ROLE OF SPARC IN GLIOMA INVASION

描述：(改编自调查人员的摘要)本病预后不良 胶质瘤患者在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，我们的初选 数据显示，SPARC促进体内侵袭。这项提案的重点是 确定SPARC表达促进脑肿瘤发生的机制 入侵。在具体目标1.我们将确定SPARC的程度 功能性地通过以下方式促进胶质瘤的侵袭：LA)表征 SPARC在肿瘤发生发展过程中对肿瘤细胞侵袭的表达 万岁。我们将使用多西环素(Dox)可调节的SPARC转基因U87胶质瘤 在裸鼠移植模型中建立克隆以评估SPARC增加的效果 表达对肿瘤细胞侵袭程度、血管生成的影响。坏死， 在肿瘤进展过程中的增殖和整体肿瘤生长。 Ib)确定肿瘤表达的SPARC是否足以引起肿瘤侵袭 在试管中。我们将评估肿瘤对正常和Sparc基因敲除胎儿的侵袭性 体外球体对峙试验中的聚集体。IC)确定 SPARC表达缺失在多大程度上抑制脑瘤的体内侵袭。 我们将使用SPARC靶向构建和同源重组来创建 C6胶质瘤细胞系中的SPARC基因敲除将被评估为 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(S)。我们将使用我们的U87T2 亲本克隆和SPARC转基因克隆(/-dox)，无血清C6(/- SPARC)，以及SPARC(-/-)和(7/-)胎儿星形胶质细胞： 2)分析SPARC如何促进粘附性的关键中间阶段。我们 将在使用不同浓度的定时研究中使用这三种细胞模型 大脑细胞外基质分子。我们将评估SPARC对1)依恋的影响，2) 3)通过其影响扩散或形成局灶性粘连和应力纤维 关于整合素和焦点黏附相关蛋白。2B)确定是否 SPARC以浓度依赖、ECM特异的方式促进迁移。我们 我将使用这三种细胞模型执行迁移分析，使用不同的 ECM的浓度。迁移将使用细胞沉降法进行评估 化验、伤害性化验和博伊登室。2C)确定SPARC是否 通过降低肿瘤的增殖率来促进侵袭。我们将评估 SPARC通过绘制三种细胞的生长曲线对增殖的影响 特定脑细胞外基质分子上的细胞模型，以及执行2) 与锚定无关的生长分析，以及3)在相同条件下的FACS分析 条件。将监测SPARC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。

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.