Cell and Tissue Research

, Volume 324, Issue 3, pp 411–422 | Cite as

Dynamic analysis of hepatoma spheroid formation: roles of E-cadherin and β1-integrin

  • Ruei-Zeng Lin
  • Li-Fang Chou
  • Chi-Chen Michael Chien
  • Hwan-You Chang
Regular Article

Abstract

A spheroid is an in vitro multicellular aggregate that provides a microenvironment resembling that of normal tissue in vivo. Although cell adhesion molecules such as integrins and cadherins have been implicated in participating in the process of spheroid formation, little is known about the timing of their action. In this study, we have employed an image-based quantitative method to investigate the compactness of cell aggregates during hepatoma spheroid formation in a dynamic fashion. By modulating β1-integrin and E-cadherin activity with specific blocking antibodies, ion chelators, and RGD-sequence-containing peptides, we show that these cell adhesion molecules mediate the formation of spheroids through the establishment of complex cell-cell and cell-extracellular matrix (ECM) interactions. The dynamics of spheroid formation can be separated into three stages. In the first stage, ECM fibers act as a long-chain linker for the attachment of dispersed single-cells to form loose aggregations through the binding of integrins. This is followed by a delay period in which cell aggregates pause in compaction, presumably because of the accumulation of sufficient amounts of E-cadherins. In the third stage, strong homophilic interaction of E-cadherins is a major factor for the morphological transition from loose cell aggregates to compact spheroids. These findings thus provide comprehensive information on the molecular mechanisms and dynamics of hepatoma spheroid formation.

Keywords

Aggregates Cadherins Dynamics Integrins Spheroids Hepatoma cell lines (Human) 

