Tumor xenograft model

All experimental protocols were approved by the Laboratory Animal Care and Use Committee of Hokkaido University and performed in accordance with the Guidelines for Animal Experiments at the Graduate School of Medicine, Hokkaido University. Nine-week-old male BALB/c athymic nude mice (Japan SLC, Inc., Hamamatsu, Japan) were housed in groups (4–5 animals) in ventilated cages. The environmental conditions were: temperature ~25°C, 40%–70% humidity, and a 12-h light/12-h dark cycle. The mice were fed a standard laboratory diet (FR-1, Sankyo Laboratory Service Corporation, Tokyo, Japan). Food and water were supplied ad libitum. A human head and neck cancer xenograft model was established using the human head and neck cancer cell line FaDu (American Type Culture Collection, Manassas, VA, USA). This cell line is an established human hypopharyngeal squamous cell carcinoma that grows as an undifferentiated carcinoma in nude mice [16]. The FaDu cells were maintained in Eagle’s Minimum Essential Medium (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum and penicillin (100 U/ml)–streptomycin (100 μg/ml) at 37°C in a humidified atmosphere of 95% air and 5% CO₂. FaDu cells (5×10⁶ cells) suspended in 100 μl phosphate-buffered saline were inoculated subcutaneously into the right flank of each mouse. Further experiments were performed after a 2-week tumor growth period. At that time, the body weights were 24.0 ± 1.2 g, and the tumor volumes were 853 ± 612 mm³, calculated using the formula: π/6 × largest diameter × (smallest diameter)². Mice were monitored for health and body condition every 3–4 days and observed not to show any abnormal symptoms such as weight loss or injury. Mice with tumor sizes of largest diameter ≥2 cm were excluded from this study. Excluded animals were euthanized by exsanguination under anesthesia with 1.5%–2.0% isoflurane. All animal manipulations were performed using sterile techniques.