Fig 1: Effects of different immunosuppressants on a murine inflammatory colon carcinogenesis model.a Animal experiments were performed according to Italian Law 116/92 and European directive 2010/63/UE. Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (Comitato Etico Scientifico per la Sperimentazione Animale) of the University of Padova, Padova, Italy. The experimental design of the mouse model of inflammation-driven colon carcinogenesis involved cohoused 12-wk-old C57Bl6/J male mice that were injected with azoxymethane (AOM, Sigma) intraperitoneally (i.p.) at a dose of 7.4 mg/kg body weight (mean weight 27 ± 1 g). After 4 weeks, the mice received 2.5% dextran sodium sulfate (DSS, Applichem) (M.W. 40,000 g/mol) in their drinking water for 5 days, followed by 2 weeks of plain water. This cycle was repeated two more times. One week after the third DSS treatment, the AOM/DSS mice were randomized (randomization based on a single sequence of computer-generated random numbers) into seven experimental groups receiving immunosuppression and an untreated control group (n = 10). All the mice alive at the end of the experiment were included in the study. Three groups were treated i.p. with 200 μg/mouse of anti-CD3 Ab (clone 145-2C11, ATCC hybridoma no. CRL-1975) (n = 7), anti-CD4 Ab (clone GK1.5, ATCC hybridoma no. TIB-207) (n = 7), or anti-CD8 (clone 2.43, ATCC hybridoma no. TIB-210) (n = 10) monthly. Mice were euthanized 4 weeks after the antibody injection for tissue collection. Cyclosporine A (10 mg/kg/day, CyA) (Sandimmun, Novartis) (n = 7), tacrolimus (1 mg/kg/day, TAC) (Prograf, Astellas) (n = 7), mycophenolate-mofetil sodium (30 mg/kg/day, MPS) (Myfortic, Novartis) (n = 7), or rapamycin (1 mg/kg/day, RAPA) (Rapamune, Pfizer) (n = 7) were administered by oral gavage (200 µl) for 4 consecutive weeks to the additional four AOM/DSS groups. The dosages were prepared according to an average weight of the experimental mice of 27 g. All mice were sacrificed 24 h after the last oral gavage for tissue collection. Sections (4 μm) from formalin-fixed and paraffin-embedded specimens were stained with hematoxylin-eosin and evaluated by a single pathologist in a blinded fashion. b Histological inflammation and dysplasia presence and extent were quantified and classified by a pathologist (S.M.) unaware of the arm of the experiment using Floren’s score26 and the Vienna classification of gastrointestinal epithelial neoplasia27. c Representative histopathological lesions observed in the inflammation-driven colon carcinogenesis model. d Murine colons were analyzed for dysplasia at high magnification (40×). The extent of dysplasia was quantified as the percentage of total bowel length represented by the length of the involved bowel. Carcinoma and adenoma frequencies were macroscopically quantified over the whole length of each mouse colon and then verified with histology. Each treatment group was compared to control mice who were treated only with AOM and DSS. Fisher’s exact test and the Mann–Whitney U test were used to compare dichotomous and continuous variables, respectively (*p < 0.05, **p < 0.01)