Animal studies and procedures

Pure strain FVBN Abcb4^−/− mice were kindly provided by Albert K. Groen (Department of Paediatrics, Center for Liver, Digestive and Metabolic Diseases, University Medical Center, Groningen, The Netherlands). FVBN/Apc^Min/+ mice were generated by crossing for more than 8 generations FVBN mice with Apc^Min/+ mice obtained from Jackson laboratory. Apc^Min/+/Mdr^+/− mice were generated by crossing Apc^Min/+ mice with Abcb4^−/− mice. Then, by intercrossing Apc^Min/+/Abcb4^+/−, we generated Apc^Min/+/Abcb4^−/− mice. Intestinal specific Lrh1^−/− mice (iLrh1^−/−) were generated by crossing villin-cre mice from Jackson laboratory with floxed Lrh1 mice that were kindly provided by Drs. Kristina Schoonians and Johan Awuerx (EPFL, Lausanne, Switzerland)¹⁴. iLrh1^−/−/Abcb4^−/− mice were generated by crossing Abcb4^−/− mice with iLrh1^−/− mice. In vivo Lrh1 agonism experiment was performed on 10 weeks old mice employing the natural phospholipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC). DLPC (Avanti Polar Lipids) was dissolved in alcohol and administered by gavage (200 mg/Kg body weight/day in PEG400/Tween80, 4:1) once daily for ten consecutive days. Animals in the control group received vehicle only. For the genetic model of CRC formation, Apc^Min/+, Apc^Min/+/Abcb4^+/− and Apc^Min/+/Abcb4^−/− were sacrificed at 6 months of age. For the chemical induced colitis carcinogenesis model⁴³, pathogen free 10–16 week old male Abcb4^+/+, Abcb4^−/−, iLrh1^−/−/Abcb4^−/− and iLrh1^+/+/Abcb4^−/− mice on a pure FVBN background were injected intraperitoneally with 12 mg/Kg body weight of azoxymethane (AOM, Sigma-Aldrich, Saint Louis, MO, USA) dissolved in NaCl 0.9%. Five days later, 3% dextran sulfate sodium (DSS) was given in the drinking water over 5 days, followed by 16 days of regular water. This cycle was repeated 3 times and the body weight was measured at the end of each cycle. To evaluate the effect of a dietary phosphatidylcholine (PC) on colon cancer formation, we fed both Abcb4^+/+, Abcb4^−/−, iLrh1^−/−/Abcb4^−/− and iLrh1^+/+/Abcb4^−/− mice with a PC-enriched diet, supplemented with soybean lecithin 2.5% w/w (DP1000 mod, PC-supplemented diet, Altromin-Rieper, Vandoies, BZ, Italy) during the entire duration of AOM/DSS protocol. A minimum of eight mice per group was employed in the different experimental settings. All animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals”. At the end of each experimental protocol, macroscopic inspection, histological analysis, and total RNA extraction were performed. Briefly, colons were removed, washed with PBS, open longitudinally and laid out. Then, they were thawed in 2.5% formalin solution at room temperature, then fixed in 70% ethanol at 4 °C for 30 min, and stained with 0.2% methylene blue for 2 min. The samples were then fixed in 10% buffered formalin, washed in 70% ethanol, and stored in this solution. Stained intestine were transferred to 2.5% formalin solution for up to 1 h and then examined in their entirety. The number of tumors was scored with a dissecting microscope by a single observer blind to the genotype of the mice or their treatment group. The results are indicated as a mean ± SEM of all the tumors counted within a single genotype group. All mice were housed under standard diet (except when indicated) provided ad libitum and examined daily. Genotyping was done using DNA extracted from tail biopsies of 2- to 3-week-old pups, and new breeding harems of 5 to 6 week-old mice were established to expand the population. All the experiments presented in this study have been carried out in male mice. The Ethical Committee of the Consorzio Mario Negri Sud approved this experimental set-up, which was also certified by the Italian Ministry of Health according with internationally accepted guidelines for the animal care.