For the chick embryo chorioallantoic membrane (CAM) in vivo CRC model 22, on embryonic day 11, 2×106 SW1222 CRC cells suspended 1:1 in culture medium with Matrigel (BD Biosciences, Bedford, MA) (100 µl final volume) were inoculated on CAMs. Tumor volume was estimated assuming the ellipsoid shape and using the formula V= 4/3.π.((l.w.h)/8), where l, w, h represent the tumor length, width and height, respectively.

For the orthotopic model of CRC liver metastasis formation, SW1222, HT29 and HCT116 cells were injected in the spleen of NOD-SCID mice (Janvier Labs, Saint Berthevin Cedex, France). All experimental procedures for this study were performed in accordance with the ARRIVE ethical guidelines 23, and were approved by the Institutional Animal Care and Ethics Committee of the University of Liège (Belgium). The study adhered to the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by National Academy Press, as well as to European and local legislation. Mice were anesthetized with 75 mg/kg of ketamine (CEVA, Brussel, BE) and 10 mg/kg of xylazine (Rompun®, Bayer, Diegem, BE), and spleen was surgically exposed for injection of 500,000 cells in 100 µL saline solution with 5 mM EDTA. Liver metastasis development was evaluated at week 6 post-injection.

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