Combination of measles virus virotherapy and radiation therapy has synergistic activity in the treatment of glioblastoma multiforme

Purpose: Glioblastoma multiforme is the most frequent primary brain tumor in adults and represents one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that an oncolytic measles virus derivative expressing soluble human carcinoembryonic antigen (MV-CEA) has significant antitumor activity against glioblastoma multiforme cell lines and xenografts. Radiation therapy (RT) represents one of the mainstays of glioma treatment. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. Experimental Design: 3-(4,5-Dimethyl-thiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) -2H-tetrazolium (MTS) and clonogenic assays were used to test cytoxicity of the combination treatment in vivo. To examine the mechanism of synergy, one-step viral growth curves, terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays, and Western blot analyses were performed. In vivo assessment of synergistic antitumor activity was conducted in a U87 glioma model. Results: MTS and clonogenic assays showed a strong synergistic interaction between MV-CEA and RT in glioblastoma multiforme cells including both primary and established glioma lines. Furthermore, significant antitumor efficacy was observed in vivo in a subcuteneous U87 xenograph model. There was significant prolongation of survival (P = 0.001) in the combination treatment group as compared with single modality- or control-treated animals. One-step viral growth curves showed increased viral burst size by up to 2 log in MV/RT combination-treated cells, as compared with single agent MV-CEA-treated glioma cells. Changes in CEA levels and expression of viral N and H protein were also consistent with increased viral production. Furthermore, TUNEL assays and Western blot analysis showed increase in apoptosis in MV/RT combination-treated cells. The pan-caspase inhibitor Z-VAD-FMK and the caspase-8 inhibitor Z-IETD-FMK, but not the caspase-9 inhibitor Z-IEHD-FMK, protected glioma cells from MV-CEA/RT-induced cleavage of poly(ADP-ribose) polymerase (PARP), indicating that the apoptotic death in combination-treated cells is mostly mediated via the extrinsic caspase pathway. The Fas/Fas ligand interaction blocking antibody NOK-1 blocked MV/RT-induced PARP cleavage whereas the Fas agonistic antibody CH11increased PARP cleavage in MV/RT combination-treated cells. Reverse transcription-PCR, fluorescence-activated cell sorting analysis and immunohistochemistry showed up-regulation of Fas in combination-treated tumor in vitro and in vivo cells. Conclusions: There is synergy between MV-CEA and RT in vitro and in vivo. The synergistic effect of the combination seems to be due to increase in viral burst size and increase in apoptotic cell death. This latter effect is mostly mediated via the extrinsic caspase-8 pathway, activated via increased signaling through the Fas death receptor pathway. These results could have translational implications in glioma therapy.