Comparison of the Efficacy of EGFR Tyrosine Kinase Inhibitors Erlotinib and Low-dose Osimertinib on a PC-9-GFP EGFR Mutant Non-small-cell Lung Cancer Growing in the Brain of Nude Mice

Materials and Methods

Cell line. GFP-expressing PC-9 (NSCLC, EGFR exon 19 deletion)-GFP cells (AntiCancer, Inc., San Diego, CA, USA) were maintained in RPMI-1640 medium (Mediatech, Inc. Manassas, VA, USA) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 37°C with 95% air and 5% CO₂, and split 48 h prior to inoculation to ensure they were in log growth phase when harvested. Then, cells were re-suspended at a concentration of 4×10⁷ cells/ml in serum-free RPMI medium. Suspended cells were well mixed with ice-thawed Matrigel at 1:1 ratio for inoculation (7).

Experimental animals. Athymic nude (nu/nu) male mice (6-8 week AntiCancer, Inc., San Diego, CA, USA) were utilized. Animal housing and their diet were as previously described (7). Mice were observed on a daily basis and humanely sacrificed by CO₂ inhalation with humane endpoint criteria as previously described (7).

Tumor stock preparation using subcutaneous models. Nude female mice (5-6) weeks were used for tumor stock. Each mouse received a subcutaneous cancer-cell inoculation in both flanks with a 0.1 ml inoculum of 2.0×10⁶ cells. Once the tumor diameter reached approximately 10 mm, the mouse was anaesthetized and euthanized in order to resect the tumor (7).

Establishment of brain implantation model. Subcutaneous tumors were harvested and cut into small fragments (2-3 mm in diameter). Mice were anesthetized with a subcutaneous ketamine mixture. An 8-mm sagittal incision of the scalp was made to expose the skull. A 5 mm v-shaped flap on the skull was made. Each tumor fragment was implanted between the bone flap and brain. The skin was closed with a surgical suture (7).

Randomization and Initiation of treatment. Group randomization took place the day prior to the dosing day (day 0) based on the condition of the animals. Treatment was started 3 weeks later in the brain models when the tumor GFP fluorescent area reached 8-60 mm².

Treatment protocol. The mice were randomized into 5 groups of 10 mice each; G1: control group [vehicle (PBS + 1% DMSO), 0.1 ml/10 g body weight, oral administration (p.o.), once daily (qd)]; G2: erlotinib 5 mg/kg + vehicle, 0.1 ml/10 g body weight, p.o., qd; G3: erlotinib 50 mg/kg + vehicle, 0.1 ml/10 g body weight, p.o., qd; G4: osimertinib 0.5 mg/kg + vehicle, 0.1 ml/10 g body weight, p.o., qd; G5: osimertinib 5 mg/kg + vehicle, 0.1 ml/10 g body weight, p.o., qd.

Mice were observed daily for general mobility, morbidity and mortality. Body weight was evaluated twice a week. Mouse deaths were assessed as tumor-related, drug-related, technical, or of unknown reason based on factors including gross observation and weight loss and number and day of death in each group. Signs of toxicity were monitored, including cachexia, diarrhea, skin rash and color. Animals were sacrificed 15 days after treatment initiation, or earlier, if mice became moribund.

Imaging. A FluorVivo Small Animal Imaging System (INDEC BioSystems, Santa Clara, CA, USA) was used for fluorescence imaging. Tumor volume was calculated by multiplying fluorescent tumor area (mm²) and fluorescence intensity (% × 1/100).

Statistical analysis. All statistical analyses were performed with EZR (http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html). Statistical analysis of tumor volume and body weight was performed using the Student's t-test to compare differences among treatment groups. The Kruskal-Wallis test was performed as a non-parametric test. A probability value of p<0.05 was considered as statistically significant.

Ethical considerations. All experiments were done at AntiCancer Inc. with an Institutional Animal Care and Use Committee (IACUC)-protocol solely approved for the study and following the principles and procedures defined in the National Institutes of Health (NIH) Guide for the Care and Use of Animals under Assurance Number A3873-1.