Analyzing network formation during brain tumour initiation

高级别胶质瘤(HGG)是一种复杂且具有破坏性的疾病，临床需求尚未得到满足。这些肿瘤抵抗多种模式的治疗，生存时间平均只有14个月。最近的研究表明，胶质瘤细胞彼此之间以及与宿主组织2-4之间存在类似神经网络的连接。肿瘤细胞被证明是通过称为肿瘤微管的长细胞过程连接起来的。肿瘤微管(Tumour Microtube，TM)对胶质瘤细胞的侵袭和增殖至关重要，并将单个肿瘤细胞连接到一个功能正常的通讯网络，极大地增强了其生长和耐药性。Sieger实验室开发了一种新的模型来分析斑马鱼幼体的胶质瘤起始阶段，并能够证明脑肿瘤起始细胞劫持机制，这种机制通常用于将小胶质细胞过程导向大脑中高活性的神经元和损伤。小胶质细胞的这种异常作用导致了早期肿瘤细胞增殖的积极促进。有趣的是，现在已经很清楚，小胶质细胞在健康大脑中神经网络的形成中扮演着几个角色，例如通过促进轴突生长、突触形成和电路功能6-9。对钙离子瞬变的测量显示，这些早期肿瘤细胞中有活跃的信号活动，这意味着这些早期网络的功能。重要的是，我们发现干扰小胶质细胞的活动会导致肿瘤微管生长受损，并减少网络中的钙信号。我的项目将专注于在HGG形成过程中建立的细胞网络。主要的长期目标是：1.使用最先进的显微镜在活体中直观地识别和描述脑TM的特征；2.使用钙离子实时成像来描绘在肿瘤形成的第一阶段建立的肿瘤钙网络；3.研究小胶质细胞在TM阳性FISH的钙信号模式中的作用及其对肿瘤细胞转录的影响。我将使用的肿瘤启动模型是基于人类癌基因AKT1在斑马鱼幼虫中的过表达，因为这之前已经被证明会导致胶质瘤的形成。癌基因的表达与荧光标记相联系，以便在体内容易识别。钙成像将基于已经建立的表达β-肌动蛋白的突变斑马鱼系：GCamp6f钙记者。参考：1.成人恶性胶质瘤。N.Engl.J.Med.359,492-507(2008)。2.Osswald，M.等人。脑瘤细胞相互连接到一个功能强大的耐药网络。《自然》528，93-98(2015)。3.Venkatesh，H.S.等人。神经胶质瘤与神经回路的电和突触整合。自然，573，1-27(2019)。4.Venkataramani，V.等人。谷氨酸能突触输入到胶质瘤细胞驱动脑瘤的进展。自然3,147(2019)。5.Chia，K.，Keatinger，M.，Mazzolini，J.&Sieger，D.脑肿瘤重新利用内源性神经元到小胶质细胞的信号机制，以促进自身的增殖。ELife 8，19(2019年)。6.宫本，A.等人。小胶质细胞接触在发育中的躯体感觉皮质诱导突触形成。自然通讯1-12(2019)。7.Cunningham，C.L.，Martinez-Cerdeno，V.&Noctor，S.C.小胶质细胞调节发育中的大脑皮质中神经前体细胞的数量。《神经科学杂志》33卷，4216-4233页(2013)。8.Ueno，M.&Yamashita，T.大脑发育中小胶质细胞的双向调节：从神经发生到神经回路形成。神经生物学的当代观点27，8-15(2014)。9.Mosser，C.-A.，Baptista，S.，Arnoux，I.&Audinat，E.CNS发育中的小胶质细胞：为未来塑造大脑。神经生物学进展149-150，1-20(2017)。

High grade gliomas (HGGs) represent a complex and devastating disease and are posing an unmet clinical need. These tumours resist multi-modal therapies and survival times are only 14 months on average1. Recent studies show that glioma cells make neural network-likeconnections with one another, and with host tissue2-4.Tumour cells have been shown to be connected by long cellular processes called tumour microtubes. Tumour microtubes (TM) are crucial for the invasion and proliferation of glioma cells and connect single tumour cells to a functional communicating network, greatly enhancing its growth and drug-resistance.The Sieger lab have developed a novel model to analyse glioma initiation stages in the larval zebrafish and were able to show that brain tumour initiating cells hijack mechanisms which are usually employed to direct microglial processes towards highly active neurons and injuries in the brain5. This aberrant role of microglia results in the active promotion of proliferation of early tumour cells. Interestingly, it is now clear that microglia play several roles in the formation of neural networks in the healthy brain, e.g. by promoting axonal growth, synapse formation and circuit function6-9. Measurements of Ca2+ transients revealed active signalling activity in these early tumour cells, implying functionality of these early networks. Importantly, we found that interfering with microglia activity resulted in impaired tumour microtube outgrowth as well as decreased Ca2+ signalling within the network.My project will focus on the cellular networks built during HGGs formation. The principal long-term aims are:1. To visually identify and characterise brain TMs in vivo using state-of-the-art microscopy;2. To delineate the tumoral calcium networks established during the first phases of tumour formation, using calcium live imaging;3. To investigate the role of microglia in the calcium signalling patterns of TM-positive fish, and their influence on tumour cells transcriptome.The tumour initiation model I will use is based on overexpression of human oncogene AKT1 in zebrafish larvae, as this has previously shown to lead to glioma formation. The oncogene expression is linked to fluorescent labelling to allow for easy identification in vivo. Calcium imaging will be based on an already established mutagenic zebrafish line expressing the beta-actin:GCamp6f calcium reporter.References:1. Wen, P. Y. & Kesari, S. Malignant gliomas in adults. N. Engl. J. Med. 359, 492-507 (2008). 2. Osswald, M. et al. Brain tumour cells interconnect to a functional and resistant network. Nature 528, 93-98 (2015). 3. Venkatesh, H. S. et al. Electrical and synaptic integration of glioma into neural circuits. Nature 573, 1-27 (2019). 4. Venkataramani, V. et al. Glutamatergic synaptic input to glioma cells drives brain tumour progression. Nature 3, 147 (2019). 5. Chia, K., Keatinge, M., Mazzolini, J. & Sieger, D. Brain tumours repurpose endogenous neuron to microglia signalling mechanisms to promote their own proliferation. Elife 8, 19 (2019). 6. Miyamoto, A. et al. Microglia contact induces synapse formation in developing somatosensory cortex. Nature Communications 1-12 (2019). 7. Cunningham, C. L., Martinez-Cerdeno, V. & Noctor, S. C. Microglia Regulate the Number of Neural Precursor Cells in the Developing Cerebral Cortex. Journal of Neuroscience 33, 4216-4233 (2013). 8. Ueno, M. & Yamashita, T. Bidirectional tuning of microglia in the developing brain: from neurogenesis to neural circuit formation. Current Opinion in Neurobiology 27, 8- 15 (2014). 9. Mosser, C.-A., Baptista, S., Arnoux, I. & Audinat, E. Microglia in CNS development: Shaping the brain for the future. Progress in Neurobiology 149-150, 1-20 (2017).