2.4. Experiments in Tumor Xenograft Mouse Models

BALB/c Nude immunodeficient male mice (unable to produce T lymphocytes) were obtained from Janvier labs (France). Ethical permission was granted by Local Ethical Committee of the Warsaw University of Life Sciences in Warsaw, reference: 432/2013. At the beginning of the experiment, mice were 3 months old. During the experiment, the animals were bred in separate, individually ventilated cages (IVC), with ad libitum access to food and water. The animals were injected subcutaneously around the neck with the mixture of tumor cells and matrigel (BD Biosciences) (1:1) with PBS (Gibco) in a final volume of 100 µL. A preliminary experiment was performed in which the optimal number of cancer cells administered giving linear tumor growth was selected [17]. After optimization, the BALB/c Nude mice xenografts were injected with 3.5 × 10⁶ HT-29 cells or with 7 × 10⁶ SK-N-MC cells. When the tumor reached 100 mm³ in volume, administration of the tested substances was started. In mice xenografted with HT-29 cells, OAT-449 was administered intraperitoneally at a dose of 5 mg/kg in 30% solutol, every day for 5 days, followed by 2 day intervals. In the same way, 30% solutol alone (vehicle) was administered as a negative control. As the positive control irinotecan (CPT-11) dissolved in 10% solutol, a final concentration of 3 mg/mL was used and administered intraperitoneally every 3 days. In mice xenografted with SK-N-MC cells, OAT-449 in 10% solutol was administered intravenously at a dose of 2.5 mg/mL, as well as vincristine (1 mg/kg every 7 days) and diluent (10% solutol, every 5 days). Measurements of tumor volume were made every 3 days using an electronic display caliper. Two dimensions were measured: length (L) for the longest dimension of a tumor and width (W) as the dimension perpendicular to the length. Tumor volume was calculated according to the formula: V = (L × W2)/2 (mm³).