ROLE OF SPARC IN GLIOMA INVASION

SPARC 在神经胶质瘤侵袭中的作用

• 批准号: 6514616
• 负责人: SANDRA ANN REMPEL
• 金额: $ 23.82万
• 依托单位: HENRY FORD HEALTH SYSTEM
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-16 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6514616
• 关键词: angiogenesis; astrocytes; cell migration; cell proliferation; collagen; cyclins; fibronectins; flow cytometry; gene targeting; genetically modified animals; glioma; hyaluronate; immunofluorescence technique; integrins; laboratory mouse; laboratory rat; laminin; necrosis; neoplasm /cancer invasiveness; neoplastic growth; osteonectin; paxillin; tenascin; vinculin; vitronectin

DESCRIPTION: (Adapted from the investigator's abstract) The poor prognosis of glioma patients is largely due to the highly invasive nature of these tumors. We have demonstrated that SPARC is highly expressed in gliomas and that it functionally contributes to invasion in vitro. Importantly, our Preliminary Data show that SPARC promotes invasion in vivo. The focus of this proposal is to determine the mechanisms by which SPARC expression promotes brain tumor invasion. In Specific Aim 1. we will determine the extent to which SPARC functionally promotes glioma invasion by: la) Characterizing the effects of SPARC expression on tumor cell invasion during the course of tumor development in viva. We will use doxycycline (dox)-regulatable SPARC-transfected U87 glioma clones in a nude rat xenograft model to evaluate the effects of increased SPARC expression on the extent of tumor cell invasion, angiogenesis. necrosis, proliferation, and overall tumor growth during the course of tumor progression. ib) Determining whether tumor-expressed SPARC is sufficient for tumor invasion in vitro. We will assess tumor invasion into normal and Sparc knockout fetal aggregates in the in vitro spheroid confrontation assay. ic) Determining the extent to which loss ol SPARC expression inhibits brain tumor invasion in viva. We will use a SPARC-targeting construct and homologous recombination to create a SPARC knockout in the C6 glioma cell line that will be assessed as in Specific Aim 1a. In Specific Aim 2, we will determine the biological mechanism(s) by which SPARC promotes tumor invasion. We will use our U87T2 parental clone and SPARC-transfected clones (+/- dox), serum-free C6 (+/- SPARC), and Sparc (-/-) and (+7+/-) fetal astrocytes to: 2a) Analyze how SPARC promotes the critical intermediate stage of adhesion. We will use the three cell models in timed studies using different concentrations of brain ECM molecules. We will assess the effect of SPARC on 1) attachment, 2) spreading, 3) or formation of focal adhesions and stress fibers via its affects on integrins and focal adhesion-associated proteins. 2b) Determine whether SPARC promotes migration in a concentration-dependent, ECM-specific manner. We will use the three cell models to perform migration assays using different concentrations of ECMs. Migration will be assessed using the cell sedimentation assay, the wounding assay, and Boyden chambers. 2c) Determine whether SPARC promotes invasion by reducing the proliferation rate of tumors. We will assess SPARC's effect on proliferation by performing 1) growth curves for the three cell models on specific brain ECM molecules, as well as perform 2) anchorage-independent growth assays, and 3) FACS analyses under the same conditions. Direct effects of SPARC on cell cycle progression will be monitored for changes in cyclin A, tk, and p107 expression and/or phosphorylation.

描述：（改编自研究者摘要） 神经胶质瘤患者的死亡主要是由于这些肿瘤的高度侵袭性。 我们已经证明，在神经胶质瘤中高表达， 在功能上有助于体外侵袭。重要的是，我们的初步 数据显示，在体内，H2 O2促进侵袭。这项建议的重点是 以确定hepatocellular carcinoma表达促进脑肿瘤的机制 入侵具体目标1。我们将确定在多大程度上 通过以下方式功能性地促进胶质瘤侵袭： 肿瘤发生发展过程中肿瘤细胞侵袭性的表达 活的我们将使用多西环素（dox）可调节的SPARC转染的U87胶质瘤 克隆在裸鼠异种移植模型中，以评估增加的细胞增殖的作用。 表达对肿瘤细胞侵袭程度、血管生成的影响。坏死， 增殖和肿瘤进展过程中的总体肿瘤生长。 ib）确定肿瘤表达的EGFR是否足以用于肿瘤侵袭 体外我们将评估肿瘤对正常和Sparc基因敲除胎儿的侵袭， 在体外球状体对抗测定中观察聚集体。c）确定 在体内，表达缺失抑制脑肿瘤侵袭的程度。 我们将使用一个SPARC靶向构建体和同源重组， 在C6神经胶质瘤细胞系中的cDNA 3基因敲除，其将如在 具体目标1a。在具体目标2中，我们将确定生物 这是一种促进肿瘤侵袭的机制。我们将使用U87 T2 亲本克隆和经SPARC转染的克隆（+/- dox）、无血清C6（+/-dox） Sparc（-/-）和（+7+/-）胎儿星形胶质细胞： 2a）分析粘合剂如何促进粘合的关键中间阶段。我们 将在不同浓度的定时研究中使用三种细胞模型， 脑细胞外基质分子我们将评估情绪对1）依恋，2） 扩散，3）或通过其影响形成局部粘连和应力纤维 对整合素和粘着斑相关蛋白的影响。2.确定是否 依达拉奉以浓度依赖性、ECM特异性方式促进迁移。我们 将使用三种细胞模型，使用不同的 ECM浓度。将使用细胞沉降法评估迁移 试验、创伤试验和Boyden室。2c）确定是否 通过降低肿瘤的增殖率来促进侵袭。我们将评估 通过执行1）三种生长曲线， 特定脑ECM分子的细胞模型，以及执行2） 锚定非依赖性生长测定，和3）在相同条件下的FACS分析。 条件将监测顺铂对细胞周期进程的直接影响 细胞周期蛋白A、tk和p107表达和/或磷酸化的变化。