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Confocal Laser Scanning Imaging of Cell Junctions in Human Colon Cancer Cells

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Intestinal Differentiated Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2650))

Abstract

The intestinal epithelium is formed by a single layer of cells. These cells originate from self-renewal stem cells that give rise to various lineages of cells: Paneth, transit-amplifying, and fully differentiated cells (as enteroendocrine, goblet cells, and enterocytes). Enterocytes, also known as absorptive epithelial cells, are the most abundant cell type in the gut. Enterocytes have the potential to polarize as well as form tight junctions with neighbor cells which altogether serve to ensure both the absorption of “good” substances into the body and the blockage of “bad” substances, among other functions. Culture cell models such as the Caco-2 cell line have been proved to be valuable tools to study the fascinating functions of the intestine. In this chapter we outline some experimental procedures to grow, differentiate, and stain intestinal Caco-2 cells, as well as image them using two modes of confocal laser scanning microscopy.

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References

  1. Kayama H, Okumura R, Takeda K (2020) Interaction between the microbiota, epithelia, and immune cells in the intestine. Annu Rev Immunol 38:23–48. https://doi.org/10.1146/annurev-immunol-070119-115104

    Article  CAS  PubMed  Google Scholar 

  2. Peterson LW, Artis D (2014) Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 14:141–153. https://doi.org/10.1038/nri3608

    Article  CAS  PubMed  Google Scholar 

  3. Conigrave AD, Young JA (1996) Function of the intestine. In: Greger R, Windhorst U (eds) Comprehensive human physiology. Springer, Berlin/Heidelberg, pp 1259–1287

    Chapter  Google Scholar 

  4. Ali A, Tan H, Kaiko GE (2020) Role of the intestinal epithelium and its interaction with the microbiota in food allergy. Front Immunol 11:604054. https://doi.org/10.3389/fimmu.2020.604054

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kong S, Zhang YH, Zhang W (2018) Regulation of intestinal epithelial cells properties and functions by amino acids. Biomed Res Int 2018:2819154. https://doi.org/10.1155/2018/2819154

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. van der Flier LG, Clevers H (2009) Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol 71:241–260. https://doi.org/10.1146/annurev.physiol.010908.163145

    Article  CAS  PubMed  Google Scholar 

  7. Spit M, Koo B-K, Maurice MM (2018) Tales from the crypt: intestinal niche signals in tissue renewal, plasticity and cancer. Open Biol 8. https://doi.org/10.1098/rsob.180120

  8. Umar S (2010) Intestinal stem cells. Curr Gastroenterol Rep 12:340–348. https://doi.org/10.1007/s11894-010-0130-3

    Article  PubMed  PubMed Central  Google Scholar 

  9. Bonis V, Rossell C, Gehart H (2021) The intestinal epithelium – fluid fate and rigid structure from crypt bottom to villus tip. Front Cell Dev Biol 9:661931. https://doi.org/10.3389/fcell.2021.661931

    Article  PubMed  PubMed Central  Google Scholar 

  10. Juanes MA (2020) Cytoskeletal control and Wnt signaling-APC’s dual contributions in stem cell division and colorectal cancer. Cancers (Basel) 12. https://doi.org/10.3390/cancers12123811

  11. McCarthy N, Kraiczy J, Shivdasani RA (2020) Cellular and molecular architecture of the intestinal stem cell niche. Nat Cell Biol 22:1033–1041. https://doi.org/10.1038/s41556-020-0567-z

    Article  CAS  PubMed  Google Scholar 

  12. Clevers H (2013) The intestinal crypt, a prototype stem cell compartment. Cell 154:274–284. https://doi.org/10.1016/j.cell.2013.07.004

    Article  CAS  PubMed  Google Scholar 

  13. Ouladan S, Gregorieff A (2021) Taking a step back: insights into the mechanisms regulating gut epithelial dedifferentiation. Int J Mol Sci 22. https://doi.org/10.3390/ijms22137043

