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

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


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) 



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.


  1. 1.
    Haggar FA, Boushey RP (2009) Colorectal cancer epidemiology: incidence, mortality, survival, and risk factors. Clin Colon Rectal Surg 22(4):191–197. Scholar
  2. 2.
    Asano TK, McLeod RS (2004) Nonsteroidal anti-inflammatory drugs and aspirin for the prevention of colorectal adenomas and cancer: a systematic review. Dis Colon Rectum 47(5):665–673. Scholar
  3. 3.
    Keerthivasan S, Aghajani K, Dose M, Molinero L, Khan MW, Venkateswaran V, Weber C, Emmanuel AO, Sun T, Bentrem DJ, Mulcahy M, Keshavarzian A, Ramos EM, Blatner N, Khazaie K, Gounari F (2014) beta-Catenin promotes colitis and colon cancer through imprinting of proinflammatory properties in T cells. Sci Transl Med 6(225):225–228. Scholar
  4. 4.
    Terzic J, Grivennikov S, Karin E, Karin M (2010) Inflammation and colon cancer. Gastroenterology 138(6):2101–2114.e5. Scholar
  5. 5.
    Wang D, Wang H, Shi Q, Katkuri S, Walhi W, Desvergne B, Das SK, Dey SK, DuBois RN (2004) Prostaglandin E(2) promotes colorectal adenoma growth via transactivation of the nuclear peroxisome proliferator-activated receptor delta. Cancer Cell 6(3):285–295. Scholar
  6. 6.
    O'Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445(7123):106–110. Scholar
  7. 7.
    Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445(7123):111–115. Scholar
  8. 8.
    De Robertis M, Massi E, Poeta ML, Carotti S, Morini S, Cecchetelli L, Signori E, Fazio VM (2011) The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies. J Carcinog 10:9. Scholar
  9. 9.
    Rosenberg DW, Giardina C, Tanaka T (2009) Mouse models for the study of colon carcinogenesis. Carcinogenesis 30(2):183–196. Scholar
  10. 10.
    Popivanova BK, Kitamura K, Wu Y, Kondo T, Kagaya T, Kaneko S, Oshima M, Fujii C, Mukaida N (2008) Blocking TNF-alpha in mice reduces colorectal carcinogenesis associated with chronic colitis. J Clin Invest 118(2):560–570. Scholar
  11. 11.
    Chassaing B, Aitken JD, Malleshappa M, Vijay-Kumar M (2014) Dextran sulfate sodium (DSS)-induced colitis in mice. In: Current protocols in immunology. John EC, et al. (Ed.). 104:Unit 15 25.
  12. 12.
    Muller S, Lory J, Corazza N, Griffiths GM, Z'Graggen K, Mazzucchelli L, Kappeler A, Mueller C (1998) Activated CD4+ and CD8+ cytotoxic cells are present in increased numbers in the intestinal mucosa from patients with active inflammatory bowel disease. Am J Pathol 152(1):261–268PubMedPubMedCentralGoogle Scholar
  13. 13.
    Weigmann B, Tubbe I, Seidel D, Nicolaev A, Becker C, Neurath MF (2007) Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nat Protoc 2(10):2307–2311. Scholar

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© 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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