4.13. Tumor Xenograft Model

All studies involving mice were conducted in accordance by a standard animal protocol approved with the Institutional Animal Care and Use Committee (Protocol #18-010-ICH-H), University of Oklahoma Health Sciences Center. Female five-week-old SCID mice were obtained from Taconic Biosciences, Inc and were maintained under a 12-h light/dark cycle under pathogen-free conditions, using sterile food and water within the cage. After acclimation period of 72 h, xenograft formation was generated by the subcutaneous injection of 10 million Ishikawa cells, suspended in 100 µL sterile normal saline. Tumor detection was assessed by palpation and once identified measurement of tumor volume was carried out using calipers three times per week. Tumor volume was calculated utilizing the hemiellipsoid formula Y=πlwh/6 [65]. After tumors reached an average volume of 50 mm³, the mice were randomized into four separate groups of 10 mice in each. Sulforaphane was purchased from LKT laboratories and dissolved in sterile normal saline. Paclitaxel was purchased from LKT laboratories and dissolved in cell grade 200 proof ethanol, purchased from Sigma-Aldrich, and then suspended in sterile normal saline. Treatment groups consisted of controls (0.9% normal saline), 50 mg/kg sulforaphane daily, 10 mg/kg paclitaxel weekly, and the combination of 50 mg/kg sulforaphane daily with 10 mg/kg paclitaxel weekly. All treatments were administered via intraperitoneal (IP) administration. Mice were weighed three times a week, and animal health was monitored daily. When the tumor sizes in the control group reached 1000 mm³ in diameter after 16 days of treatment, all mice were euthanized by CO₂ inhalation followed by cervical dislocation to assure death. Tumors were collected at necropsy and a portion of each tumor from each animal was fixed in paraformaldehyde and embedded in paraffin, while another portion was snap-frozen in liquid nitrogen.