By serial transplantation of human glioblastoma biopsies into the brain of immunodeficient nude rats, two different tumor phenotypes were obtained. Initially, the transplanted xenografts displayed a highly invasive phenotype that showed no signs of angiogenesis. By serial transplantation in animals, the tumors changed to a less invasive, predominantly angiogenic phenotype. To identify novel proteins related to the invasive phenotype, the xenografts were analyzed using a global proteomics approach. One of the identified proteins was protein disulfide isomerase (PDI) A6 precursor. PDI is a chaperone protein that mediates integrin-dependent cell adhesion. It is both present in the cytosol and at the cell surface. We show that PDI is strongly expressed on invasive glioma cells, in both xenografts and at the invasive front of human glioblastomas. Using an in vitro migration assay, we also show that PDI is expressed on migrating glioma cells. To determine the functional significance of PDI in cell migration, we tested the effect of a PDI inhibitor, bacitracin, and a PDI monoclonal antibody on glioma cell migration and invasion in vitro. Both tumor spheroids derived from human glioblastoma xenografts in nude rat brain and cell line spheroids were used. The PDI antibody, as well as bacitracin, inhibited tumor cell migration and invasion. The anti-invasive effect of bacitracin was reversible after withdrawal of the inhibitor, indicating a specific, nontoxic effect. In conclusion, using a global proteomics approach, PDI was identified to play an important role in glioma cell invasion, and its action was effectively inhibited by bacitracin. (Cancer Res 2006; 66(20): 9895-902)

In this first study of the influence of SFM on primary glioma spheroids, the conditions favored an in vivo-like phenotype with increased expression of CD133. More vascular structures were found in SFM, suggesting that the close relationship between blood vessels and tumor stem-like cells was better preserved in this medium.

We have previously established two distinct glioma phenotypes by serial xenotransplantation of human glioblastoma (GBM) biopsies in nude rats. These tumors undergo a gradual transition from a highly invasive nonangiogenic to a less-invasive angiogenic phenotype. In a protein screen to identify molecular markers associated with the infiltrative phenotype, we identified ␣-basic-crystallin (␣Bc) , a small heatshock protein with cytoprotective properties. Its increased expression in the infiltrative phenotype was validated by immunohistochemistry and Western blots , confirming its identity to be tumor-derived and not from the host. Stereotactic human GBM biopsies taken from MRI-defined areas verified stronger ␣Bc expression in the infiltrative edge compared to the tumor core. Cell migration assays and immunofluorescence staining showed ␣Bc to be expressed by migrating cells in vitro. To determine ␣Bc function, we altered its expression levels. ␣Bc siRNA depletion caused a loss of migrating tumor cells from biopsy spheroids and delayed monolayer wound closure. In contrast, glioma cell migration in a Boyden chamber assay was unaffected by either ␣Bc knockdown or overexpression, indicating that ␣Bc is not functionally linked to the cell migration machinery. However, after siRNA ␣Bc depletion, a significant sensitization of cells to various apoptotic inducers was observed (actinomycin, tumor necrosis factor ␣, and TNF-related apoptosisinducing ligand [TRAIL]). In conclusion, ␣Bc is overexpressed by highly migratory glioma cells where it plays a functional role in apoptosis resistance. (Am J Pathol