2.3. Experimental Mouse Models

The carcinogenic agent azoxymethane (AOM, Sigma-Aldrich, Darmstadt, Germany). induces precancerous and cancerous lesions similar to those of sporadic colon cancer [24]; dextran sulfate sodium (DSS, 40 kDa, TdB Consultancy) induces inflammation, and together with AOM, produces colitis-associated adenocarcinomas [25]. Colonic lesions were induced in mice according to the experimental design shown in Figure 1. Aberrant crypt foci (ACF), polyps and adenocarcinomas were induced in 2-month-old C57BL6/J mice via the intraperitoneal injection of 10 mg AOM/kg body weight dissolved in PBS: mice received 6 weekly doses followed by an interval of either 4 weeks without treatment before the sacrifice for ACF induction or 24 weeks for polyps. Sporadic adenocarcinomas were induced with 8 weekly AOM doses and a period of 24 weeks without treatment [26]. Colitis-associated adenocarcinoma was induced in 3-month-old C57BL6/J mice following the protocol of [27]: Briefly, 10 mg AOM/kg body weight was first administered via a single intraperitoneal injection followed by 3 cycles in a 4-day period with 1% (wt/vol) DSS in drinking water. Between DSS periods, the animals drank normal water for 14 days. Acute colitis was induced in 3-month-old B6C3Fe wild-type and reeler mice by administering 3% (wt/vol) DSS in the drinking water for 9 days. Control group (untreated mice) received normal drinking water. The progression of the colon inflammation of the mice receiving DSS treatment was assessed by determining the disease activity index (DAI), histological score and mRNA levels of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), as described previously [19]. For DAI evaluation, throughout the DSS treatment, animals were monitored daily for weight loss, stool consistency and blood in the feces (0–3 scale). Histological score (0–3 scale) was based on destruction of epithelium, dilatation of crypts, loss of goblet cells, inflammatory cell infiltrate and edema.

Mouse model of colon cancer progression. Experimental design of the treatments to induce lesions in the colon. Dextran sulfate sodium (DSS) was administered in the drinking water (3%) for 9 days to induce acute colitis. Azoxymethane (AOM) dissolved in PBS was administered by intraperitoneal injection at the point indicated by the arrowheads. DSS was administered in the drinking water (1%) in 4-day periods. Control groups (untreated mice) received PBS intraperitoneally and water orally. Representative photographs of mice colonic mucosa along colon cancer initiation and progression. Macroscopic (fresh tissue or stained with methylene blue solution in the case of aberrant crypt foci) and microscopic images (eosin–hematoxylin-stained sections) of mice colon with colitis, aberrant crypt foci, polyps, sporadic adenocarcinomas, colitis-associated adenocarcinomas and healthy colons (control groups). The lesions are indicated by red arrows. For each mouse model, the number of animals per experimental group was ten. White scale bars represent 1 cm, the green scale bar represents 50 µm and black scale bars represent 200 µm.

Control groups (untreated mice) received injections of phosphate buffer solution (PBS; in mM, 137 NaCl, 2.7 KCl, 10 Na₂HPO₄ and 1.8 KH₂PO₄; pH7.4) and normal drinking water.