Skip to main content
SpringerLink
Log in

Studying Cell Polarity Dynamics During Cancer Initiation Using Inducible 3D Organotypic Cultures

  • Protocol
  • First Online:
Cell Polarity Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2438))

Abstract

Epithelial tissues are highly organized structures that are structured at both the cellular and tissue levels. Individual cells are characterized by an apical membrane facing a central lumen, and a basolateral membrane that contacts adjacent cells and the basement membrane. The maintenance of apical-basal polarity is crucial for maintaining epithelial homeostasis and is considered a barrier to carcinogenesis. Apical-basal cell polarity is compromised in many epithelial cancers, such as breast, lung, and prostate, and has been associated with disease progression. Three-dimensional (3D) organotypic cultures recapitulate the 3D tissue architecture and mechanical properties found in vivo. This chapter describes methods to establish 3D organoids from human cell lines or mouse primary cells with inducible oncogene expression in polarized epithelial structures to investigate mechanisms of tumor initiation, luminal filling, and growth. The method is versatile, and simple modifications can be made to study diverse cell/tissue types and oncogenes.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 199.99
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. Rodriguez-Boulan E, Macara IG (2014) Organization and execution of the epithelial polarity programme. Nat Rev Mol Cell Biol 15(4):225–242. https://doi.org/10.1038/nrm3775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Laprise P, Lau KM, Harris KP, Silva-Gagliardi NF, Paul SM, Beronja S, Beitel GJ, McGlade CJ, Tepass U (2009) Yurt, Coracle, Neurexin IV and the Na(+),K(+)-ATPase form a novel group of epithelial polarity proteins. Nature 459(7250):1141–1145. https://doi.org/10.1038/nature08067

    Article  CAS  PubMed  Google Scholar 

  3. Roman-Fernandez A, Bryant DM (2016) Complex polarity: building multicellular tissues through apical membrane traffic. Traffic 17(12):1244–1261. https://doi.org/10.1111/tra.12417

    Article  CAS  PubMed  Google Scholar 

  4. Halaoui R, Rejon C, Chatterjee SJ, Szymborski J, Meterissian S, Muller WJ, Omeroglu A, McCaffrey L (2017) Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma. Genes Dev 31(15):1573–1587. https://doi.org/10.1101/gad.300566.117

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Catterall R, Lelarge V, McCaffrey L (2020) Genetic alterations of epithelial polarity genes are associated with loss of polarity in invasive breast cancer. Int J Cancer 146(6):1578–1591. https://doi.org/10.1002/ijc.32691

    Article  CAS  PubMed  Google Scholar 

  6. Huang L, Muthuswamy SK (2010) Polarity protein alterations in carcinoma: a focus on emerging roles for polarity regulators. Curr Opin Genet Dev 20(1):41–50. https://doi.org/10.1016/j.gde.2009.12.001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Saito Y, Desai RR, Muthuswamy SK (2018) Reinterpreting polarity and cancer: the changing landscape from tumor suppression to tumor promotion. Biochim Biophys Acta Rev Cancer 1869(2):103–116. https://doi.org/10.1016/j.bbcan.2017.12.001

    Article  CAS  PubMed  Google Scholar 

  8. Halaoui R, Rejon C, Chatterjee SJ, Szymborski J, Meterissian S, Muller WJ, Omeroglu A, McCaffrey L (2017) Progressive polarity loss and luminal collapse disrupt tissue organization in carcinoma. Genes Dev 31(15):1573–1587

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Saito Y, Li L, Coyaud E, Luna A, Sander C, Raught B, Asara JM, Brown M, Muthuswamy SK (2019) LLGL2 rescues nutrient stress by promoting leucine uptake in ER(+) breast cancer. Nature 569(7755):275–279. https://doi.org/10.1038/s41586-019-1126-2

    Article  CAS  PubMed  Google Scholar 

  10. Mrozowska PS, Fukuda M (2016) Regulation of podocalyxin trafficking by Rab small GTPases in 2D and 3D epithelial cell cultures. J Cell Biol 213(3):355–369. https://doi.org/10.1083/jcb.201512024

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Takagi A, Watanabe M, Ishii Y, Morita J, Hirokawa Y, Matsuzaki T, Shiraishi T (2007) Three-dimensional cellular spheroid formation provides human prostate tumor cells with tissue-like features. Anticancer Res 27(1a):45–53

    CAS  PubMed  Google Scholar 

  12. Desoize B, Jardillier J-C (2000) Multicellular resistance: a paradigm for clinical resistance? Crit Rev Oncol Hematol 36(2):193–207. https://doi.org/10.1016/S1040-8428(00)00086-X

