ROLE OF SPARC IN GLIOMA INVASION

SPARC 在神经胶质瘤侵袭中的作用

• 批准号: 6325364
• 负责人: SANDRA ANN REMPEL
• 金额: $ 23.71万
• 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/6325364
• 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：（改编自研究者的摘要）预后不良 神经胶质瘤患者的发病很大程度上是由于这些肿瘤的高度侵袭性。 我们已经证明 SPARC 在神经胶质瘤中高度表达，并且它 在功能上有助于体外侵袭。重要的是，我们的初步 数据显示SPARC促进体内侵袭。该提案的重点是 确定 SPARC 表达促进脑肿瘤的机制 入侵。在具体目标 1 中，我们将确定 SPARC 的程度 通过以下方式在功能上促进神经胶质瘤侵袭：la) 表征 肿瘤发展过程中SPARC表达对肿瘤细胞侵袭的影响 在维瓦。我们将使用多西环素 (dox) 调节的 SPARC 转染 U87 神经胶质瘤 在裸鼠异种移植模型中进行克隆以评估增加的 SPARC 的效果 表达对肿瘤细胞侵袭程度、血管生成的影响。坏死， 肿瘤进展过程中的增殖和总体肿瘤生长。 ib) 确定肿瘤表达的 SPARC 是否足以进行肿瘤侵袭 体外。我们将评估肿瘤对正常和 Sparc 敲除胎儿的侵袭 体外球体对抗测定中的聚集体。 ic) 确定 SPARC表达缺失在体内抑制脑肿瘤侵袭的程度。 我们将使用 SPARC 靶向构建体和同源重组来创建 C6 神经胶质瘤细胞系中的 SPARC 敲除将按如下方式进行评估 具体目标 1a。在具体目标 2 中，我们将确定生物 SPARC 促进肿瘤侵袭的机制。我们将使用我们的 U87T2 亲本克隆和 SPARC 转染克隆 (+/- dox)、无血清 C6 (+/- SPARC)、Sparc (-/-) 和 (+7+/-) 胎儿星形胶质细胞： 2a) 分析 SPARC 如何促进粘附的关键中间阶段。我们 将在不同浓度的定时研究中使用这三种细胞模型 脑 ECM 分子。我们将评估 SPARC 对 1) 依恋、2) 的影响 通过其影响扩散，3) 或形成粘着斑和应力纤维 整合素和粘着斑相关蛋白。 2b) 确定是否 SPARC 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来进行不同的迁移测定 ECM 的浓度。将使用细胞沉降来评估迁移 化验、伤害化验和博伊登室。 2c) 确定是否SPARC 通过降低肿瘤的增殖率来促进侵袭。我们将评估 SPARC 通过执行 1) 三种生长曲线对增殖的影响 特定大脑 ECM 分子的细胞模型，以及执行 2) 不依赖贴壁的生长测定，以及3)相同条件下的FACS分析 状况。将监测 SPARC 对细胞周期进展的直接影响 用于检测细胞周期蛋白 A、tk 和 p107 表达和/或磷酸化的变化。