Fig. 1.

A comparison of TMZ and a combination of EGFR plus TNF inhibition in GBMs lacking MGMT. (A) EGFR expression in the Mayo PDX lines. (B) GBM12 cells were exposed to dimethyl sulfoxide (vehicle control), TMZ (1 µM), or afatinib (1 µM) with or without etanercept (100 µg/mL), or thalidomide (1 µM) for 72 hours, followed by alamarBlue cell viability assay. (C–E) Similar experiments were conducted in GBM9, GBM22, and GBM39 cells. (F) GBM12 cells were treated with TMZ (1 μM), afatinib (1 µM) and/or etanercept (100 μg/mL), thalidomide (1μM) and/or afatinib for 72 hours followed by annexin–fluorescence activated cell sorting assay. (G) GBM12 cells were transfected with TNF receptor 1 siRNA, and after 48 hours cells were treated with afatinib for 72 hours followed by alamarBlue assay. (H) Western blotting confirming siRNA knockdown of TNF receptor 1. Data are presented as mean ± SEM of at least 3 independent experiments. (I) Both TMZ and combined treatment of afatinib plus thalidomide prolonged survival in a GBM12 orthotopic model. Ten days after intracranial injection, mice were divided into 5 groups: vehicle, afatinib (50 mg/kg), thalidomide (150 mg/kg), TMZ (50 mg/kg), afatinib plus thalidomide (n = 6). (J) A similar experiment was undertaken in a GBM9 orthotopic model (n = 6). Kaplan–Meier survival curves were calculated using GraphPad Prism 7. Statistical significance was verified by the log rank test. *P < 0.05, **P < 0.01,***P < 0.001; n.s., not significant.