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To reveal molecular drivers of glioma invasion, two distinct glioblastoma (GBM) cell phenotypes (invading cells and tumor core cells) were collected from 19 GBM specimens using laser capture microdissection. Isolated RNA underwent whole human genome expression profiling to identify differentially expressed genes. Pathway enrichment analysis highlighted the bidirectional receptor/ligand tyrosine kinase system, EphB/ephrin-B, as the most tightly linked system to the invading cell phenotype. Clinical relevance of ephrin-B genes was confirmed in a clinically annotated expression data set of 195 brain biopsy specimens. Levels of ephrin-B1 and -B2 mRNA were significantly higher in GBM (n = 82) than in normal brain (n = 24). Kaplan–Meier analysis demonstrated ephrin-B2, but not ephrin-B1, expression levels were significantly associated with short term survival in malignant astrocytomas (n = 97, p = 0.016). In human brain tumor specimens, the production and phosphorylation of ephrin-B2 were high in GBM. Immunohistochemistry demonstrated ephrin-B2 localization primarily in GBM cells but not in normal brain. A highly invasive glioma cell line, U87, expressed high levels of ephrin-B2 compared with relatively less invasive cell lines. Treatment with EphB2/Fc chimera further enhanced migration and invasion of U87 cells, whereas treatment with an ephrin-B2 blocking antibody significantly slowed migration and invasion. Forced expression of ephrin-B2 in the U251 cell line stimulated migration and invasion in vitro and ex vivo, concomitant with tyrosine phosphorylation of ephrin-B2. These results demonstrate that high expression of ephrin-B2 is a strong predictor of short-term survival and that ephrin-B2 plays a critical role in glioma invasion rendering this signaling pathway as a potential therapeutic target.