Evaluating Gefitinib for Lung Cancer Prevention

Two animal studies were carried out. In both study, all mice were monitored for their well-being, including body weight (BW), general appearance (e.g., skin, hair [rough hair coat], eyes, nose, breathing, locomotion), clinical signs (e.g., rapid weight loss, diarrhea, bleeding), behavior changes (e.g., eating, drinking, or lethargy), breathing abnormalities, and posture (e.g., hunched posture or body hunching).
In experiment 1, mice were given a single i.p. injection of B(a)P at 100 mg/kg body weight in 0.2 ml of tricaprylin. Six-week-old AJ/p53^val135/wt mice were randomized into 6 groups with 18 mice per group (Figure 1A). Gefitinib was freshly prepared in Mazola corn oil before gavage administration. Oral gavage was administered once a day 5 times a week for the duration of the study. Control animals were treated with 0.2 ml corn oil throughout the study. The treatment was begun 2 weeks after the B(a)P injection and continued for 18 consecutive weeks. The daily dose of Gefitinib was 80 mg/kg body weight; oral gavage once a day, 5 days a week. The weekly dose of Gefitinib was 400 mg/kg body weight; oral gavage once weekly. The intermittent dosing of Gefitinib was 400 mg/kg body weight; oral gavage once weekly on weeks 3, 4, 5, 9, 10, 11, 15, 16, 17 with final sacrifice at week 20.
In experiment 2, Swiss/p53^val135/wt mice at 8 weeks of age were given the first dose of NTCU and this time point is noted as “week 0”. The dorsal skin of each mouse was shaved 24 to 48 hours prior to the first dose of NTCU. For the application of NTCU, 100-microliter drops of 0.04 M NTCU was applied to the shaved skin with a micro-pipette. This process was repeated twice a week with a 3.5-day interval for 30 consecutive weeks (Figure 2A).
Two weeks after the first dose of NTCU when mice were ∼10 weeks old, mice were divided into the 4 groups and treated with Gefitinib using both daily and weekly dosing regimens. Gefitinib daily (80 mg/kg body weight/day i.g., 5 days a week) and weekly dosing (5 × 80 mg = 400 mg/kg body weight i.g. oral gavage once weekly) were used.
Animals were housed with wood chip bedding in an environmentally controlled, clean-air room with a 12-hour light-dark cycle and a relative humidity of 50%. Drinking water and diet were supplied ad libitum. The study was approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin.
For both experiments, body weight was recorded weekly. Mice were euthanized by carbon dioxide (CO₂) asphyxiation. Lungs of each mouse were fixed in zinc formalin solution overnight then stored in 70% ethanol [17 (link)]. For the lung AD model, the fixed lungs were evaluated under a dissecting microscope to obtain the surface tumor count and individual tumor diameter. Tumor volume was calculated based on the formula: V = 4πr³/3. The total tumor volume in each mouse was calculated from the sum of all tumors. Tumor load was determined by averaging the total tumor volume of each mouse in each group. For the SCC model, mice were euthanized by CO₂ asphyxiation at 32 weeks after the first dose of NTCU treatment. Lungs from all mice were fixed in Zinc formalin overnight then stored in 70% ethanol. Histopathological evaluation of the lung tumors was carried out using the following method [18 (link)]: approximately 100 serial tissue sections (4 μm each) were cut from formalin fixed lung, and one in every 20 sections (approximately 100 μm apart) was stained with hematoxylin and eosin (H&E). The lung SCCs area/lung lobe area ratio was evaluated using NanoZoomer Digital Pathology Virtual Slide Viewer software (Hamamatsu Photonic Co.). H&E-stained slides were scanned with the NanoZoomer HT slide scanner (Hamamatsu Photonics), and virtual slides were analyzed and quantified.