  14. Seishima R, Barker N (2019) A contemporary snapshot of intestinal stem cells and their regulation. Differentiation 108:3–7. https://doi.org/10.1016/j.diff.2019.01.004

    Article  CAS  PubMed  Google Scholar 

  15. Beumer J, Clevers H (2021) Cell fate specification and differentiation in the adult mammalian intestine. Nat Rev Mol Cell Biol 22:39–53. https://doi.org/10.1038/s41580-020-0278-0

    Article  CAS  PubMed  Google Scholar 

  16. Klunder LJ, Faber KN, Dijkstra G, van Ijzendoorn SCD (2017) Mechanisms of cell polarity-controlled epithelial homeostasis and immunity in the intestine. Cold Spring Harb Perspect Biol 9. https://doi.org/10.1101/cshperspect.a027888

  17. Crawley SW, Mooseker MS, Tyska MJ (2014) Shaping the intestinal brush border. J Cell Biol 207:441–451. https://doi.org/10.1083/jcb.201407015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ding X, Hu X, Chen Y et al (2021) Differentiated Caco-2 cell models in food-intestine interaction study: current applications and future trends. Trends Food Sci Technol 107:455–465. https://doi.org/10.1016/j.tifs.2020.11.015

    Article  CAS  Google Scholar 

  19. Chantret I, Barbat A, Dussaulx E et al (1988) Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res 48:1936–1942

    CAS  PubMed  Google Scholar 

  20. Simon-Assmann P, Turck N, Sidhoum-Jenny M et al (2007) In vitro models of intestinal epithelial cell differentiation. Cell Biol Toxicol 23:241–256. https://doi.org/10.1007/s10565-006-0175-0

    Article  CAS  PubMed  Google Scholar 

  21. van Klinken BJ, Oussoren E, Weenink JJ et al (1996) The human intestinal cell lines Caco-2 and LS174T as models to study cell-type specific mucin expression. Glycoconj J 13:757–768

    Article  PubMed  Google Scholar 

  22. Natoli M, Leoni BD, D’Agnano I et al (2012) Good Caco-2 cell culture practices. Toxicol In Vitro 26:1243–1246. https://doi.org/10.1016/j.tiv.2012.03.009

    Article  CAS  PubMed  Google Scholar 

  23. Fatmawati NND, Goto K, Mayura IPB et al (2020) Caco-2 cells monolayer as an in-vitro model for probiotic strain translocation. Bali Med J 9:137. https://doi.org/10.15562/bmj.v9i1.1633

    Article  Google Scholar 

  24. Hidalgo IJ, Raub TJ, Borchardt RT (1989) Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96:736–749. https://doi.org/10.1016/0016-5085(89)90897-4

    Article  CAS  PubMed  Google Scholar 

  25. Kenny B, Dean P (2013) Do Caco-2 subclones provide more appropriate in vitro models for understanding how human enteric pathogens cause disease? Future Microbiol 8:701–703. https://doi.org/10.2217/fmb.13.51

    Article  CAS  PubMed  Google Scholar 

  26. Ferruzza S, Rossi C, Scarino ML, Sambuy Y (2012) A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing medium. Toxicol In Vitro 26:1252–1255. https://doi.org/10.1016/j.tiv.2012.01.008

    Article  CAS  PubMed  Google Scholar 

  27. Ferruzza S, Scacchi M, Scarino ML, Sambuy Y (2002) Iron and copper alter tight junction permeability in human intestinal Caco-2 cells by distinct mechanisms. Toxicol In Vitro 16:399–404. https://doi.org/10.1016/S0887-2333(02)00020-6

    Article  CAS  PubMed  Google Scholar 

  28. Noben M, Vanhove W, Arnauts K et al (2017) Human intestinal epithelium in a dish: current models for research into gastrointestinal pathophysiology. United European Gastroenterol J 5:1073–1081. https://doi.org/10.1177/2050640617722903

