We used an orthotopic nude mouse model of HNSCC because its host microenvironment is more similar to that of patients with HNSCC than that of subcutaneous xenograft models of HNSCC (25 (
link)). OSC-19-luc, OSC-19, and HN5 cells were harvested from subconfluent cultures by trypsinization and washed with PBS. An orthotopic nude mouse model of an oral tongue tumor was established by injecting OSC-19-luc (1 × 10
5), OSC-19 (1 × 10
5), or HN5 (2 × 10
5) cells suspended in 30 µL of serum-free DMEM into the tongues of mice as described previously (26 (
link)).
Eight to 10 d after the cells were injected, the mice were randomly assigned to 1 of 8 treatment groups (7 or 8 mice per group): (1) control; (2) cisplatin; (3) vandetanib; (4) vandetanib plus cisplatin; (5) radiation; (6) cisplatin plus radiation; (7) vandetanib plus radiation; (8) vandetanib plus cisplatin and radiation. Cisplatin was administered intravenously once a week for 2 weeks at a dose of 1 mg/kg, and vandetanib was administered by oral gavage once a day for 2 weeks at a dose of 20 mg/kg. Control mice were given 200 µL of 1% Tween 80 by oral gavage once daily for 2 weeks and/or 200 µL PBS intraperitoneally once weekly for 2 weeks.
Mice bearing tumors in the tongue were locally irradiated with a single dose of 5 Gy using a small-animal irradiator (γ-rays using a cesium-137 source, 4.762 Gy/min). Sodium pentobarbital was administered by intraperitoneal injection at a dose of 50 mg/kg prior to radiation treatment. The mice were immobilized on a customized jig during irradiation with the tumor centered in the 3-cm diameter circular irradiation field. When cisplatin and radiation were combined, cisplatin was given 1 h before single-dose irradiation (27 (
link)). When vandetanib and radiation were combined, vandetanib was given 4 h before single-dose irradiation (28 (
link)).
Mice were examined twice a week for tumor size and weight loss. Tongue tumor size was measured with microcalipers. Tumor volume was calculated as (A)(B
2)π/6, where A is the longest dimension of the tumor and B is the dimension of the tumor perpendicular to A. The degree of growth delay was expressed as the absolute tumor growth delay (AGD), defined as the time in days required for OSC-19-luc tumors HN5 tumors to grow to 40 mm
3 minus the time in days for the tumors in the untreated control group to reach the same sizes; or the normalized growth delay (NGD), defined as the time in d required for OSC-19-luc tumors and HN5 tumors to grow to 40 mm
3 minus the time to reach the same size in mice treated with drug (s) alone. Treatment enhancement factors (EFs) were obtained by dividing the NGD in mice treated with drugs plus radiation by the AGD in mice treated with radiation alone (29 (
link)).
We used bioluminescence imaging to monitor orthotopic tumor growth
in vivo. Bioluminescence was quantified using Living Image software 3.2 (Xenogen, Alameda, CA) as described previously (30 (
link)). Animals were anesthetized with 2% isoflurane (Abbott, Abbott Park, IL), and an aqueous solution of luciferin (Xenogen) at 150 mg/kg in a volume of 0.1 mL was injected intraperitoneally 5 min prior to imaging. We used an IVIS 200 Imaging System (Xenogen) to image the animals and Living Image software (Xenogen) to quantify the photons emitted from luciferase-expressing cells. Photon flux was calculated using a rectangular region of interest encompassing the head and neck region of each mouse while in a dorsal position. Animals were imaged on an almost weekly basis. Before engineered OSC-19-luc cells were used
in vivo, we used the IVIS imaging system to confirm
in vitro that the cells homogeneously expressed high levels of luciferase.
We euthanized mice by CO
2 asphyxiation when they lost more than 20% of their preinjection body weight or at 50 d after cell injection. Half of the mouse tumors were fixed in formalin and embedded in paraffin for immunohistochemical and hematoxylin-and-eosin staining; the other half were embedded in optimal cutting temperature compound (Miles, Inc., Elkhart, IN), rapidly frozen in liquid nitrogen, and stored at −80°C. Cervical lymph nodes were resected, embedded in paraffin, sectioned, stained with hematoxylin and eosin. The presence of cervical lymph node metastasis was evaluated histologically using one H&E slide which was from a paraffin block prepared for each animal.
Sano D., Matsumoto F., Valdecanas D.R., Zhao M., Molkentine D.P., Takahashi Y., Hanna E.Y., Papadimitrakopoulou V., Heymach J., Milas L, & Myers J.N. (2011). Vandetanib Restores Head and Neck Squamous Cell Carcinoma Cells’ Sensitivity to Cisplatin and Radiation In Vivo and In Vitro. Clinical cancer research : an official journal of the American Association for Cancer Research, 17(7), 1815-1827.
