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Use of Organoids to Characterize Signaling Pathways in Cancer Initiation

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Colorectal Cancer

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1765))

Abstract

The development of intestinal organoid technology has greatly accelerated research in the field of colorectal cancer. Contrary to traditional cancer cell lines, organoids are composed of multiple cell types arranged in 3D structures highly reminiscent of their native tissues. Thus, organoids provide a near-physiological and readily accessible model to study tissue morphogenesis, adult stem cell behavior and tumorigenesis. Here, we provide protocols for establishing intestinal organoid cultures from genetically modified mouse lines and describe methods to overexpress and knockout genes of interest using lentiviral-based approaches.

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References

  1. Holland JD, Klaus A, Garratt AN, Birchmeier W (2013) Wnt signaling in stem and cancer stem cells. Curr Opin Cell Biol 25(2):254–264. https://doi.org/10.1016/j.ceb.2013.01.004

    Article  CAS  PubMed  Google Scholar 

  2. Krausova M, Korinek V (2014) Wnt signaling in adult intestinal stem cells and cancer. Cell Signal 26(3):570–579. https://doi.org/10.1016/j.cellsig.2013.11.032

    Article  CAS  PubMed  Google Scholar 

  3. Fearon ER (2011) Molecular genetics of colorectal cancer. Annu Rev Pathol 6:479–507. https://doi.org/10.1146/annurev-pathol-011110-130235

    Article  CAS  PubMed  Google Scholar 

  4. Jackstadt R, Sansom OJ (2016) Mouse models of intestinal cancer. J Pathol 238(2):141–151. https://doi.org/10.1002/path.4645

    Article  PubMed  Google Scholar 

  5. Sansom OJ, Griffiths DF, Reed KR, Winton DJ, Clarke AR (2005) Apc deficiency predisposes to renal carcinoma in the mouse. Oncogene 24(55):8205–8210. https://doi.org/10.1038/sj.onc.1208956

    Article  CAS  PubMed  Google Scholar 

  6. Ashton GH, Morton JP, Myant K, Phesse TJ, Ridgway RA, Marsh V, Wilkins JA, Athineos D, Muncan V, Kemp R, Neufeld K, Clevers H, Brunton V, Winton DJ, Wang X, Sears RC, Clarke AR, Frame MC, Sansom OJ (2010) Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling. Dev Cell 19(2):259–269. https://doi.org/10.1016/j.devcel.2010.07.015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Buller NV, Rosekrans SL, Metcalfe C, Heijmans J, van Dop WA, Fessler E, Jansen M, Ahn C, Vermeulen JL, Westendorp BF, Robanus-Maandag EC, Offerhaus GJ, Medema JP, D'Haens GR, Wildenberg ME, de Sauvage FJ, Muncan V, van den Brink GR (2015) Stromal Indian hedgehog signaling is required for intestinal adenoma formation in mice. Gastroenterology 148(1):170–180 e176. https://doi.org/10.1053/j.gastro.2014.10.006

    Article  PubMed  Google Scholar 

  8. Cordero JB, Ridgway RA, Valeri N, Nixon C, Frame MC, Muller WJ, Vidal M, Sansom OJ (2014) c-Src drives intestinal regeneration and transformation. EMBO J 33(13):1474–1491. https://doi.org/10.1002/embj.201387454

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Marsh V, Winton DJ, Williams GT, Dubois N, Trumpp A, Sansom OJ, Clarke AR (2008) Epithelial Pten is dispensable for intestinal homeostasis but suppresses adenoma development and progression after Apc mutation. Nat Genet 40(12):1436–1444. https://doi.org/10.1038/ng.256

    Article  CAS  PubMed  Google Scholar 

  10. Sansom OJ, Meniel VS, Muncan V, Phesse TJ, Wilkins JA, Reed KR, Vass JK, Athineos D, Clevers H, Clarke AR (2007) Myc deletion rescues Apc deficiency in the small intestine. Nature 446(7136):676–679. https://doi.org/10.1038/nature05674

    Article  CAS  PubMed  Google Scholar 

  11. Gregorieff A, Liu Y, Inanlou MR, Khomchuk Y, Wrana JL (2015) Yap-dependent reprogramming of Lgr5(+) stem cells drives intestinal regeneration and cancer. Nature 526(7575):715–718. https://doi.org/10.1038/nature15382

