Evaluation of a novel in vitro model of sonic hedgehog medulloblastoma by single cell transcriptomics

研究背景：起源于小脑的脑细胞引起最常见的儿童恶性脑肿瘤，称为髓母细胞瘤。虽然据报道治愈率高达70%，但这通常是一种毁灭性的疾病，伴随着由多模式治疗引起的相当大的不良反应，包括生长迟缓，癫痫发作和中风。与大多数其他癌症一样，治疗进展关键取决于重新投降人类疾病的肿瘤模型，因此有希望的实验性治疗在患者中有很高的成功机会。目前的髓母细胞瘤模型是不够的，有几个原因。首先，患者的髓母细胞瘤细胞很难在正常环境外生长，这阻碍了实验性疗法的测试。对于已经离体生长的少数肿瘤细胞系，它们被剥夺了正常环境，这是癌症进展的一个重要因素，因为它影响肿瘤表达的基因类型。肿瘤基因表达反过来决定了对治疗的反应，这解释了为什么实验性治疗在患者中大多失败。其次，尝试使用髓母细胞瘤的体内模型重建肿瘤环境，无论是基于遗传小鼠模型，还是患者来源的肿瘤移植到小鼠中（称为PDX模型）也有局限性。这些模型是昂贵的，物种差异限制了治疗向临床的转化，并且在PDX模型的情况下，与原始肿瘤的遗传差异仍然存在。最后，肿瘤在患者之间存在遗传差异，并且肿瘤异质性在个体内也很明显。后者的发现来自于新技术，该技术可以检测在单细胞分辨率下表达的基因类型的差异。以前对肿瘤细胞进行的基因检测无法检测到这些差异，这些差异决定了肿瘤的生长模式并影响治疗的成功。因此，患者的治疗反应可能不一致。总之，这些缺点是负责缺乏新的治疗方法在过去的30 years.We假设，肿瘤环境的存在更好地模拟体内肿瘤生长条件。为了测试这一点，我们之前已经胁迫了一种称为人类诱导多能干细胞（hiPSC）的人类干细胞，它可以使体内任何类型的细胞在体外生长成微型小脑结构，称为小脑类器官。在一种新的方法中，我们目前正在人类小脑类器官中培养人类髓母细胞瘤细胞系，以模拟肿瘤环境。目的和目标：我将调查是否为髓母细胞瘤的最普遍的遗传亚型之一，称为音刺猬髓母细胞瘤，我们的新模型促进肿瘤特征，更好地类似于人类肿瘤。为此，我将使用肿瘤细胞的基因表达作为肿瘤行为的代表。通过在我们的体外模型中分别确定数千个髓母细胞瘤细胞基因组中的基因表达，我可以构建其异质性的图像，并确定我们的体外模型与其体内模型的紧密对齐程度。潜在应用和益处：在小脑类器官上培养新鲜获得的髓母细胞瘤细胞可以克服目前髓母细胞瘤建模的困难，并提供一个新的改进平台来测试患者的神经功能。特异性治疗，包括对正常小脑细胞的药物毒性。单细胞分析可用于生成音刺猬髓母细胞瘤多样性的细胞图谱，其进而可用于鉴定患者特异性可药物化分子靶标。

CONTEXT OF THE RESEARCH: Brain cells that originate in the cerebellum give rise to the commonest childhood malignant brain tumour, termed medulloblastoma. Although cure rates of up to 70% have been reported, this is typically a devastating disease that is accompanied by considerable adverse effects resulting from multi-modal treatments including growth retardation, seizures and strokes. Like most other cancers, treatment advances crucially depend on tumour models that re-capitulate human disease, so that promising experimental treatments have a high chance of success in patients. Current medulloblastoma models are inadequate for several reasons. First, medulloblastoma cells from patients are hard to grow outside their normal environment, which prevents testing of experimental therapies. For the few tumour cell lines that have been grown ex vivo, they are deprived of their normal environment, which is an important factor in cancer progression as it influences the types of genes expressed by the tumour. Tumour gene expression in turn determines the response to treatments which explains why experimental treatments mostly fail in patients. Second, attempts to re-create the tumour environment using in vivo models of medulloblastoma, either based on genetic mouse models, or patient-derived tumour grafting into the mouse (termed PDX models) also have limitations. These models are costly, species differences limit the translation of therapies to the clinic, and in the case of PDX models, genetic divergence from the original tumour still occurs. Lastly, tumours differ genetically between patients and tumour heterogeneity is also evident within an individual. The latter discoveries have arisen from new technologies which can detect differences in the types of genes expressed at single cell resolution. Previous genetic assays of tumour cells in bulk were incapable of detecting these differences, which determine tumour growth patterns and influence treatment success. Consequently, treatment responses can be patchy across patients. Together, these shortcomings are responsible for the lack of new treatments for the last 30 years.We hypothesise that the presence of the tumour environment better simulates in vivo tumour growth conditions. To test this, we have previously coerced a type of human stem cell, called human induced pluripotent stem cells (hiPSCs), which can make any cell type in the body, to grow into miniature cerebellar structures, termed cerebellar organoids, in vitro. In a novel approach, we are currently growing human medulloblastoma cell lines within human cerebellar organoids to mimic the tumour environment. AIMS AND OBJECTIVES: I will investigate whether for one of the most prevalent genetic subtypes of medulloblastoma, termed sonic hedgehog medulloblastoma, our new model promotes tumour characteristics that better resemble human tumours. To this end, I will use the expression of genes by tumour cells as a proxy for tumour behaviour. By determining gene expression across the genome of thousands of medulloblastoma cells in our in vitro model individually, I can build a picture of their heterogeneity and determine how closely aligned our in vitro model is with its in vivo counterpart.POTENTIAL APPLICATIONS AND BENEFITS: Growing freshly obtained medulloblastoma cells on cerebellar organoids could overcome current medulloblastoma modelling difficulties and provide a new improved platform to test patient-specific therapies, including drug toxicity on normal cerebellar cells. Single cell analysis can be used to generate a cellular atlas of sonic hedgehog medulloblastoma diversity, which in turn can be used to identify patient-specific druggable molecular targets.