    Article  PubMed  PubMed Central  Google Scholar 

  29. Ponce de León-Rodríguez MDC, Guyot J-P, Laurent-Babot C (2019) Intestinal in vitro cell culture models and their potential to study the effect of food components on intestinal inflammation. Crit Rev Food Sci Nutr 59:3648–3666. https://doi.org/10.1080/10408398.2018.1506734

    Article  CAS  PubMed  Google Scholar 

  30. Riedl A, Schlederer M, Pudelko K et al (2017) Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6. J Cell Sci 130(1):203–218. Epub 2016 Sep 23.PMID: 27663511. https://doi.org/10.1242/jcs.188102

  31. Juanes MA, Bouguenina H, Eskin JA et al (2017) Adenomatous polyposis coli nucleates actin assembly to drive cell migration and microtubule-induced focal adhesion turnover. J Cell Biol 216:2859–2875. https://doi.org/10.1083/jcb.201702007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Juanes MA, Isnardon D, Badache A et al (2019) The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover. J Cell Biol 218:3415–3435. https://doi.org/10.1083/jcb.201904165

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Juanes MA, Fees C, Hoeprich GJ et al (2020) EB1 directly regulates APC-mediated actin nucleation. Curr Biol 30:4763

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sanderson MJ, Smith I, Parker I, Bootman MD (2014) Fluorescence microscopy. Cold Spring Harb Protoc 2014:pdb.top071795. https://doi.org/10.1101/pdb.top071795

  35. Downloads.leica-microsystems.com/LeicaTCSSP8/Brochures/SP8-Lightning-Product-Flyer-201910-EN.pdf

  36. Wang YL, Grooms NWF, Civale SC, Chung SH (2021) Confocal imaging capacity on a widefield microscope using a spatial light modulator. PLoS One 16:e0244034. https://doi.org/10.1371/journal.pone.0244034

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Wang X, Kress A, Brasselet S, Ferrand P (2013) High frame-rate fluorescence confocal angle-resolved linear dichroism microscopy. Rev Sci Instrum 84:053708. https://doi.org/10.1063/1.4807318

    Article  CAS  PubMed  Google Scholar 

  38. SP8 LIGHTNING Confocal Microscope | Products | Leica Microsystems. https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8/media/. Accessed 7 July 2020

  39. Ferraretto A, Gravaghi C, Donetti E et al (2007) New methodological approach to induce a differentiation phenotype in Caco-2 cells prior to post-confluence stage. Anticancer Res 27:3919–3925

    PubMed  Google Scholar 

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Acknowledgments

Caco-2 cells were kindly provided by Dr. Paloma Ordoñez-Morán from the University of Nottingham, UK. This work was supported by grants from the Academy of Medical Science/the Wellcome Trust/the Government Department of Business, Energy and Industrial Strategy/the British Heart Foundation/Diabetes UK AMS Springboard Award [SBF006\1070], and the CIDEGENT Excellence Research Program from the Valencian regional goverment CIDEGENT/2021/026 to M.A.J.

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Authors and Affiliations

  1. School of Health and Life Science, Teesside University, Middlesbrough, UK

    Peixun Zhou & M. Angeles Juanes

  2. National Horizons Centre, Teesside University, Darlington, UK

    Peixun Zhou & M. Angeles Juanes

  3. Centro de Investigación Príncipe Felipe, Valencia, Spain

    M. Angeles Juanes

Authors
  1. Peixun Zhou
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  2. M. Angeles Juanes
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Correspondence to M. Angeles Juanes .

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Editors and Affiliations

  1. Biodiscovery Inst- 3, University Park, University of Nottingham, Nottingham, UK

    Paloma Ordóñez-Morán

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Zhou, P., Juanes, M.A. (2023). Confocal Laser Scanning Imaging of Cell Junctions in Human Colon Cancer Cells. In: Ordóñez-Morán, P. (eds) Intestinal Differentiated Cells. Methods in Molecular Biology, vol 2650. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3076-1_19

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  • DOI: https://doi.org/10.1007/978-1-0716-3076-1_19

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3075-4

  • Online ISBN: 978-1-0716-3076-1

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