Neural stem cells proteomic contribution to glioblastoma malignancy

神经干细胞蛋白质组对胶质母细胞瘤恶性肿瘤的贡献

• 批准号: 10458526
• 负责人: Emily Shannon Norton-Ramos
• 金额: $ 3.88万
• 依托单位: MAYO CLINIC JACKSONVILLE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-02 至 2023-04-13
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10458526
• 关键词: Adult; Brain Neoplasms; Cell Communication; Cells; Cerebrospinal Fluid; Coculture Techniques; Disease; Distal; Enzyme-Linked Immunosorbent Assay; Ependymal Cell; Excision; Gene Expression; Gene Expression Profile; Gene Proteins; Glioblastoma; Goals; Homeostasis; Human; Immunohistochemistry; In Vitro; Intracranial Neoplasms; Label; Laboratories; Lateral; Lentivirus; Life; Life Expectancy; Link; Location; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of brain; Mammals; Measures; Methionine-tRNA Ligase; Modeling; Mus; Nature; Outcome; Pathway interactions; Patient-Focused Outcomes; Patients; Phenotype; Play; Primary Brain Neoplasms; Prognosis; Proteins; Proteome; Proteomics; Recurrence; Rodent; Rodent Model; Role; Sampling; Site; Source; System; TAL1 gene; Tamoxifen; Testing; Therapeutic; Western Blotting; Work; base; brain repair; experimental study; improved; in vivo; lateral ventricle; migration; monolayer; mouse model; mutant; neoplastic cell; nerve stem cell; nestin protein; new therapeutic target; novel; organizational structure; patient prognosis; patient stratification; promoter; protein expression; release factor; response; stem; stem cell proliferation; stemness; subventricular zone; survival outcome; tandem mass spectrometry; tumor

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults, with a median life expectancy of only 15 months and a recurrence rate approaching 100%. Tumors in contact with the lateral ventricles (LVs) account for over 50% of GBM cases and result in worse patient prognosis and increased distal recurrence. Despite worse prognosis for patients, there are no significant changes in gene or protein expression signatures in the bulk tumor signature when stratifying samples based on LV contact. Therefore, patient outcome may be due in part to the interaction of GBM with the subventricular zone (SVZ) microenvironment. The SVZ is located along the lateral walls of the LVs and contains dynamic neural stem cells (NSCs) that contribute to brain repair and homeostasis throughout life. Through my preliminary experiments, I have developed a novel rodent model reproducing the increased proliferation and decreased survival of patients with LV-proximal tumors. I have observed that culturing patient GBM cells with human NSCs increases the levels of proliferation and malignant protein expression. Additionally, I have observed decreased levels of NSC proliferation and increased stem gene expression when stimulated with GBM cells. I hypothesize that GBM alters the native NSC proteome and secretome, ultimately resulting in increased tumor malignancy. My long-term goal is to determine whether SVZ factors are responsible for the worse prognosis of LV-associated GBM and to target this interaction therapeutically. To evaluate this hypothesis, I propose the two specific aims. In specific aim 1, I will characterize changes in the NSC-specific proteome and secreted proteins in response to LV-proximal GBM. This will be done using the L274G mutant methionyl-tRNA synthetase (MetRS*) protein labeling system in vitro and in vivo. For in vitro studies, human NSCs will be transduced to express MetRS* and co-cultured with patient-derived GBM cells. In vivo, the mutant MetRS* will be expressed under the Nestin promoter upon induction with tamoxifen, leading to specific proteomic labeling in NSCs. For both approaches, lysate proteins and secreted factors will be isolated and analyzed by tandem mass spectrometry, and identified targets will be confirmed using western blot, ELISA, and immunohistochemistry. In specific aim 2, I will determine the effect of NSC-secreted components on GBM malignancy. I will co-culture GBM cells with their NSC counterparts and measure GBM malignancy through changes in proliferation, migration, and stemness. I will also co-inject GBM cells with NSC secreted factors into a mouse model and analyze brains for tumor size, proliferation, cell invasiveness, and survival outcome. Through the identification of novel, NSC-derived proteomic targets that promote GBM, this work will contribute to my long-term goal of determining whether SVZ-derived factors are responsible for the worse prognosis of LV-proximal GBM and to target this interaction therapeutically.

摘要 胶质母细胞瘤（GBM）是成人中最常见和最具侵袭性的原发性脑肿瘤， 预期寿命只有15个月，复发率接近100%。肿瘤与外侧接触 脑室（LV）占GBM病例的50%以上，导致患者预后较差，远端出血增加。 复发尽管患者预后较差，但基因或蛋白质没有显著变化， 当基于LV接触对样品进行分层时，在本体肿瘤标记中的表达标记。因此，我们认为， 患者结局可能部分归因于GBM与脑室下区（SVZ）的相互作用 微环境SVZ沿着LV的侧壁分布，含有动力神经干 神经干细胞（NSC）有助于大脑修复和整个生命的稳态。通过我的初步调查 在实验中，我开发了一种新的啮齿动物模型，重现了增殖增加和减少的情况。 左心室近端肿瘤患者的生存率。我已经观察到，用人GBM细胞培养患者GBM细胞， 神经干细胞的增殖和恶性蛋白表达水平增加。另外，我观察到 当用GBM细胞刺激时，NSC增殖水平降低，干细胞基因表达增加。 我假设GBM改变了天然NSC蛋白质组和分泌组，最终导致 肿瘤恶性程度增加。我的长期目标是确定SVZ因素是否是导致 LV相关GBM的预后更差，并在治疗上靶向这种相互作用。评价这一 假设，我提出了两个具体的目标。在具体目标1中，我将描述神经干细胞特异性 蛋白质组和分泌的蛋白质对LV近端GBM的反应。这将使用L274 G突变体完成 甲硫氨酰-tRNA合成酶（MetRS*）蛋白标记系统的体外和体内研究。对于体外研究， NSC将被转导以表达MetRS*，并与患者来源的GBM细胞共培养。在体内 突变MetRS* 在用他莫昔芬诱导后将在巢蛋白启动子下表达，导致特异性的 神经干细胞中的蛋白质组标记。对于这两种方法，裂解物蛋白和分泌因子将被分离， 通过串联质谱分析，并使用蛋白质印迹，ELISA， 和免疫组织在具体目标2中，我将确定神经干细胞分泌的组分对GBM的影响 恶性肿瘤我将GBM细胞与它们的NSC对应物共培养，并通过 增殖、迁移和干性的变化。我还将GBM细胞与NSC分泌的因子共注射到 小鼠模型，并分析大脑的肿瘤大小，增殖，细胞侵袭性和生存结果。 通过鉴定促进GBM的新的神经干细胞衍生的蛋白质组学靶点，这项工作将有助于 我的长期目标是确定SVZ衍生因素是否是导致预后不良的原因。 LV-近端GBM和治疗靶向这种相互作用。

项目成果

Glioblastoma disrupts the ependymal wall and extracellular matrix structures of the subventricular zone.

• DOI: 10.1186/s12987-022-00354-8
• 发表时间: 2022-07-11
• 期刊: FLUIDS AND BARRIERS OF THE CNS
• 影响因子: 7.3
• 作者: Norton, Emily S.;Whaley, Lauren A.;Ulloa-Navas, Maria Jose;Garcia-Tarraga, Patricia;Meneses, Kayleah M.;Lara-Velazquez, Montserrat;Zarco, Natanael;Carrano, Anna;Quinones-Hinojosa, Alfredo;Garcia-Verdugo, Jose Manuel;Guerrero-Cazares, Hugo
• 通讯作者: Guerrero-Cazares, Hugo