References

  1. Abu-Absi SF, Friend JR, Hansen LK, Hu WS (2002) Structural polarity and functional bile canaliculi in rat hepatocyte spheroids. Exp Cell Res 274:56–67PubMedCrossRefGoogle Scholar
  2. Alpaugh ML, Tomlinson JS, Ye Y, Barsky SH (2002) Relationship of sialyl-Lewis(x/a) underexpression and E-cadherin overexpression in the lymphovascular embolus of inflammatory breast carcinoma. Am J Pathol 161:619–628PubMedGoogle Scholar
  3. Angres B, Barth A, Nelson WJ (1996) Mechanism for transition from initial to stable cell-cell adhesion: kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay. J Cell Biol 134:549–557PubMedCrossRefGoogle Scholar
  4. Angst BD, Marcozzi C, Magee AI (2001) The cadherin superfamily: diversity in form and function. J Cell Sci 114:629–641PubMedGoogle Scholar
  5. Arusha O, Timothy M, Shoukat D (2004) Regulation of E-cadherin expression and β-catenin/Tcf transcriptional activity by the integrin-linked kinase. Biochem Biophys Acta 1691:1–15CrossRefGoogle Scholar
  6. Bates RC, Edwards NS, Yates JD (2000) Spheroids and cell survival. Crit Rev Oncol Hematol 36:61–74PubMedCrossRefGoogle Scholar
  7. Byers SW, Sommers CL, Hoxter B, Mercurio AM, Tozeren A (1995) Role of E-cadherin in the response of tumor cell aggregates to lymphatic, venous and arterial flow: measurement of cell-cell adhesion strength. J Cell Sci 108:2053–2064PubMedGoogle Scholar
  8. Casey RC, Burleson KM, Skubitz KM, Pambuccian SE, Oegema TR Jr, Ruff LE, Skubitz AP (2001) Beta 1-integrins regulate the formation and adhesion of ovarian carcinoma multicellular spheroids. Am J Pathol 159:2071–2080PubMedGoogle Scholar
  9. Cavallaro U, Christofori G (2004) Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat Rev Cancer 4:118–132PubMedGoogle Scholar
  10. Conacci-Sorrell M, Simcha I, Ben-Yedidia T, Blechman J, Savagner P, Ben-Ze’ev A (2003) Autoregulation of E-cadherin expression by cadherin-cadherin interactions: the roles of beta-catenin signaling, Slug, and MAPK. J Cell Biol 163:847–857PubMedCrossRefGoogle Scholar
  11. Desoize B (2000) Contribution of three-dimensional culture to cancer research. Crit Rev Oncol Hematol 36:59–60PubMedCrossRefGoogle Scholar
  12. Enmon RM Jr, O’Connor KC, Lacks DJ, Schwartz DK, Dotson RS (2001) Dynamics of spheroid self-assembly in liquid-overlay culture of DU 145 human prostate cancer cells. Biotechnol Bioeng 72:579–591PubMedCrossRefGoogle Scholar
  13. Enmon RM Jr, O’Connor KC, Song H, Lacks DJ, Schwartz DK (2002) Aggregation kinetics of well and poorly differentiated human prostate cancer cells. Biotechnol Bioeng 80:580–588PubMedCrossRefGoogle Scholar
  14. Frisch SM, Ruoslahti E (1997) Integrins and anoikis. Curr Opin Cell Biol 9:701–706PubMedCrossRefGoogle Scholar
  15. Frisch SM, Screaton RA (2001) Anoikis mechanisms. Curr Opin Cell Biol 13:555–562PubMedCrossRefGoogle Scholar
  16. Frixen UH, Behrens J, Sachs M, Eberle G, Voss B, Warda A, Lochner D, Birchmeier W (1991) E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J Cell Biol 113:173–185PubMedCrossRefGoogle Scholar
  17. Fukuda J, Mizumoto H, Nakazawa K, Kajiwara T, Funatsu K (2004) Hepatocyte organoid culture in elliptic hollow fibers to develop a hybrid artificial liver. Int J Artif Organs 27:1091–1099PubMedGoogle Scholar
  18. Glinsky VV, Glinsky GV, Glinskii OV, Huxley VH, Turk JR, Mossine VV, Deutscher SL, Pienta KJ, Quinn TP (2003) Intravascular metastatic cancer cell homotypic aggregation at the sites of primary attachment to the endothelium. Cancer Res 63:3805–3811PubMedGoogle Scholar
  19. Guo W, Giancotti FG (2004) Integrin signalling during tumour progression. Nat Rev Mol Cell Biol 5:816–826PubMedCrossRefGoogle Scholar
  20. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70PubMedCrossRefGoogle Scholar
  21. Holtfreter J (1944) A study of the mechanics of gastrulation. J Exp Zool 95:171–212CrossRefGoogle Scholar
  22. Islam S, Carey TE, Wolf GT, Wheelock MJ, Johnson KR (1996) Expression of N-cadherin by human squamous carcinoma cells induces a scattered fibroblastic phenotype with disrupted cell-cell adhesion. J Cell Biol 135:1643–1654PubMedCrossRefGoogle Scholar
  23. Jakab K, Neagu A, Mironov V, Markwald RR, Forgacs G (2004) Engineering biological structures of prescribed shape using self-assembling multicellular systems. Proc Natl Acad Sci USA 101:2864–2869PubMedCrossRefGoogle Scholar
  24. Kale S, Biermann S, Edwards C, Tarnowski C, Morris M, Long MW (2000) Three-dimensional cellular development is essential for ex vivo formation of human bone. Nat Biotechnol 18:954–958PubMedCrossRefGoogle Scholar
