并提供一种研究 GBM 细胞作用的方法。该模型的有效性将使用几个标准来确定：屏障紧密度；星形胶质细胞端足；基因表达；将评估药物交叉和紧密连接蛋白表达。最后，使用转录组学，将新型体外 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着减少动物使用，并且从长远来看有可能取代 GBM 异种移植模型。我们估计英国每年至少使用 4,500 只啮齿动物进行 GBM 异种移植实验。对于啮齿动物 GBM 异种移植模型在全球范围内的使用情况，一项搜索显示，去年发表的近 800 篇使用 GBM 异种移植模型的主要出版物。据此，我们估计全世界每年至少有 60,000 只啮齿动物被用于 GBM 异种移植研究。使用体外模型作为新型药物疗法的主要筛选将导致动物工作量估计减少 20%，因此每年大约有 12,000 只啮齿动物不会被用于 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。