A novel hexameric CD95 ligand as a potent agonist of apoptosis in human glioma cells in vitro and in vivo

Glioblastoma is the most aggressive primary human brain tumor. One feature of the malignant phenotype of gliomas is resistance to CD95-induced apoptosis. CD95/Fas/APO-1 is a member of the tumor necrosis factor (TNF) receptor family. Activation of CD95 leads to caspase-dependent apoptotic cell death in sensitive cells. The soluble form of monomeric CD95 ligand fails to initiate this death cascade but it becomes active upon cross linking. Here we report the pro-apoptotic potency of a novel hexameric CD95 ligand (FasL) called MegaFas ligand (MFL) in glioma cell lines and primary glioma cell cultures in vitro and in an orthotopic xenograft mouse model in vivo. MFL is a recombinant protein obtained by fusion of the collagen-like domain of human adiponectin (ACRP30) to the extracellular portion of FasL. The potency of MFL was compared with a (1) flag-tagged soluble FasL cross linked with a monoclonal antibody (sFasL) and (2) an agonist monoclonal anti-CD95 antibody (clone CH-11). In U88MG and LNT-229 glioma cells, MFL was a potent inducer of apoptosis with a mean EC50 of 5ng/ml and 50ng/ml, respectively, as measured by crystal violet assay. MFL induced apoptosis, as verified by measuring DEVD-amc cleavage and by assessment of Annexin-V staining in flow cytometry. sFasL showed an EC50 of 100ng/ml in U88 cells and only induced low levels of cell death in LNT-229 glioma cells. The anti-CD95 agonist antibody failed to induce relevant apoptosis in these cell lines. Notably, MFL also induced apoptosis in 8 out of 10 randomly selected ex vivo primary human glioma cell cultures. Induction of apoptosis by MFL was again superior to sFasL. To investigate the action of MFL in vivo, U88MG glioma cells were implanted stereotactically in the right striatum of athymic nude mice. Treatment of xenografted mice by local administration of MFL resulted in a clear prolongation of survival and induction of apoptosis as assessed by TUNEL- and caspase 3- staining in immunohistochemistry.

In summary, MFL is a novel promising agent for local treatment of glioblastomas. Nevertheless, further studies are needed to investigate the safety and optimal dosage for local administration of MFL.