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: 10311686
• 负责人: Emily Shannon Norton-Ramos
• 金额: $ 4.6万
• 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 2023-08-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10311686
• 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 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.

抽象的 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，中位 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，远端增加 复发。尽管患者预后较差，但基因或蛋白质没有显着变化 基于 LV 接触对样本进行分层时，大量肿瘤特征中的表达特征。所以， 患者的结果可能部分归因于 GBM 与室下区 (SVZ) 的相互作用 微环境。 SVZ 位于左心室侧壁，包含动态神经干 终生有助于大脑修复和体内平衡的细胞（NSC）。通过我的初步 实验中，我开发了一种新的啮齿动物模型，可以再现增殖增加和减少 左心室近端肿瘤患者的生存率。我观察到用人类培养患者 GBM 细胞 NSC 增加增殖水平和恶性蛋白表达。另外，我观察到 当 GBM 细胞刺激时，NSC 增殖水平降低，干基因表达增加。 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 因素是否对 LV 相关 GBM 的预后较差，并针对这种相互作用进行治疗。为了评价这一点 假设，我提出了两个具体目标。在具体目标 1 中，我将描述 NSC 特定的变化 左心室近端 GBM 响应的蛋白质组和分泌蛋白。这将使用 L274G 突变体来完成 体外和体内甲硫氨酰-tRNA 合成酶 (MetRS*) 蛋白质标记系统。对于体外研究，人类 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， 在用他莫昔芬诱导后，突变型 MetRS* 将在 Nestin 启动子下表达，从而产生特定的 NSC 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都将被分离并 通过串联质谱分析，并使用蛋白质印迹、ELISA、 和免疫组织化学。在具体目标 2 中，我将确定 NSC 分泌成分对 GBM 的影响 恶性肿瘤。我将与 NSC 细胞共培养 GBM 细胞，并通过以下方式测量 GBM 恶性程度： 增殖、迁移和干性的变化。我还将 GBM 细胞与 NSC 分泌因子共同注射到 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。 通过鉴定新的 NSC 衍生的促进 GBM 的蛋白质组靶标，这项工作将有助于 我的长期目标是确定 SVZ 衍生因素是否导致较差的预后 左心室近端 GBM 并针对这种相互作用进行治疗。