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.
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