Abstract
Colonic adenocarcinoma-derived Caco-2 and T84 epithelial cell lines are frequently used as in vitro model systems of functional epithelial barriers. Both are utilised interchangeably despite evidence that differentiated Caco-2 cells are more reminiscent of small intestinal enterocytes than of colonocytes, whereas differentiated T84 cells are less well characterised. The aim of this study was, therefore, to further characterise and compare differentiated Caco-2 and T84 cells. The objectives were to (1) compare the brush border morphology, (2) measure the expression of enterocyte- and colonocyte-specific genes and (3) compare their response to butyrate, which is dependent on the monocarboxylate transporter 1 (MCT1), an apical protein expressed primarily in colonocytes. T84 microvilli were significantly shorter than those of Caco-2 cells, which is a characteristic difference between small intestinal enterocytes and colonocytes. Also, enterocyte-associated brush border enzymes expressed in differentiated Caco-2 cells were not increased during T84 maturation, whereas colonic markers such as MCT1 were more abundant in differentiated T84 cells compared to differentiated Caco-2 cells. Consequently, T84 cells displayed a dose-responsive improvement of barrier function towards butyrate, which was absent in Caco-2 cells. On the other hand, differences in epithelial toll-like receptor expression between Caco-2 and T84 monolayers did not result in a corresponding differential functional response. We conclude that differentiated Caco-2 and T84 cells have distinct morphological, biochemical and functional characteristics, suggesting that T84 cells do not acquire the biochemical signature of mature small intestinal enterocytes like Caco-2 cells, but retain much of their original colonic characteristics throughout differentiation. These findings can help investigators select the appropriate intestinal epithelial cell line for specific in vitro research purposes.
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Abbreviations
- ALPI:
-
Alkaline phosphatase, intestinal
- ANPEP:
-
Alanyl aminopeptidase, membrane
- CFTR:
-
Cystic fibrosis transmembrane conductance regulator
- DDP4:
-
Dipeptidyl peptidase 4
- DMEM:
-
Dulbecco’s Modified Eagle Medium
- FBS:
-
Fetal bovine serum
- FLG:
-
Flagellin
- HMBS:
-
Hydroxymethyl-bilane synthase
- IL-8:
-
Interleukin-8
- LCT:
-
Lactase
- LPS:
-
Lipopolysaccharide
- MCT1:
-
Monocarboxylate transporter 1
- MGAM:
-
Maltase-glucoamylase
- MS4A12:
-
Membrane spanning 4-domains A12
- NaB:
-
Sodium butyrate
- NRQs:
-
Normalised relative quantities
- OPLS:
-
Orthogonal partial least squares
- PCA:
-
Principle component analysis
- qRT-PCR:
-
Quantitative real-time PCR
- SEM:
-
Standard error of the mean
- SI:
-
Sucrase-isomaltase
- TEER:
-
Transepithelial electrical resistance
- TEM:
-
Transmission electron microscopy
- TLR:
-
Toll-like receptor
- VIP:
-
Variable importance for the projection.
References
Abreu MT, Vora P, Faure E, Thomas LS, Arnold ET, Arditi M (2001) Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide. J Immunol 167(3):1609–1616
Artursson P, Palm K, Luthman K (2001) Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv Drug Deliv Rev 46(1–3):27–43
Bolte G, Wolburg H, Beuermann K, Stocker S, Stern M (1998) Specific interaction of food proteins with apical membranes of the human intestinal cell lines Caco-2 and T84. Clin Chim Acta 270(2):151–167
Brabletz T (2012) EMT and MET in metastasis: where are the cancer stem cells? Cancer Cell 22(6):699–701
Chantret I, Barbat A, Dussaulx E, Brattain MG, Zweibaum A (1988) Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res 48(7):1936–1942
Dharmsathaphorn K, McRoberts JA, Mandel KG, Tisdale LD, Masui H (1984) A human colonic tumor cell line that maintains vectorial electrolyte transport. Am J Physiol 246(2 Pt 1):G204–G208
Engle MJ, Goetz GS, Alpers DH (1998) Caco-2 cells express a combination of colonocyte and enterocyte phenotypes. J Cell Physiol 174(3):362–369
Ferruzza S, Rossi C, Scarino ML, Sambuy Y (2012) A protocol for in situ enzyme assays to assess the differentiation of human intestinal Caco-2 cells. Toxicol In Vitro 26(8):1247–1251
