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，并伴有多种治疗方式造成的相当大的不良反应，包括生长迟缓、癫痫发作和中风。像大多数其他癌症一样，治疗的进展主要依赖于再现人类疾病的肿瘤模型，因此有希望的实验性治疗在患者身上有很大的成功机会。由于几个原因，目前的成神经管细胞瘤模型是不充分的。首先，患者的髓母细胞瘤细胞很难在正常环境外生长，这阻碍了实验疗法的测试。对于少数体外培养的肿瘤细胞系，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，利用髓母细胞瘤体内模型重建肿瘤环境的尝试，无论是基于小鼠遗传模型，还是患者来源的肿瘤移植到小鼠（称为PDX模型），也有局限性。这些模型是昂贵的，物种差异限制了将治疗方法转化为临床，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，患者之间的肿瘤基因不同，个体内肿瘤异质性也很明显。后者的发现源于新技术，该技术可以检测单细胞分辨率下表达的基因类型的差异。以前对肿瘤细胞的大量遗传分析无法检测到这些差异，而这些差异决定了肿瘤的生长模式并影响了治疗的成功。因此，不同患者的治疗反应可能参差不齐。总之，这些缺点是过去30年来缺乏新治疗方法的原因。我们假设肿瘤环境的存在更好地模拟了体内肿瘤的生长条件。为了验证这一点，我们之前强迫一种人类干细胞，称为人类诱导多能干细胞（hiPSCs），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成神经管细胞瘤细胞系，以模拟肿瘤环境。目的和目的：我将研究一种最普遍的髓母细胞瘤遗传亚型，称为超音刺猬髓母细胞瘤，我们的新模型是否促进肿瘤特征更像人类肿瘤。为此，我将使用肿瘤细胞的基因表达作为肿瘤行为的代理。通过在我们的体外模型中单独测定数千个髓母细胞瘤细胞基因组中的基因表达，我可以构建出它们异质性的图像，并确定我们的体外模型与体内模型的密切程度。潜在的应用和益处：在小脑类器官上生长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声波刺猬髓母细胞瘤多样性的细胞图谱，进而可用于识别患者特异性药物分子靶点。

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.