  25. Kelm JM, Fussenegger M (2004) Microscale tissue engineering using gravity-enforced cell assembly. Trends Biotechnol 22:195–202PubMedCrossRefGoogle Scholar
  26. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:173–180PubMedCrossRefGoogle Scholar
  27. Korff T, Augustin HG (1998) Integration of endothelial cells in multicellular spheroids prevents apoptosis and induces differentiation. J Cell Biol 143:1341–1352PubMedCrossRefGoogle Scholar
  28. Kunz-Schughart LA, Kreutz M, Knuechel R (1998) Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology. Int J Exp Pathol 79:1–23PubMedCrossRefGoogle Scholar
  29. Landry J, Bernier D, Ouellet C, Goyette R, Marceau N (1985) Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J Cell Biol 101:914–923PubMedCrossRefGoogle Scholar
  30. Larue L, Antos C, Butz S, Huber O, Delmas V, Dominis M, Kemler R (1996) A role for cadherins in tissue formation. Development 122:3185–3194PubMedGoogle Scholar
  31. Lazar A, Peshwa MV, Wu FJ, Chi CM, Cerra FB, Hu WS (1995) Formation of porcine hepatocyte spheroids for use in a bioartificial liver. Cell Transplant 4:259–268PubMedCrossRefGoogle Scholar
  32. Marrs JA, Andersson-Fisone C, Jeong MC, Cohen-Gould L, Zurzolo C, Nabi IR, Rodriguez-Boulan E, Nelson WJ (1995) Plasticity in epithelial cell phenotype: modulation by expression of different cadherin cell adhesion molecules. J Cell Biol 129:507–519PubMedCrossRefGoogle Scholar
  33. Moscona A, Moscona H (1952) The dissociation and aggregation of cells from organ rudiments of the early chick embryo. J Anat 86:287–301PubMedGoogle Scholar
  34. Mueller-Klieser W (1997) Three-dimensional cell cultures: from molecular mechanisms to clinical applications. Am J Physiol 273:C1109–C1123PubMedGoogle Scholar
  35. Nagar B, Overduin M, Ikura M, Rini JM (1996) Structural basis of calcium-induced E-cadherin rigidification and dimerization. Nature 380:360–364PubMedCrossRefGoogle Scholar
  36. Nakamura M, Shinji T, Ujike K, Hirasaki S, Koide N, Tsuji T (2002) Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation. Acta Med Okayama 56:43–50PubMedGoogle Scholar
  37. Narayanan RA, Rink A, Beattie CW, Hu WS (2002) Differential gene expression analysis during porcine hepatocyte spheroid formation. Mamm Genome 13:515–523PubMedCrossRefGoogle Scholar
  38. Neelamegham S, Munn LL, Zygourakis K (1997) A model for the kinetics of homotypic cellular aggregation under static conditions. Biophys J 72:51–64PubMedCrossRefGoogle Scholar
  39. O’Brien LE, Zegers MM, Mostov KE (2002) Building epithelial architecture: insights from three-dimensional culture models. Nat Rev Mol Cell Biol 3:531–537PubMedCrossRefGoogle Scholar
  40. Robinson EE, Zazzali KM, Corbett SA, Foty RA (2003) Alpha5beta1 integrin mediates strong tissue cohesion. J Cell Sci 116:377–386PubMedCrossRefGoogle Scholar
  41. Robinson EE, Foty RA, Corbett SA (2004) Fibronectin matrix assembly regulates alpha5beta1-mediated cell cohesion. Mol Biol Cell 15:973–981PubMedCrossRefGoogle Scholar
  42. Shimaoka M, Takagi J, Springer TA (2002) Conformational regulation of integrin structure and function. Annu Rev Biophys Biomol Struct 31:485–516PubMedCrossRefGoogle Scholar
  43. Shimazui T, Schalken JA, Kawai K, Kawamoto R, Bockhoven A van, Oosterwijk E, Akaza H (2004) Role of complex cadherins in cell-cell adhesion evaluated by spheroid formation in renal cell carcinoma cell lines. Oncol Rep 11:357–360PubMedGoogle Scholar
  44. Tomlinson JS, Alpaugh ML, Barsky SH (2001) An intact overexpressed E-cadherin/alpha,beta-catenin axis characterizes the lymphovascular emboli of inflammatory breast carcinoma. Cancer Res 61:5231–5241PubMedGoogle Scholar
  45. Wong CW, Song C, Grimes MM, Fu W, Dewhirst MW, Muschel RJ, Al-Mehdi AB (2002) Intravascular location of breast cancer cells after spontaneous metastasis to the lung. Am J Pathol 161:749–753PubMedGoogle Scholar
  46. Wong BW, Luk JM, Ng IO, Hu MY, Liu KD, Fan ST (2003) Identification of liver-intestine cadherin in hepatocellular carcinoma—a potential disease marker. Biochem Biophys Res Commun 311:618–624PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Ruei-Zeng Lin
    • 1
  • Li-Fang Chou
    • 1
  • Chi-Chen Michael Chien
    • 2
  • Hwan-You Chang
    • 1
  1. 1.Institute of Molecular MedicineNational Tsing Hua UniversityHsin ChuRepublic of China
  2. 2.Department of Obstetrics and GynecologyTon-Yen General HospitalChubeiRepublic of China

Personalised recommendations