Our recent studies have focused on identifying invasion-promoting genes that are expressed early in brain tumor progression. We identified and characterized SPARC (secreted protein acidic and rich in cysteine) as a potential candidate. To determine whether increased SPARC expression functionally promotes brain tumor invasion, SPARC was transfected into U87MG glioblastoma cells using the tetracycline-off gene expression system. The parental cell line (U87MG), the parental transactivator-transfected clone (U87T2) and three selected U87T2-SPARC-transfected clones (A2bi, A2b2 and C2a4) were characterized for endogenous and transfected SPARC expression. In comparison to the parental or U87T2 cell lines, the SPARC-transfected clones demonstrated: (1) morphological changes, (2) increased SPARC transcript and protein abundances that were down-regulated by the tetracycline analog doxycycline, (3) perinuclear localization of the transfected SPARC (consistent with reported localization of SPARC in normal cells in culture) and (4) altered adhesion and increased invasion as assessed by the spheroid confrontation assay. These data indicate that increased SPARC expression contributes to U87 glioblastoma tumor invasion in vitro and that these cell lines will serve as useful reagents to investigate the mechanism(s) by which SPARC promotes this phenotype in vitro and in vivo.

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In an attempt to identify genetic alterations occurring early in astrocytoma progression, we performed subtractive hybridization between astrocytoma and glioblastoma cDNA libraries. We identified secreted protein acidic and rich in cysteine (SPARC), a protein implicated in cell-matrix interactions, as a gene overexpressed early in progression. Northern blot and immunohistochemical analyses indicated that transcript and protein were both elevated in all tumor specimens (grades II-IV) examined when compared with levels in normal brain. The level of SPARC expression was found to be tumor-dependent rather than grade-related. Immunohistochemically, SPARC protein was found to be overexpressed in 1) cells in the less cellularly dense regions within the tumor mass, 2) histomorphologically neoplastic-looking cells in adjacent normal brain at the tumor/brain interface, 3) neovessel endothelial cells in both the tumor and adjacent normal brain, and 4) reactive astrocytes in normal brain adjacent to tumor. Using a combination of DNA in situ hybridization and protein immunohistochemical analyses of the human/rat xenograft, SPARC expression was observed in the human glioma cells within the tumor mass, and in cells that invaded along vascular basement membranes and individually into the rat brain parenchyma, suggesting it may be an invasion-related gene. While it remains to be determined whether SPARC functionally contributes to tumor cell invasion, these data suggest that the early onset of increased SPARC expression, though complex, may serve as a signal indicative of neoplastic astrocytic transformation and reactive response to tumor-induced stress.