Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Summary

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Abstract

Individuals with inflammatory bowel disease (IBD), such as Crohn’s disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.¹ Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.² While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.³ This distinction argues the need for specific animal models of CAC.

Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.⁴ Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).⁵

The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.^{6, 7} Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.⁸

Protocol

1. Colitis-associated Cancer Induction Set aside cages of sex and age-matched 6-8 week old mice to be used for experimental and control groups. Mice can be individually labeled with tail markings or ear clips. On day 0, record baseline weights and inject each mouse intraperitoneally (IP) with 10 mg/kg of AOM working solution (1 mg/ml in isotonic saline, diluted from 10 mg/ml stock solution in dH2O kept at -20 deg C). Based on experience, this dose can be adjusted between 7-14 mg/kg and/or repea…

Discussion

Treatment of mice with AOM and DSS rapidly and effectively models human colitis-associated cancer. Hypotheses regarding heritable factors contributing to colitis-associated cancers can be easily studied with genetically engineered mice.^{13, 16} Alternatively, the effect of pharmacologic targets in colitis-associated cancer can be studied by employing wild-type mice.

While this model is highly valued by those interested in the study of colon tumor development in the setting of inflamma…

Materials

Name of the reagent	Company	Catalogue number	Comments
C57BL/6J Mice	Jackson Laboratory	000664
Azoxymethane (AOM)	Sigma Aldrich	A5486-100MG	Stock solution: dilute to 10 mg/ml in distilled water to be kept at -20 °C as 0.5 – 1 ml aliquots. Working solution: dilute stock to 1 mg/ml in isotonic (0.9%) saline
Dextran Sulfate Sodium (DSS)	TdB Consultancy	DB001	MW 40 kDa (36-50 kDa preparations from other sources are acceptable; The same lot should be used for a single experiment)6
Coloview miniendoscopic system	Karl Storz	Multiple	See Becker et al. for detailed explanation of equipment and setup.11
TPP Rapid FILTERMAX 500 ml Bottle-Filter, 0.22 μm PES	Midwest Scientific	TP99500	Any standard tissue culture filter is acceptable
Ethyl Alcohol 200 Proof ASC/USP	Pharmaco-AAPER (or other)	11ACS200	Dilute to 70% in distilled water
Isoflurane, USP	Butler Animal Health Supply	4029405	Place mouse in glass jar with gauze or a small cloth soaked in anesthetic
18G Straight Gavage Needle	Braintree Scientific	N-008
Phosphate Buffered Saline (PBS)	Sigma Aldrich	P5493	Dilute to 1X (0.01 M) in distilled water
Cold Tray (Tissue Tek II Cold Plate)	Fisher Scientific	NC9491941	Store at -20 °C
ImageJ Software	NIH (free download)		http://rsbweb.nih.gov/ij/
Formaldehyde (37%)	Fisher Scientific	F79-500	Dilute to 10% in PBS
BD Bacto Agar	Fisher Scientific	DF0140-01-0	Use hotplate to create 2% solution in distilled water
Miltex Eye Dressing Forceps	MedPlus Inc.	18-780
Miltex Eye Scissors	MedPlus Inc.	18-1430	Curved points prevent damage to colon during opening.
Alcian Blue 8GX (powder)	Sigma Aldrich	A5268	Add 1 g powder to 100 ml 3% acetic acid (3 ml glacial acetic acid + 97 ml distilled water)
1 mL Tuberculin syringe with attached 26 G x 3/8 in intradermal bevel needle	BD	305946	For injection of AOM