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TMZ + LEV + IFN-α further obviously increased TMZ + LEV or TMZ + IFN-α efficiency in MGMT-positive cell lines, while not in negative cell lines (SKMG-4, U87, U373, and U251) in vitro, which were also observed in subcutaneous mice models (U138, GSC-1 compared to SKMG-4, U87) and orthotopic models (GSC-1) in vivo. Strikingly, the combination of LEV and IFN-α together with TMZ significantly prolonged the survival of mice with orthotopic GSC-1 glioma. Furthermore, we confirmed that the combination of LEV and IFN-α enhanced the inhibition of