Induction of colorectal carcinogenesis in the C57BL/6J and A/J mouse strains with a reduced DSS dose in the AOM/DSS model

Abstract

Background: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers worldwide and thus mouse models of CRC are of significant value to study the pathogenesis. The Azoxymethane/Dextran sulfate sodium (AOM/DSS) model is a widely used, robust initiation-promotion model for chemical induction of colitis-associated CRC in rodents. However, the dosage of chemicals, treatment regimens and outcome measures vary greatly among studies employing this model. Thus, the aim of this study was to examine an AOM/DSS model involving a reduced (1%) dose of DSS for induction of carcinogenesis in A/J and C57BL/6J (B6) mice.

Results: We show that colonic preneoplastic lesions can be reliably detected in A/J and B6 mice by use of a AOM/DSS model involving a single injection of 10 mg/kg AOM followed by three 7-day cycles of a low-dose (1%) DSS administration. Supporting existing evidence of A/J mice exhibiting higher susceptibility to AOM than B6 mice, our AOM/DSS-treated A/J mice developed the highest number of large colonic lesions. Clinical symptoms in both strains subjected to the AOM/DSS treatment did not persist in-between treatment cycles, demonstrating that the animals tolerated the treatment well.

Conclusions: Our findings suggest that a reduced dose of DSS in the AOM/DSS model can be considered in future studies of early phase colorectal carcinogenesis in the A/J and B6 mouse strains using preneoplastic lesions as an outcome measure, and that such regimen may reduce the risk of early trial terminations to accommodate human endpoints. Overall, our data emphasize the importance of devoting attention towards choice of protocol, outcome measures and mouse strain in studies of CRC in mice according to the study purpose.

Keywords: AOM/DSS; Azoxymethane; Colorectal cancer; Dextran sulfate sodium; Disease models; Mouse models.

Fig. 1

Experimental design. Figure shows grouping and timeline of the experiment. A/J and C57BL/6J (B6) mice were randomized to groups administered NaCl injection (−), AOM-injection (+), or AOM-injection combined with three cycles of 1% DSS treatment (++). All animals were sacrificed at week 12. Figure was created with elements from BioRender.com

Fig. 2

Assessment of body weight, fluid intake and symptoms following AOM/DSS treatment. (A) Body weight curves for AOM/DSS (++) and control (−) treated B6 and A/J mice, presented as mean. DSS/H₂O treatment periods are highlighted with grey background. Statistical differences were determined over the timepoints where data was collected for all groups, using repeated-measures two-way ANOVA with Greenhouse-Geisser correction and Tukey’s multiple comparisons tests (A/J++ vs B6++ *p ≤ 0.05; B6++ vs B6- #p ≤ 0.05, ##p ≤ 0.01). n = 5 in each group. (B) DSS intake for AOM/DSS (++) treated B6 and A/J mice. Bars represent estimated average intake per animal, calculated based on drinking bottle volumes for each cage, presented as mean (SD) of mean intake for the whole treatment regimen. (C) Number of individuals in the AOM/DSS (++) treated B6 (left) and A/J (right) mice exhibiting clinical symptoms of abnormal appearance, rectal bleeding (including blood in feces) and/or rectal prolapse at different timepoints during the trial. DSS treatment periods are highlighted with grey background

Fig. 3

Scoring of intestinal lesions. (A, B) Number, load and average size of colonic lesions in B6 or A/J mice treated with AOM/DSS (++) or AOM only (+). Lesions were categorized as either ACFs (≤30 abnormal crypts) or tumors (≥30 abnormal crypts). Box plots show median (line), mean (+), IQR (box) and minimum to maximum (whiskers). Statistical significance between strains treated with AOM/DSS or AOM only was determined by Mann-Whitney U tests (**p ≤ 0.01). (C) Size distribution of lesions in colons of B6 and A/J mice. Data is presented as median number of lesions in each lesion size category for each group, plotted by treatment. The smallest size category includes lesions with 1–4 aberrant crypts, while lesions are considered tumors if they contain ≥30 abnormal crypts per lesion (0.4 mm²). (D) Number, load and average size of small intestinal lesions in B6 or A/J mice treated with AOM/DSS (++) or AOM only (+). Box plots show median (line), mean (+), IQR (box) and minimum to maximum (whiskers)

Fig. 4

Examples of morphological features of colonic lesions. HE-stained Swiss roll sections showing (A) a small area with crypt cell hyperplasia (arrow), (B) a moderately sized adenoma (ad) located in the lamina propria, and (C) large, continuous adenoma (ad) present in the mucosa. The presented pictures were taken of colons from one AOM-treated A/J mouse (A, B) and one AOM/DSS-treated A/J mouse (C)