A 3D in vitro glioblastoma cell culture system for identification and evaluation of novel radiosensitisers reducing rodent xenograft studies

胶质母细胞瘤是最常见的原发性脑肿瘤，目前无法治愈。尽管积极的治疗包括手术，放疗和化疗，胶质母细胞瘤患者的平均预期寿命约为一年。在过去的十年里，许多大型的国际临床试验已经测试了新的治疗方法，但没有一个是成功的。这尤其令人失望，因为在这些试验中测试的许多新的“靶向”药物在实验室测试时似乎是有效的。当胶质母细胞瘤细胞在实验室中培养时，它们通常在塑料烧瓶中作为单层细胞生长。这些二维（2D）细胞培养条件导致肿瘤细胞的形状和行为发生显著变化，影响它们对癌症治疗（包括放射治疗和靶向药物）的反应方式。由于许多新药似乎在2D细胞培养中有效，因此它们随后在植入胶质母细胞瘤细胞的小鼠和大鼠中进行测试。一些药物在这些动物实验中显示出有希望的结果，但在患者身上测试时仍然无效。因此，我们需要一种不同的方法来开发和测试新的治疗方法。首先，我们需要了解为什么胶质母细胞瘤对放疗、化疗和新的靶向药物具有抗药性。然后我们需要开发新的治疗方法来克服这些耐药机制。为了避免用无效的药物治疗大量动物，我们还需要在开始进行动物实验之前更有信心地相信药物对患者有效。为了解决这些问题，我们开发了一种新的胶质母细胞瘤3D模型，可以在实验室中使用胶质母细胞瘤患者的肿瘤细胞进行生长。这些细胞生长在涂有胶质母细胞瘤中发现的特殊蛋白质的聚苯乙烯支架上，并用胶质母细胞瘤中也存在的特殊生长因子滋养。我们已经证明，这个3D模型包含了我们在患者肿瘤中看到的许多特征。更重要的是，我们已经证明，对患者有效的药物在3D模型中也有效，而对患者无效的药物在3D模型中没有效果。其中一些药物对二维和三维条件下生长的细胞有相反的作用。因为我们对三维模型有信心，我们相信寻找在三维细胞接受放射治疗时被打开的基因和蛋白质，然后测试可以阻断这些基因和蛋白质作用的新药是有价值的。我们已经在3D细胞中发现了几个被放射治疗打开的基因，而不是2D细胞，我们早期的实验表明，我们可以通过靶向这些基因来克服对放射治疗的抵抗。通过这种方式，我们将确定更有可能对患者有效的新药。然而，我们意识到，我们目前的3D模型相当简单，胶质母细胞瘤中的许多其他细胞和结构可能很重要。肿瘤血管特别有影响力，因为它们的细胞（内皮细胞）产生的化学物质滋养肿瘤细胞，使它们对放射治疗有抵抗力。因此，我们将开发一个由胶质母细胞瘤细胞和人脑微血管内皮细胞组成的更复杂的3D模型，看看新药在这种新的“多细胞”模型中是否也有效。与此同时，我们将研究不同类型的细胞如何相互作用，以及这如何导致对治疗的抵抗。最后，我们将把新的3D模型转换成一种允许新药“高通量筛选”的格式。这将使我们和世界各地的其他研究人员能够尽可能有效地测试大量新药。总的来说，我们的目标是改善胶质母细胞瘤患者的治疗，同时减少动物实验的数量。我们将通过开发一种新的胶质母细胞瘤3D模型来实现这些目标，该模型可以准确预测哪些新药对患者有效。

Glioblastoma is the most common primary brain tumour and is currently incurable. Despite aggressive treatment involving surgery, radiotherapy and chemotherapy, average life expectancy for glioblastoma patients is about one year. Over the past ten years many large, international clinical trials have tested new treatments but none of them has been successful. This is particularly disappointing because many of the new 'targeted' drugs being tested in these trials seemed to be effective when tested in the laboratory. When glioblastoma cells are cultured in the laboratory they are usually grown as single layers of cells in plastic flasks. These 2-dimensional (2D) cell culture conditions cause marked changes in the shape and behaviour of the tumour cells which affect the way they respond to cancer treatments including radiotherapy and targeted drugs. Because many new drugs appear to be effective in 2D cell cultures, they are then tested in mice and rats implanted with glioblastoma cells. Some drugs show promising results in these animal experiments but are still not effective when tested in patients. So, we need a different method for developing and testing new treatments. First we need to understand why glioblastomas are resistant to radiotherapy, chemotherapy and the new targeted drugs. Then we need to develop new treatments that overcome these mechanisms of resistance. To avoid treating large numbers of animals with ineffective drugs, we also need to be more confident that drugs will work in patients before we start performing animal experiments. To address these issues we have developed a new, 3D model of glioblastoma that can be grown in the laboratory using tumour cells from patients with glioblastoma. These cells are grown on polystyrene scaffolds coated with special proteins found in glioblastomas, and are nourished with specialised growth factors that are also present in glioblastomas. We have shown that this 3D model contains many of the features that we see in tumours in patients. More importantly, we have shown that drugs which work in patients also work in the 3D model, while drugs which don't work in patients have no effect in the 3D model. Some of these drugs had opposite effects on cells grown in 2D and 3D conditions.Because we have confidence in the 3D model, we believe it will be valuable to look for genes and proteins that are switched on when 3D cells are treated with radiotherapy, and then test new drugs that can block the effects of these genes and proteins. We have already found several genes that are switched on by radiotherapy in 3D but not 2D cells, and our early experiments suggest that we can overcome resistance to radiotherapy by targeting these genes. in this way we will identify new drugs that are much more likely to be effective in patients.However we realise that our current 3D model is rather simple and that lots of other cells and structures in glioblastoma might be important. Tumour blood vessels are particularly influential because the cells that line them (endothelial cells) produce chemicals that nourish the tumour cells and make them resistant to radiotherapy. We will therefore develop a more complex 3D model composed of glioblastoma cells and human brain microvascular endothelial cells and see if the new drugs are also effective in this new 'multicellular' model. At the same time we will investigate how the the different cell types interact and how this causes resistance to treatment. Finally, we will convert the new 3D model into a format that allows 'high throughput screening' of new drugs. This will allow us and other researchers around the world to test very large numbers of new drugs as efficiently as possible. Overall we aim to improve treatments for glioblastoma patients while reducing the number of animal experiments. We will achieve these aims by developing a new 3D model of glioblastoma that accurately predicts which new drugs will be effective in patients.