AOM/DSS Induced Colitis-Associated Colorectal Cancer in 14-Month-Old Female Balb/C and C57/Bl6 Mice-A Pilot Study

Abstract

Colitis is a major risk factor for the development of colorectal cancer, leading to colitis-associated colorectal cancer (CAC). The most commonly used animal model to study CAC is the azoxymethane-dextran sulphate-sodium (AOM/DSS) model. The ideal experimental conditions of this model depend on several factors, including the used mouse strain. No data on feasibility and conditions for older mice, e.g., for aging studies, have yet been reported. Thus, we conducted a descriptive, observational pilot study where CAC was induced in 14-month-old female Balb/C and C57/Bl6 mice using 12.5 mg/kg AOM i.p. and three different concentrations of DSS (1, 2, and 3%) in drinking water (ad. lib.). The mice were monitored regularly during the three-month experimental phase. After euthanasia, the colons of the mice were evaluated macroscopically and microscopically. Both the mouse strains showed a DSS-concentration-dependent induction of CAC. Carcinomas were only observed at 3% DSS. The DSS dose was found to be significantly correlated with the histology score and % Ki67 positive cells only in C57/Bl6 mice but not in Balb/C mice, which showed a variable response to the CAC induction. No differences in colon length, weight, or mucin content were observed. Optimal conditions for CAC induction in these aged animals are thus considered to be 3% DSS, as carcinomas did not develop when 2% DSS was used. On the other hand, Balb/C mice reacted severely to 3% DSS, indicating that 2.5% DSS may be the "sweet spot" for future experiments comparing CAC in aged Balb/C and C57/Bl6 mice. This model will allow investigation of the effect of aging on CAC development and therapy.

Keywords: AOM; DSS; aged mice; colitis associated colorectal cancer; mouse strain differences; pilot study.

Figure 1

Effect of DSS treatment on body weight (a,b), histology score (c,d) and colon macroscopic parameters (e–j) of 14-month-old female C57/Bl6 and Balb/C mice with CAC induced by 3 different concentrations of DSS. Control = untreated control mice of the same age. n = 2–4. Histology score: 0—no abnormalities, 1—minimal changes such as aberrant crypt foci, 2—high-grade dysplasia, 3—intramucosal carcinoma. ** p < 0.01 Kruskal–Wallis with Dunn post hoc test vs. control for C57/Bl6. Statistical correlation analysis is provided in Figure 3.

Figure 2

Effect of DSS treatment on percentage of Ki67 positive cells (a,b), and mucin content (c,d) in colons of 14-month-old female C57/Bl6 and Balb/C mice with CAC induced by 3 different concentrations of DSS. Control = untreated control mice of the same age. N = 2–4. * p < 0.05, ANOVA with Dunnett post hoc test vs. Control for C57/Bl6. Statistical correlation analysis is provided in Figure 3.

Figure 3

Spearman correlation matrix displaying Spearman correlation coefficient r (r = 1: 100% positive correlation, r = 0: no correlation, r = −1: 100% negative correlation) for all pairs of variables measured in C57/Bl6 (n = 15) and Balb/C (n = 12) mice (including non-treated control mice). Blue = positive correlation, white = no correlation, red = negative correlation. * p < 0.05, ** p < 0.01, *** p < 0.001 significance for correlation.

Figure 4

Schematic representation of the CAC induction protocol. In total, 12.5 mg/kg azoxymethane (AOM) were i.p. injected into the mice at the start of the protocol. In the second, fifth and eighth week, the drinking water of the mice was supplemented with 1, 2, or 3% (w/v) dextran sulphate sodium (DSS) for 5 consecutive days. 12 weeks after the AOM injection, the mice were euthanized.