Modeling Tissue Morphogenesis and Cancer in 3D
2007; Cell Press; Volume: 130; Issue: 4 Linguagem: Inglês
10.1016/j.cell.2007.08.006
ISSN1097-4172
AutoresKenneth M. Yamada, Edna Cukierman,
Tópico(s)Cancer Cells and Metastasis
ResumoThree-dimensional (3D) in vitro models span the gap between two-dimensional cell cultures and whole-animal systems. By mimicking features of the in vivo environment and taking advantage of the same tools used to study cells in traditional cell culture, 3D models provide unique perspectives on the behavior of stem cells, developing tissues and organs, and tumors. These models may help to accelerate translational research in cancer biology and tissue engineering. Three-dimensional (3D) in vitro models span the gap between two-dimensional cell cultures and whole-animal systems. By mimicking features of the in vivo environment and taking advantage of the same tools used to study cells in traditional cell culture, 3D models provide unique perspectives on the behavior of stem cells, developing tissues and organs, and tumors. These models may help to accelerate translational research in cancer biology and tissue engineering. Tissues and organs are three dimensional (3D). However, our ability to understand their formation, function, and pathology has often depended on two-dimensional (2D) cell culture studies or on animal model systems. Studies in standard cell culture have produced many important conceptual advances. Nevertheless, cells grown on flat 2D tissue culture substrates can differ considerably in their morphology, cell-cell and cell-matrix interactions, and differentiation from those growing in more physiological 3D environments (Birgersdotter et al., 2005Birgersdotter A. Sandberg R. Ernberg I. Gene expression perturbation in vitro–a growing case for three-dimensional (3D) culture systems.Semin. Cancer Biol. 2005; 15: 405-412Crossref PubMed Scopus (384) Google Scholar, Cukierman et al., 2002Cukierman E. Pankov R. Yamada K.M. Cell interactions with three-dimensional matrices.Curr. Opin. Cell Biol. 2002; 14: 633-639Crossref PubMed Scopus (734) Google Scholar, Griffith and Swartz, 2006Griffith L.G. Swartz M.A. Capturing complex 3D tissue physiology in vitro.Nat. Rev. Mol. Cell Biol. 2006; 7: 211-224Crossref PubMed Scopus (1676) Google Scholar, Nelson and Bissell, 2006Nelson C.M. Bissell M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer.Annu. Rev. Cell Dev. Biol. 2006; 22: 287-309Crossref PubMed Scopus (789) Google Scholar). At the other end of the experimental continuum, animal models frequently provide definitive tests of the importance of specific molecules and processes. However, there also can be puzzling discrepancies between conclusions from gene ablation studies and studies using chemical genetics approaches to interfere with the function of specific proteins (Knight and Shokat, 2007Knight Z.A. Shokat K.M. Chemical genetics: where genetics and pharmacology meet.Cell. 2007; 128: 425-430Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In addition, animal models may not adequately reproduce features of, for example, human tumors, drug therapeutic responses, autoimmune diseases, and stem cell differentiation. In vitro 3D tissue models provide a third approach that bridges the gap between traditional cell culture and animal models (Griffith and Swartz, 2006Griffith L.G. Swartz M.A. Capturing complex 3D tissue physiology in vitro.Nat. Rev. Mol. Cell Biol. 2006; 7: 211-224Crossref PubMed Scopus (1676) Google Scholar, Rangarajan et al., 2004Rangarajan A. Hong S.J. Gifford A. Weinberg R.A. Species- and cell type-specific requirements for cellular transformation.Cancer Cell. 2004; 6: 171-183Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). These in vitro 3D tissue models fulfill a need for reductionist approaches to understand in vivo molecular mechanisms. Moreover, the powerful tools of cell and molecular biology currently used in traditional cell cultures can often by applied to 3D tissue models. Increased use of 3D models that mimic specific tissues should promote advances in tissue engineering and could also facilitate the development and screening of new therapeutics. Our Review focuses on general principles, ideas, and caveats concerning the use of in vitro 3D model systems for studying tissue morphogenesis and tumorigenesis. We present recent