Forty-eight female nude rats (6–8-week old) were used. The U251 cell line was chosen due to its clinically relevant level of EGFR expression. The animals were anesthetized using isoflurane (2 % and 1 l/min oxygen) and an incision was made in the scalp, with the brain accessed by a 1-mm rotary drill to create a burr hole. Guided using a stereotaxic frame (Stoelting Co, Wood Dale, IL, USA), the 1 × 106 cells in 5 μl phosphate-buffered saline (PBS) were injected at a 3-mm depth into the left cerebral hemisphere of the rats, 3 mm posterior to the bregma, using a Hamilton syringe (Hamilton Company, Reno, NV). The cells were injected over a 5-min period, and the needle slowly retracted from the brain. Bone wax (Ethicon, Inc., Piscataway, NJ, USA) was used to close the hole in the skull and the incision in the scalp closed, using 5–0 sterile non-absorbable suture material (Ethicon, Inc., Piscataway, NJ, USA).