    Article  CAS  PubMed  Google Scholar 

  12. Schepers AG, Snippert HJ, Stange DE, van den Born M, van Es JH, van de Wetering M, Clevers H (2012) Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337(6095):730–735. https://doi.org/10.1126/science.1224676

    Article  CAS  PubMed  Google Scholar 

  13. Vermeulen L, Morrissey E, van der Heijden M, Nicholson AM, Sottoriva A, Buczacki S, Kemp R, Tavare S, Winton DJ (2013) Defining stem cell dynamics in models of intestinal tumor initiation. Science 342(6161):995–998. https://doi.org/10.1126/science.1243148

    Article  CAS  PubMed  Google Scholar 

  14. Date S, Sato T (2015) Mini-gut organoids: reconstitution of the stem cell niche. Annu Rev Cell Dev Biol 31:269–289. https://doi.org/10.1146/annurev-cellbio-100814-125218

    Article  CAS  PubMed  Google Scholar 

  15. Leushacke M, Barker N (2014) Ex vivo culture of the intestinal epithelium: strategies and applications. Gut 63(8):1345–1354. https://doi.org/10.1136/gutjnl-2014-307204

    Article  CAS  PubMed  Google Scholar 

  16. Drost J, van Jaarsveld RH, Ponsioen B, Zimberlin C, van Boxtel R, Buijs A, Sachs N, Overmeer RM, Offerhaus GJ, Begthel H, Korving J, van de Wetering M, Schwank G, Logtenberg M, Cuppen E, Snippert HJ, Medema JP, Kops GJ, Clevers H (2015) Sequential cancer mutations in cultured human intestinal stem cells. Nature 521(7550):43–47. https://doi.org/10.1038/nature14415

    Article  CAS  PubMed  Google Scholar 

  17. Matano M, Date S, Shimokawa M, Takano A, Fujii M, Ohta Y, Watanabe T, Kanai T, Sato T (2015) Modeling colorectal cancer using CRISPR-Cas9-mediated engineering of human intestinal organoids. Nat Med 21(3):256–262. https://doi.org/10.1038/nm.3802

    Article  CAS  PubMed  Google Scholar 

  18. Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34(2):184–191. https://doi.org/10.1038/nbt.3437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Koo BK, Stange DE, Sato T, Karthaus W, Farin HF, Huch M, van Es JH, Clevers H (2011) Controlled gene expression in primary Lgr5 organoid cultures. Nat Methods 9(1):81–83. https://doi.org/10.1038/nmeth.1802

    Article  PubMed  Google Scholar 

  20. Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784. https://doi.org/10.1038/nmeth.3047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Shalem O, Sanjana NE, Hartenian E, Shi X, Scott DA, Mikkelsen TS, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F (2014) Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343(6166):84–87. https://doi.org/10.1126/science.1247005

    Article  CAS  PubMed  Google Scholar 

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Author information

Author notes

  1. Alex Gregorieff

    Present address: Department of Pathology, McGill University, Montreal, Canada

  2. Alex Gregorieff

    Present address: Cancer Research Program of the Research Institute of McGill University Health Centre, Montreal, Canada

Authors and Affiliations

  1. Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Toronto, Canada

    Christina Oatway, Calley L. Hirsch & Alex Gregorieff

  2. Department of Pathology, McGill University, Montreal, Canada

    Alex Gregorieff

  3. Cancer Research Program of the Research Institute of McGill University Health Centre, Montreal, Canada

    Alex Gregorieff

Authors
  1. Christina Oatway
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  2. Calley L. Hirsch
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  3. Alex Gregorieff
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Corresponding author

Correspondence to Alex Gregorieff .

Editor information

Editors and Affiliations

  1. Laboratory of Intestinal Physiopathology, Department of Anatomy and Cell Biology, Faculty of Medicine and Health Science, Université de Sherbrooke, Sherbrooke, Québec, Canada

    Jean-François Beaulieu

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Oatway, C., Hirsch, C.L., Gregorieff, A. (2018). Use of Organoids to Characterize Signaling Pathways in Cancer Initiation. In: Beaulieu, JF. (eds) Colorectal Cancer. Methods in Molecular Biology, vol 1765. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7765-9_21

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  • DOI: https://doi.org/10.1007/978-1-4939-7765-9_21

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7764-2

  • Online ISBN: 978-1-4939-7765-9

  • eBook Packages: Springer Protocols

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