Analyzing network formation during brain tumour initiation

高级别胶质瘤（HGGs）是一种复杂和毁灭性的疾病，并提出了未满足的临床需求。这些肿瘤抵抗多模式治疗，存活时间平均只有14个月1。最近的研究表明，胶质瘤细胞之间以及与宿主组织之间形成神经网络般的连接。肿瘤细胞已被证明通过称为肿瘤微管的长细胞过程连接在一起。肿瘤微管（tumor microtubes， TM）对胶质瘤细胞的侵袭和增殖至关重要，它将单个肿瘤细胞连接到一个功能性的通讯网络中，极大地增强了肿瘤细胞的生长和耐药性。西格尔实验室已经开发出一种新的模型来分析斑马鱼幼体中神经胶质瘤的形成阶段，并且能够证明脑肿瘤的形成细胞劫持机制，这种机制通常用于将小胶质细胞过程导向大脑中高度活跃的神经元和损伤。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，现在已经清楚小胶质细胞在健康大脑中神经网络的形成中起着多种作用，例如通过促进轴突生长、突触形成和回路功能6-9。Ca2+瞬态的测量揭示了这些早期肿瘤细胞中活跃的信号活动，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活性导致肿瘤微管生长受损以及网络内Ca2+信号减少。我的项目将集中在hgg形成过程中建立的蜂窝网络。主要的长期目标是：1。使用最先进的显微镜在体内视觉识别和表征脑TMs；2 .利用钙活显像技术描绘肿瘤形成初期建立的肿瘤钙网络；探讨小胶质细胞在tm阳性鱼类钙信号通路中的作用及其对肿瘤细胞转录组的影响。我将使用的肿瘤起始模型是基于斑马鱼幼体中人类致癌基因AKT1的过度表达，因为这之前已被证明会导致胶质瘤的形成。致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GCamp6f钙报告蛋白的诱变斑马鱼系。Wen， P. Y.和Kesari， S.成人恶性胶质瘤。心血管病。中华医学杂志，359,492-507（2008）。2. Osswald， M.等。脑肿瘤细胞相互连接到一个功能性和抗性的网络。自然，28,93-98（2015）。3. Venkatesh, h.s.等人。神经胶质瘤在神经回路中的电和突触整合。《自然》（英文版），2016年第1期。4. Venkataramani， V.等。神经胶质瘤细胞的谷氨酸能突触输入驱动脑肿瘤进展。《自然》第3期，147（2019）。5. Chia， K, Keatinge， 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来促进自身的增殖。生命8,19（2019）。6. 宫本，A.等。小胶质细胞接触诱导发育中的体感觉皮层突触形成。自然通讯1-12（2019）。7. Cunningham, c.l, Martinez-Cerdeno， V.和Noctor， s.c.。小胶质细胞调节发育中的大脑皮层神经前体细胞的数量。中国生物医学工程学报（英文版），2013(1)。8. Ueno， M.和Yamashita， T.发育中的小胶质细胞的双向调节：从神经发生到神经回路形成。神经生物学现状，27,8- 15（2014）。9. 莫瑟C.-A。，巴普蒂斯塔，S., Arnoux， I.和Audinat， E.中枢神经系统发育中的小胶质细胞：塑造未来的大脑。神经生物学进展49-150,1-20（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).