    Article  CAS  PubMed  Google Scholar 

  13. Riedl A, Schlederer M, Pudelko K, Stadler M, Walter S, Unterleuthner D, Unger C, Kramer N, Hengstschläger M, Kenner L, Pfeiffer D, Krupitza G, Dolznig H (2017) Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT–mTOR–S6K signaling and drug responses. J Cell Sci 130(1):203–218. https://doi.org/10.1242/jcs.188102

    Article  CAS  PubMed  Google Scholar 

  14. Baker BM, Chen CS (2012) Deconstructing the third dimension: how 3D culture microenvironments alter cellular cues. J Cell Sci 125(Pt 13):3015–3024. https://doi.org/10.1242/jcs.079509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ewald AJ (2017) 3D cell biology—the expanding frontier. J Cell Sci 130(1):1. https://doi.org/10.1242/jcs.200543

    Article  CAS  PubMed  Google Scholar 

  16. Rao T, Ranger JJ, Smith HW, Lam SH, Chodosh L, Muller WJ (2014) Inducible and coupled expression of the polyomavirus middle T antigen and Cre recombinase in transgenic mice: an in vivo model for synthetic viability in mammary tumour progression. Breast Cancer Res 16(1):R11. https://doi.org/10.1186/bcr3603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Benskey MJ, Manfredsson FP (2016) Lentivirus production and purification. Methods Mol Biol 1382:107–114. https://doi.org/10.1007/978-1-4939-3271-9_8

    Article  CAS  PubMed  Google Scholar 

  18. Nasri M, Karimi A, Allahbakhshian Farsani M (2014) Production, purification and titration of a lentivirus-based vector for gene delivery purposes. Cytotechnology 66(6):1031–1038. https://doi.org/10.1007/s10616-013-9652-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang B, Metharom P, Jullie H, Ellem KA, Cleghorn G, West MJ, Wei MQ (2004) The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events. Genet Vaccine Ther 2(1):6. https://doi.org/10.1186/1479-0556-2-6

    Article  CAS  Google Scholar 

  20. Wörsdörfer P, Dalda N, Kern A, Krüger S, Wagner N, Kwok CK, Henke E, Ergün S (2019) Generation of complex human organoid models including vascular networks by incorporation of mesodermal progenitor cells. Sci Rep 9(1):15663. https://doi.org/10.1038/s41598-019-52204-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Yuki K, Cheng N, Nakano M, Kuo CJ (2020) Organoid models of tumor immunology. Trends Immunol 41(8):652–664. https://doi.org/10.1016/j.it.2020.06.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Dekkers JF, Alieva M, Wellens LM, Ariese HCR, Jamieson PR, Vonk AM, Amatngalim GD, Hu H, Oost KC, Snippert HJG, Beekman JM, Wehrens EJ, Visvader JE, Clevers H, Rios AC (2019) High-resolution 3D imaging of fixed and cleared organoids. Nat Protoc 14(6):1756–1771. https://doi.org/10.1038/s41596-019-0160-8

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by a Canadian Institutes of Health Research grant (PJT-156271) to L.M. L.M. is a Fonds Recherche du Québec—Santé Research Scholar. R.C. is supported by a Canderel Studentship. R.K. is supported by Fonds de recherche du Québec—Santé (FRQS). We thank Li-Ting Wang for images in Fig. 1.

Author information

Author notes

  1. Rachel Catterall and Reem Kurdieh contributed equally with all other contributors.

Authors and Affiliations

  1. Rosalind and Morris Goodman Cancer Institute, McGill University, Montreal, QC, Canada

    Rachel Catterall, Reem Kurdieh & Luke McCaffrey

  2. Gerald Bronfman Department of Oncology, McGill University, Montreal, QC, Canada

    Luke McCaffrey

  3. Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC, Canada

    Rachel Catterall, Reem Kurdieh & Luke McCaffrey

  4. Department of Biochemistry, McGill University, Montreal, QC, Canada

    Luke McCaffrey

Authors
  1. Rachel Catterall
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Reem Kurdieh
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Luke McCaffrey
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luke McCaffrey .

Editor information

Editors and Affiliations

  1. Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA

    Chenbei Chang

  2. Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA

    Jianbo Wang

Rights and permissions

Reprints and permissions

Copyright information

© 2022 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Catterall, R., Kurdieh, R., McCaffrey, L. (2022). Studying Cell Polarity Dynamics During Cancer Initiation Using Inducible 3D Organotypic Cultures. In: Chang, C., Wang, J. (eds) Cell Polarity Signaling. Methods in Molecular Biology, vol 2438. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2035-9_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2035-9_26

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2034-2

  • Online ISBN: 978-1-0716-2035-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation