4.7. Ectopic Subcutaneous Syngeneic CT26 Colon Cancer Allograft Mouse Model

The studies were carried out in accordance with European regulations on animal protection (Directive 86/609) and the Guide for the Care and Use of Laboratory Animals, US National Institute of Health (NIH Publication № 85–23, 1996). The protocol was approved by the Institutional Animal Care and Ethical Committee, Institute of Marine Sciences (ICIMAR), Havana, Cuba (Protocol number 1501, approved: 12 January 2015). Male Balb/c mice (6–8 weeks of age, 18–22 g) were purchased at the National Laboratory Animal Production Center, Havana, Cuba. They were supplied with water and food ad libitum, under an environment controlled for temperature (22 ± 2 °C), humidity (77 ± 3%), and cycles of 12 h light/dark. The animals were inoculated subcutaneously on the right dorsal side with 1 × 10⁵ CT26 cells/mouse in 100 μL of PBS. Once tumors grew up to 30 mm³, mice were randomly divided into five groups (10 animals/group) as follows: distilled water (negative control), TTE 10, 50, and 100 mg/kg, cisplatin 5 mg/kg (positive control). TTE was administered daily intragastrically for 14 days and cisplatin every 5 days by intraperitoneal injections. The tumor growth and size were measured by length and width with calipers every 2 days. The tumor volume was calculated (V, mm3=length × width2× 0.5). At the end, all mice were sacrificed; samples of blood and tumors were taken for the analysis. Percentages of inhibition in growth of the tumor were calculated using the following equation: Inhibition %=1−T/C∗100, where T and C are the tumor volumes at the end point for each group.

Another experiment with the same setting was performed to determinate the survival time of the animals. The dates of death of the animals were recorded daily to establish the survival curve. Based on the standard animal protocol, the mice with tumors exceeding 2000 mm³ and exhibiting signs of serious illness (i.e., tumor ulceration) were sacrificed.