44. Combination of Oncolytic Measles Virus Strains and Radiation Therapy Has Synergistic Activity in the Treatment of Glioblastoma Multiforme

Abstract

Background: Glioblastoma Multiforme (GBM), the most frequent primary brain tumor in adults, is one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that oncolytic measles virus derivatives expressing soluble human carcinoembryonic antigen (MV-CEA) have significant antitumor activity against GBM cell lines and xenografts. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas.Methods: MTS (Promega) and clonogenic assays were performed in U87 and U251glioma cell lines (ATCC) to assess the impact of the different treatment modalities on cell proliferation and survival. One-step viral growth curves, Western blots to assess expression of viral proteins, TUNEL assays, immunostaining for Fas/caspase-8 and western immunobloting for caspase-8, caspase-9, caspase-3 and PARP were employed to investigate the mechanism of the synergistic interaction between MV-CEA and RT in GBM cell lines.Results: MTS and clonogenic assays revealed a synergistic interaction between MV-CEA and RT in GBM cells. The optimal combination index for the RT/MV-CEA combination was 0.524 for U87 (0.01MOI/10GY) and 0.529 for U251 (0.05MOI/7.5GY), indicating strong synergy. Morphologic changes of U251 and U87 cells in response to the combination treatment demonstrated significant augmentation of cytopathic effect. In clonogenic assays, colony formation was significantly decreased in the MV-CEA/RT group as compared to either MV-CEA or RT alone. One-step viral growth curves demonstrated increased MV-CEA propagation by at least 2 logs in MV-CEA/RT combination treated cells, as compared to glioma cells treated with single agent MV-CEA. Changes in CEA levels and expression of viral N protein were also consistent with viral titer changes. TUNEL staining showed increased positivity in RT/MV-CEA group as compared to MV-CEA or RT alone. Furthermore, Western blot analysis demonstrated an increase in activated caspase-8, caspase-3 and PARP expression levels after combination treatment. The pan-caspase inhibitor Z-VAD-FMK completely protected cells from MV/RT induced cleavage of PARP, while the caspase-8 inhibitor (Z-IETD-FMK) partially protected glioma cells from MV-CEA/RT induced cleavage of PARP, and the caspase-9 inhibitor Z-IEHD-FMK showed no protection.Conclusions: Our results indicate that there is significant synergy between MV-CEA and RT in vitro. The synergistic effect of the combination appears to be due to increased viral bust size and increase in apoptotic death. This latter effect is mostly mediated via the extrinsic caspase 8 pathway. Experiments in primary glioma lines and animal experiments are ongoing. These results could have immediate translational implications in glioma therapy. Background: Glioblastoma Multiforme (GBM), the most frequent primary brain tumor in adults, is one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that oncolytic measles virus derivatives expressing soluble human carcinoembryonic antigen (MV-CEA) have significant antitumor activity against GBM cell lines and xenografts. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. Methods: MTS (Promega) and clonogenic assays were performed in U87 and U251glioma cell lines (ATCC) to assess the impact of the different treatment modalities on cell proliferation and survival. One-step viral growth curves, Western blots to assess expression of viral proteins, TUNEL assays, immunostaining for Fas/caspase-8 and western immunobloting for caspase-8, caspase-9, caspase-3 and PARP were employed to investigate the mechanism of the synergistic interaction between MV-CEA and RT in GBM cell lines. Results: MTS and clonogenic assays revealed a synergistic interaction between MV-CEA and RT in GBM cells. The optimal combination index for the RT/MV-CEA combination was 0.524 for U87 (0.01MOI/10GY) and 0.529 for U251 (0.05MOI/7.5GY), indicating strong synergy. Morphologic changes of U251 and U87 cells in response to the combination treatment demonstrated significant augmentation of cytopathic effect. In clonogenic assays, colony formation was significantly decreased in the MV-CEA/RT group as compared to either MV-CEA or RT alone. One-step viral growth curves demonstrated increased MV-CEA propagation by at least 2 logs in MV-CEA/RT combination treated cells, as compared to glioma cells treated with single agent MV-CEA. Changes in CEA levels and expression of viral N protein were also consistent with viral titer changes. TUNEL staining showed increased positivity in RT/MV-CEA group as compared to MV-CEA or RT alone. Furthermore, Western blot analysis demonstrated an increase in activated caspase-8, caspase-3 and PARP expression levels after combination treatment. The pan-caspase inhibitor Z-VAD-FMK completely protected cells from MV/RT induced cleavage of PARP, while the caspase-8 inhibitor (Z-IETD-FMK) partially protected glioma cells from MV-CEA/RT induced cleavage of PARP, and the caspase-9 inhibitor Z-IEHD-FMK showed no protection. Conclusions: Our results indicate that there is significant synergy between MV-CEA and RT in vitro. The synergistic effect of the combination appears to be due to increased viral bust size and increase in apoptotic death. This latter effect is mostly mediated via the extrinsic caspase 8 pathway. Experiments in primary glioma lines and animal experiments are ongoing. These results could have immediate translational implications in glioma therapy.