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The animal experiments were approved by the Norwegian National Animal Research Authority and were done according to the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing, and Education. Cells and multicellular spheroids A 07 and R 18 human melanoma cells were constitu tively transfected buy inhibitor with green fluorescence protein by lipofection. The transfected cells used in the present experiments were obtained from our frozen stock and grown as monolayers in RPMI 1640 supplemented with 13% bovine calf serum, 250 ug mL penicillin, 50 ug mL strepto mycin, and 700 ug mL or 2200 ug mL genetecin. Multicellular spheroids were produced by seeding approximately 106 cells in 30 mL medium in plas tic tissue culture flasks coated with a thin layer of 1% agar.
The flasks were agitated for 2 h using a tilting platform, and aggregates of approximately 50 um in diam eter were formed. The spheroids were then allowed to grow in coated culture flasks before implantation in win dow chambers. Cells and spheroid cultures were incubated at 37 C in a humidified atmosphere of 5% CO2 in air and subcultured twice a week. Anesthesia Window chamber implantation and intravital micros copy examinations were carried out with anesthetized mice. Fentanyl citrate, fluanisone, and mida zolam were administered intraperitoneally in doses of 0. 63 mg kg, 20 mg kg, and 10 mg kg, respectively. After surgery, the mice were given a single injection of buprenorphine intrape ritoneally in a dose of 0. 12 mg kg to relieve pain.
Window chamber preparations Window chambers were implanted into the dorsal skin fold as described previously. Briefly, the chamber consisted of two parallel frames, and after implantation, the frames sandwiched an extended double layer of skin. Before the chamber was implanted, a circular hole with a diameter of approximately 6. 0 mm was made in one of the skin layers. A plastic window with a diameter of 6. 0 mm was attached to the frame on the surgical side with a clip to provide visual access to the fascial side of the opposite skin layer. Tumors were initiated by implanting spheroids or tumor specimens with a dia meter of 200 to 400 um onto the exposed skin layer. Sunitinib treatment Sunitinb L malate was dissolved in hydrochloric acid, polysorbate 80, polyethylene Glycol 300, sodium hydro xide, and sterile water.
Mice were treated with 20 or 40 mg kg day sunitinib or vehicle for 4 or 8 days, by oral administration. Intravital microscopy Intravital microscopy was performed before initiation of sunitinib treatment, and 1, 2, and 4 days after the start of treatment, or before, and 2, 4, 6 and 8 days after the start of treatment. The mice were kept in a specially constructed holder that fixed the window chamber to the microscope stage during intravital microscopy. The body core temperature was kept at 37 to 38 C by using a hot air gen erator.