Optimized Mouse Model to Induce Colitis-Associated Colon Cancer using Azoxymethane and Dextran Sulfate Sodium for Tumor Immunology and Therapy Studies

Azoxymethane (AOM, a DNA-damaging agent) and dextran sulfate sodium (DSS, a colitis-promoting agent) can induce tumors in the large intestine of mice with high penetrance. The murine colitis-associated colon cancer (CAC) model induced by AOM/DSS stands as a gold standard in investigating inflammation-related colon cancer, given its accuracy in recapitulating human CAC clinical characteristics. The model's advantages include its origin and development from endogenous tissue, immune competence preserving a full repertoire of tumor-immune interactions, adaptability to numerous mouse strains, convenience of a 3-month latency, and relatively low costs. Despite these advantages, several challenges limit the protocol's effectiveness. Variability in DSS dosage and in tumor penetrance across studies leads to inconsistent observations. Additionally, variations in gut microbiota contribute to increased experimental variability, negatively impacting statistical power. Sex differences in response to AOM/DSS further complicate the interpretation of results. Moreover, DSS lot-to-lot variability hinders the reproducibility of findings. Addressing these challenges is crucial for enhancing the reliability and applicability of this model in preclinical research. To resolve this, using C57BL/6J mice as a model, we refined the established protocol with strategies for effective optimization to enhance reproducibility and harmonization between studies. These include recommendations for DSS dose titration in both male and female mice, implementation of microbiota homogenization prior to each DSS cycle to mitigate cage-to-cage gut flora heterogeneity. These steps significantly reduced variability and improved reproducibility, resulting in robust induction of CAC. Recognizing the pivotal role of inflammation and cellular immune mechanisms in CAC development, the protocol also describes tissue processing procedures suitable for isolation of RNA or protein and for profiling inflammatory cytokines from colon tumors and surrounding colon tissue. Ultimately, the use of this method ensures a more rigorous, robust, and reproducible CAC production.