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.