Skip to main content

Modeling Intestinal Carcinogenesis Using In Vitro Organoid Cultures

  • Protocol
  • First Online:
Inflammation and Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2691))

Abstract

Mouse models of intestinal carcinogenesis are very powerful tools for studying the impact of specific mutations on tumor initiation and progression. Mutations can be studied both singularly and in combination using conditional alleles that can be induced in a temporal manner. The steps in intestinal carcinogenesis are complex and can be challenging to image in live animals at a cellular level. The ability to culture intestinal epithelial tissue in three-dimensional organoids in vitro provides an accessible system that can be genetically manipulated and easily visualized to assess specific biological impacts in living tissue. Here, we describe methodology for conditional mutation of genes in organoids from genetically modified mice via induction of Cre recombinase induced by tamoxifen or by transient exposure to TAT-Cre protein and subsequent phenotyping of the organoids. This methodology provides a rapid platform for assessing the cellular changes induced by specific mutations in intestinal tissue.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Sato T, Vries RG, Snippert HJ et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265

    Article  CAS  PubMed  Google Scholar 

  2. Nefzger CM, Jarde T, Rossello FJ et al (2016) A versatile strategy for isolating a highly enriched population of intestinal stem cells. Stem Cell Rep 6:321–329

    Article  CAS  Google Scholar 

  3. Lindemans CA, Calafiore M, Mertelsmann AM et al (2015) Interleukin-22 promotes intestinal-stem-cell-mediated epithelial regeneration. Nature 528:560–564

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Jarde T, Chan WH, Rossello FJ et al (2020) Mesenchymal niche-derived neuregulin-1 drives intestinal stem cell proliferation and regeneration of damaged epithelium. Cell Stem Cell 27:646–662

    Article  CAS  PubMed  Google Scholar 

  5. Mileto SJ, Jarde T, Childress KO et al (2020) Clostridioides difficile infection damages colonic stem cells via TcdB, impairing epithelial repair and recovery from disease. Proc Natl Acad Sci USA 117:8064–8073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Holik AZ, Krzystyniak J, Young M et al (2013) Brg1 is required for stem cell maintenance in the murine intestinal epithelium in a tissue-specific manner. Stem Cells 31:2457–2466

    Article  CAS  PubMed  Google Scholar 

  7. Horvay K, Jarde T, Casagranda F et al (2015) Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium. EMBO J 34:1319–1335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. el Marjou F, Janssen KP, Chang BH et al (2004) Tissue-specific and inducible Cre-mediated recombination in the gut epithelium. Genesis 39:186–193

    Article  PubMed  Google Scholar 

  9. Peitz M, Pfannkuche K, Rajewsky K et al (2002) Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc Natl Acad Sci USA 99:4489–4494

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Jarde T, Evans RJ, McQuillan KL et al (2013) In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-ODeltaN89beta-catenin system. Oncogene 32:883–893

    Article  CAS  PubMed  Google Scholar 

  11. Morin PJ, Sparks AB, Korinek V (1997) Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275:1787–1790

    Article  CAS  PubMed  Google Scholar 

  12. Hill DR, Spence JR (2017) Gastrointestinal organoids: understanding the molecular basis of the host-microbe interface cell mol. Gastroenterol Hepatol 3:138–149

    Google Scholar 

  13. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71

    Article  CAS  PubMed  Google Scholar 

  14. Shibata H, Toyama K, Shioya H et al (1997) Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene. Science 278:120–123

    Article  CAS  PubMed  Google Scholar 

  15. Madisen L, Zwingman TA, Sunkin SM et al (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13:133–140

    Article  CAS  PubMed  Google Scholar 

  16. Engel RM, Chan WH, Nickless D et al (2020) Patient-derived colorectal cancer organoids upregulate revival stem cell marker genes following chemotherapeutic treatment. J Clin Med 9:128

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Nefzger CM, Jarde T, Srivastava A et al (2022) Intestinal stem cell aging signature reveals a reprogramming strategy to enhance regenerative potential. NPJ Regen Med 7:31

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia grants (1100531, 1188689 and 2003693) and an Australian Research Council grant (DP200103589). Authors wish to acknowledge support from the Monash Biomedicine Discovery Institute Organoid Program, Monash FlowCore, and Monash Microimaging platform facilities.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Thierry Jardé or Helen E. Abud .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Chan, W.H. et al. (2023). Modeling Intestinal Carcinogenesis Using In Vitro Organoid Cultures. In: Jenkins, B.J. (eds) Inflammation and Cancer. Methods in Molecular Biology, vol 2691. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3331-1_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3331-1_5

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3330-4

  • Online ISBN: 978-1-0716-3331-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics