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Mouse Model of Colitis-Associated Colorectal Cancer (CAC): Isolation and Characterization of Mucosal-Associated Lymphoid Cells

  • Llipsy Santiago
  • Marta Castro
  • Julián Pardo
  • Maykel Arias
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1884)

Abstract

Colorectal cancer (CRC) is the third most common malignancy worldwide presenting high mortality due to low treatment efficacy. Existing evidence indicates that inflammatory bowel disease (IBD) is associated with a higher risk of developing CRC. Many murine models of inflammation-related colon carcinogenesis (CAC) have been developed to study colon carcinogenesis and novel treatments. A commonly used model involves the combination of a single dose of azoxymethane (AOM), together with three cycles of the inflammatory agent dextran sodium sulfate (DSS) (5 days in drinking water followed by a two-week rest). Following this protocol, around 50% of the animals develop CRCs after 45 days and almost 100% of animals after 60 days. During CAC development, immune cells, cytokines, and other immune mediators are involved in both tumorigenesis and the elimination of cancer cells during immunotherapy. Thus, the study of mucosal immune responses (including lamina propria mononuclear cells and intraepithelial lymphocytes) is important to understand the role of the immune system during development and therapy in CRC. Single immune cell suspensions from lamina propria and epithelium can be purified combining selective tissue digestion and Percoll gradient centrifugation. Isolated cells can be characterized using flow cytometry by analyzing surface antigens or intracellular cytokines and cytotoxic mediators or employed for further investigations like comparative studies of mRNA expression, cell-proliferation assay, protein analysis, or even functional cytotoxicity assays. The CAC model is useful to study the involvement of immune cells not only during the carcinogenesis process but, in addition, during the treatment with novel immunotherapy protocols.

Key words

Colitis-associated cancer (CAC) Azoxymethane (AOM) Lamina propria Monocyte cells (LPMCs) Intraepithelial lymphocytes (IELs) Dextran sodium sulfate (DSS) 

Notes

Acknowledgments

This work was supported by Fondo Social Europeo (FSE), grant SAF2014-54763-C2-1-R from Spanish Ministry of Economy and Competitiveness and grant AEI-010500-2015-161 from the Spanish Ministry of Industry, Energy and Tourism. MA and LS were supported by a PhD fellowship from University of Zaragoza/Santander Bank Foundatio and FPI from Spanish Ministry of Economy and Competitiveness (LS). JP was supported by ARAID Foundation.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Llipsy Santiago
    • 1
  • Marta Castro
    • 2
  • Julián Pardo
    • 1
    • 3
    • 4
    • 5
    • 6
  • Maykel Arias
    • 1
  1. 1.Fundación Instituto de Investigación Sanitaria Aragón (IIS Aragón), Biomedical Research Centre of Aragon (CIBA)ZaragozaSpain
  2. 2.Faculty of Veterinary, Department of PhysiologyUniversity of ZaragozaZaragozaSpain
  3. 3.Department of Biochemistry and Molecular and Cell BiologyUniversity of ZaragozaZaragozaSpain
  4. 4.Department of Microbiology, Preventive Medicine and Public HealthUniversity of ZaragozaZaragozaSpain
  5. 5.Aragon I+D Foundation (ARAID)ZaragozaSpain
  6. 6.Nanoscience Institute of Aragon (INA), University of ZaragozaZaragozaSpain

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