2.1. Experimental tumor models

Five‐week‐old male Wistar rats (for the AH‐130 tumor model), C57BL/6 mice (for LLC tumour model) or Balb/C mice (for C26 tumour model) weighing about 20 g (all from Harlan, Barcelona, Spain) were housed in individual cages and maintained at a constant temperature of 22 ± 2°C with a regular dark‐light cycle (light from 08:00 am to 20:00 pm), with free access to food and water during the whole experimental period. All animal manipulations were made in accordance with the European Community guidelines for the use of laboratory animals. They were cared for in compliance with the Policy on Humane Care and Use of Laboratory Animals (ILAR 2011) and in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. The animals were kept in individual cages for a week prior to the beginning of the experimental protocol (adaptation period). No hyperphagia was observed during this period. All the animals were alive at the end of the experiments. On the day of sacrifice, the animals were weighed and anesthetized with an ip injection of ketamine/xylazine mixture (3:1) (Imalgene® and Rompun® respectively). Tissues were rapidly excised, weighed, and frozen in liquid nitrogen.

Wistar rats were randomized and divided into two groups, namely controls (C) and tumor bearers (TB). Experimental cachexia was obtained through ip injection of 10⁸ AH‐130 Yoshida ascites hepatoma cells, obtained from exponential tumours, as described previously,¹⁰ into male Wistar rats. On days 2, 4, 6, 8, 10 and 11 after tumor transplantation, the animals were sacrificed and the blood and tissues were rapidly excised for further analysis. Five control rats and six tumor‐bearing rats were sacrificed in each time point.

C57BL/6 mice were randomized and divided into two groups, namely controls (C) and tumor bearers (TB). Mice received an intramuscular (hind leg) inoculum of 5 × 10⁵ Lewis lung carcinoma cells obtained from exponential tumors. The Lewis lung carcinoma is a highly cachectic, rapidly growing mouse tumor containing poorly differentiated cells, with a relatively short doubling time.¹¹ On days 4, 6, 10, 14, 18 and 20 after tumor transplantation, the animals were sacrificed and blood and tissues were rapidly excised for further analysis. Five control mice and six tumor‐bearing mice were sacrificed at each time point.

Balb/C mice were randomized and divided into two groups, namely controls (C) and tumor bearers (TB). Tumor‐bearing mice were inoculated subcutaneously in the back with 5 × 10⁵ Colon26 carcinoma cells.¹² Colon26 cells were maintained in vitro in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL sodium pyruvate, 2 mmol/L L‐glutamine, at 37°C in a humidified atmosphere of 5% CO₂ in air. The day of tumor implantation the cells were trypsinized, re‐suspended in sterile saline and subsequently implanted in the back of the animals at the concentration indicated above. Mice were sacrificed under anesthesia at days 6, 8, 14, 15, 20 and 24 after tumor implantation. Five control mice and six tumor‐bearing mice were sacrificed at each time point.