Gill RK, Saksena S, Alrefai WA, Sarwar Z, Goldstein JL, Carroll RE, Ramaswamy K, Dudeja PK (2005) Expression and membrane localization of MCT isoforms along the length of the human intestine. Am J Physiol Cell Physiol 289(4):C846–C852
Guilloteau P, Martin L, Eeckhaut V, Ducatelle R, Zabielski R, Van Immerseel F (2010) From the gut to the peripheral tissues: the multiple effects of butyrate. Nutr Res Rev 23(2):366–384
Jumarie C, Malo C (1991) Caco-2 cells cultured in serum-free medium as a model for the study of enterocytic differentiation in vitro. J Cell Physiol 149(1):24–33
Junqueira LC, Carneiro J (2005) Basic histology. McGraw-Hill, New York
Koslowski M, Sahin U, Dhaene K, Huber C, Tureci O (2008) MS4A12 is a colon-selective store-operated calcium channel promoting malignant cell processes. Cancer Res 68(9):3458–3466
Lewis K, Lutgendorff F, Phan V, Soderholm JD, Sherman PM, McKay DM (2010) Enhanced translocation of bacteria across metabolically stressed epithelia is reduced by butyrate. Inflamm Bowel Dis 16(7):1138–1148
Liu Z, Zhang P, Zhou Y, Qin H, Shen T (2010) Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3. Braz J Med Biol Res 43(5):451–459
Madara JL, Stafford J, Dharmsathaphorn K, Carlson S (1987) Structural analysis of a human intestinal epithelial cell line. Gastroenterology 92(5 Pt 1):1133–1145
Natoli M, Leoni BD, D’Agnano I, D’Onofrio M, Brandi R, Arisi I, Zucco F, Felsani A (2011) Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer. J Cell Physiol 226(6):1531–1543
Peng L, He Z, Chen W, Holzman IR, Lin J (2007) Effects of butyrate on intestinal barrier function in a Caco-2 cell monolayer model of intestinal barrier. Pediatr Res 61(1):37–41
Peng L, Li ZR, Green RS, Holzman IR, Lin J (2009) Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in Caco-2 cell monolayers. J Nutr 139(9):1619–1625
Pinto M, Robine-Leon S, Appay MD, Kedinger M, Triadou N, Dussaulx E, Lacroix B, Simon-Assmann P, Haffen K, Fogh J, Zweibaum A (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol Cell 47:323–330
Pryde SE, Duncan SH, Hold GL, Stewart CS, Flint HJ (2002) The microbiology of butyrate formation in the human colon. FEMS Microbiol Lett 217(2):133–139
Sambuy Y, De Angelis I, Ranaldi G, Scarino ML, Stammati A, Zucco F (2005) The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biol Toxicol 21(1):1–26
Schuerer-Maly CC, Eckmann L, Kagnoff MF, Falco MT, Maly FE (1994) Colonic epithelial cell lines as a source of interleukin-8: stimulation by inflammatory cytokines and bacterial lipopolysaccharide. Immunology 81(1):85–91
Sood R, Bear C, Auerbach W, Reyes E, Jensen T, Kartner N, Riordan JR, Buchwald M (1992) Regulation of CFTR expression and function during differentiation of intestinal epithelial cells. EMBO J 11(7):2487–2494
Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139(5):871–890
Triba MN, Le Moyec L, Amathieu R, Goossens C, Bouchemal N, Nahon P, Rutledge DN, Savarin P (2015) PLS/OPLS models in metabolomics: the impact of permutation of dataset rows on the K-fold cross-validation quality parameters. Mol Biosyst 11(1):13–19
Vereecke L, Beyaert R, van Loo G (2011) Enterocyte death and intestinal barrier maintenance in homeostasis and disease. Trends Mol Med 17(10):584–593
Wang HB, Wang PY, Wang X, Wan YL, Liu YC (2012) Butyrate enhances intestinal epithelial barrier function via up-regulation of tight junction protein Claudin-1 transcription. Dig Dis Sci 57(12):3126–3135
Acknowledgements
The authors would like to thank Petra Van Wassenhove, Griet Driesschaert and Jelle De Medts for technical support, and Ran Rumes for electron microscopy.
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This study was funded by the Flemish Agency for Innovation by Science and Technology (Grant Number SBO-100016). LVDB, TH and DL are paid by grants from the Research Fund—Flanders (FWO 11J9915N, 13/ASP/145 and 1298213N).
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The authors declare that they have no conflict of interest.
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Devriese, S., Van den Bossche, L., Van Welden, S. et al. T84 monolayers are superior to Caco-2 as a model system of colonocytes. Histochem Cell Biol 148, 85–93 (2017). https://doi.org/10.1007/s00418-017-1539-7
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DOI: https://doi.org/10.1007/s00418-017-1539-7