Eph receptor tyrosine kinases and their ligands, ephrins, mediate 
neurodevelopmental processes such as boundary formation, axon guidance, 
vasculogenesis, and cell migration. We determined the expression profiles of the 
Eph family members in five glioma cell lines under migrating and nonmigrating 
conditions. EphB2 mRNA was overexpressed in all five during migration 
(1.2-2.8-fold). We found abundant EphB2 protein as well as strong 
phosphorylation of EphB2 in migrating U87 cells. Confocal imaging showed EphB2 
localized in lamellipodia of motile U87 cells. Treatment with ephrin-B1/Fc 
chimera stimulated migration and invasion of U87, whereas treatment with a 
blocking EphB2 antibody significantly inhibited migration and invasion. Forced 
expression of EphB2 in U251 cells stimulated cell migration and invasion and 
diminished adhesion concomitant with the tyrosine phosphorylation of EphB2. U251 
stably transfected with EphB2 showed more scattered and more pronounced invasive 
growth in an ex vivo rat brain slice. In human brain tumor specimens, EphB2 
expression was higher in glioblastomas than in low-grade astrocytomas or normal 
brain; patterns of phosphorylated EphB2 matched the expression levels. Laser 
capture microdissection of invading glioblastoma cells revealed elevated EphB2 
mRNA (1.5-3.5-fold) in 7 of 7 biopsy specimens. Immunohistochemistry 
demonstrated EphB2 localization primarily in glioblastoma cells (56 of 62 cases) 
and not in normal brain. This is the first demonstration that migrating 
glioblastoma cells overexpress EphB2 in vitro and in vivo; glioma migration and 
invasion are promoted by activation of EphB2 or inhibited by blocking EphB2. 
Dysregulation of EphB2 expression or function may underlie glioma invasion.