Investigating the role of glutamine metabolism in breast tumor innervation and brain metastasis

项目总结--项目负责人英慧松 这个项目的总体目标是了解改变的谷氨酰胺代谢在乳腺肿瘤中的作用。 神经支配及其与脑转移的联系。最近对临床数据的分析表明，神经纤维 在浸润性导管癌中的表达显著高于导管原位癌和正常组织 乳房组织。有证据表明谷氨酰胺代谢可能促进乳腺肿瘤的神经和脑功能 转移：1)乳腺癌细胞表达高水平的谷氨酰胺酶，通过转化为 谷氨酰胺到谷氨酸，2)癌症相关成纤维细胞上调谷氨酰胺合成酶表达以满足 乳腺癌细胞对谷氨酰胺的需求，3)糖酵解和谷氨酰胺分解之间的协同作用促进癌症 细胞将谷氨酰胺转化为乳酸，刺激脑源性神经营养因子(BDNF)的分泌和 4)脑转移的乳腺癌细胞表达突触后标记PSD-95和 脑源性神经营养因子受体TrkB。然而，抑制这几个关键代谢途径和减少转移的可能性 三阴性乳腺癌的表型尚不清楚。为此，我们假设 三阴性乳腺癌细胞谷氨酰胺代谢升高促进乳腺肿瘤神经支配和 脑部转移。为了验证这一假设，我们将对3D培养平台进行生物工程，使其接近于乳房 肿瘤微环境。为此，我们将开发一种乳腺肿瘤的组织工程模型 以脱细胞乳腺组织为支架培养4T1小鼠三重阴性乳腺的微环境 癌细胞、小鼠原代脂肪基质细胞(ASCs)和小鼠初级背根节(DRG)(AIM 1)。我们将分析乳腺肿瘤神经支配的三维模型的谷氨酰胺代谢分布。 -ART代谢谱工具和开发代谢辅因子的内源性自体荧光(AIM 2)。最后，我们将通过免疫荧光来评估乳腺肿瘤神经支配对脑转移的影响。 体外和体内植入DRG轴突条件下的4T1和ASCs(目标3)。谷氨酰胺抑制剂 合成、消耗和谷氨酸生产/功能将被测试其阻断神经支配和功能的能力 结果将在《数据科学核心》杂志上进行分析，以评估谷氨酰胺对乳腺肿瘤神经支配的影响 在试管中。我们将利用三个核心(用于数据分析的数据科学核心、成像和光谱核心 用于3D体积延时共聚焦和多光子成像，以及用于代谢分析的生物能学核心 体外培养)，以达到研究设计、执行、分析和解释的高度严谨性，并改进 我们对乳腺肿瘤的神经支配和脑转移的理解。与蒂姆博士的指导相结合 马尔登和罗伯特·格里芬博士以及AIMRC的高级指导委员会和其他中心领导 宋博士将为科学独立建立坚实的道路，导致在#年末提交R01 第二年，重点是与目标3的重点和长期项目目标相呼应的全面研究。

Project Summary – Project Leader Young Hye Song The overall goal of this project is to understand the role of altered glutamine metabolism in breast tumor innervation and its connection to brain metastasis. Recent analyses of clinical data have shown that nerve fibers are significantly more present in invasive ductal carcinoma compared to both ductal carcinoma in situ and normal breast tissue. There exists evidence that glutamine metabolism may promote breast tumor innervation and brain metastasis: 1) breast cancer cells express high levels of glutaminase that initiates glutaminolysis by converting glutamine to glutamate, 2) cancer-associated fibroblasts upregulate glutamine synthetase expression to meet glutamine demands of breast cancer cells, 3) a synergy between glycolysis and glutaminolysis enhances cancer cell conversion of glutamine to lactate, which stimulates brain-derived neurotrophic factor (BDNF) secretion and drive neurite extension, 4) brain metastasizing breast cancer cells express post-synaptic marker PSD-95 and BDNF receptor TrkB. Yet, the potential to inhibit these several key metabolic pathways and reduce metastatic phenotypes in triple negative breast cancer have not been clearly elucidated. To this end, we hypothesize that elevated glutamine metabolism by triple negative breast cancer cells promotes breast tumor innervation and brain metastasis. To test this hypothesis, we will bioengineer 3D culture platforms to closely mimic the mammary tumor microenvironment. To do so, we will develop a tissue engineered model of the mammary tumor microenvironment using decellularized mammary tissue as a scaffold to culture 4T1 mouse triple negative breast cancer cells, mouse primary adipose stromal cells (ASCs) and mouse primary dorsal root ganglia (DRG) (Aim 1). We will analyze glutamine metabolic profile of the 3D model of breast tumor innervation by using state-of- the-art metabolism profiling tools and exploiting the endogenous autofluorescence of metabolic cofactors (Aim 2). Finally we will assess the influence of breast tumor innervation on brain metastasis via immunofluorescence in vitro and in vivo implantation of DRG neurite-conditioned 4T1s and ASCs (Aim 3). Inhibitors of glutamine synthesis, consumption and glutamate production/function will be tested for their ability to block innervation and the results will be analyzed in the Data Science Core to evaluate glutamine effects on breast tumor innervation in vitro. We will utilize the three cores (Data Science Core for data analysis, Imaging & Spectroscopy Core for 3D volumetric time-lapse confocal and multiphoton imaging, and Bioenergetics Core for metabolic profiling of in vitro cultures) to achieve strong rigor in study design, execution, analysis and interpretation and improve our understanding of breast tumor innervation and brain metastasis. Combined with mentoring by Dr. Tim Muldoon and Dr. Robert Griffin, as well as the AIMRC’s senior mentoring committee and other center leaders Dr. Song will establish a solid pathway for scientific independence leading to an R01 submission at the end of year 2 that focuses on a full scope study echoing the focus of Aim 3 and the long-term project goals.