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.