examples chosen to illustrate key concepts. We also discuss exciting opportunities for further fundamental and translational research on cancer, stem cells, and tissue engineering. One commonly used approach makes use of tissues harvested in vivo (microscopic embryonic organs or intact tissue slices), which are then explanted and cultured in vitro. They often retain their original 3D architecture in culture. This approach has been particularly effective in relatively short-term cultures for experimental analysis of numerous tissues including brain and embryonic glands (Gahwiler et al., 1997Gahwiler B.H. Capogna M. Debanne D. McKinney R.A. Thompson S.M. Organotypic slice cultures: a technique has come of age.Trends Neurosci. 1997; 20: 471-477Abstract Full Text Full Text PDF PubMed Scopus (709) Google Scholar, Sakai et al., 2003Sakai T. Larsen M. Yamada K.M. Fibronectin requirement in branching morphogenesis.Nature. 2003; 423: 876-881Crossref PubMed Scopus (388) Google Scholar). For most studies, the tissues must be thin enough to permit adequate oxygenation and nutrition of the tissue interior, e.g., less than ∼0.3 mm thick. On the other hand, studies of tumor biology have benefited from systems that mimic the internal nutrient insufficiency of tumors to induce necrosis, which is important in studying tumor-host interactions such as the induction of tumor angiogenesis and resistance to chemotherapeutic drugs (Hicks et al., 2006Hicks K.O. Pruijn F.B. Secomb T.W. Hay M.P. Hsu R. Brown J.M. Denny W.A. Dewhirst M.W. Wilson W.R. Use of three-dimensional tissue cultures to model extravascular transport and predict in vivo activity of hypoxia-targeted anticancer drugs.J. Natl. Cancer Inst. 2006; 98: 1118-1128Crossref PubMed Scopus (120) Google Scholar). Many 3D models have been established starting from isolated cells, e.g., from cell lines, dissociated tissues, or stem cells. A widely used strategy is to propagate cells in tissue culture and then implant them in a 3D matrix scaffold as either single cells or as tissue-like aggregates. 3D scaffolds have been generated from purified molecules such as collagen I, synthetic biomaterials, and even from native extracellular matrices from which living cells were previously extracted (Table S1). Another approach is to use more than one type of isolated cell or a tissue fragment in combination with another cell type. An example of this is a 3D tissue model of human skin that combines keratinocytes and fibroblasts with cancer cells to simulate human melanoma (Smalley et al., 2006Smalley K.S. Lioni M. Herlyn M. Life isn't flat: taking cancer biology to the next dimension.In Vitro Cell. Dev. Biol. Anim. 2006; 42: 242-247Crossref PubMed Scopus (229) Google Scholar). Table 1 lists major advantages and disadvantages of current 3D systems compared to regular cell culture and animal models. Comparisons of 2D and 3D models reveal that the latter are better, but not exact, models of in vivo tissues. Table 2 compares specific biological properties and their regulation in 2D and 3D systems, and details are presented in Tables S1 and S2. For example, glandular epithelial cell organization, signaling, and secretion are more similar to what occurs in vivo in 3D settings than comparable 2D approaches (Debnath and Brugge, 2005Debnath J. Brugge J.S. Modelling glandular epithelial cancers in three-dimensional cultures.Nat. Rev. Cancer. 2005; 5: 675-688Crossref PubMed Scopus (789) Google Scholar, Nelson and Bissell, 2006Nelson C.M. Bissell M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer.Annu. Rev. Cell Dev. Biol. 2006; 22: 287-309Crossref PubMed Scopus (789) Google Scholar). The morphologies of fibroblasts, including cytoskeletal organization and types of cell adhesions, are also more similar to their in vivo behavior when the fibroblasts are grown in a 3D matrix than when grown in 2D (Figure 1). This is also true of their intracellular signaling characteristics (Cukierman et al., 2001Cukierman E. Pankov R. Stevens D.R. Yamada K.M. Taking cell-matrix adhesions to the third dimension.Science. 2001; 294: 1708-1712Crossref PubMed Scopus (2321) Google Scholar, Cukierman et al., 2002Cukierman E. Pankov R. Yamada K.M. Cell interactions with three-dimensional matrices.Curr. Opin. Cell Biol. 2002; 14: 633-639Crossref PubMed Scopus (734) Google Scholar, Grinnell, 2003Grinnell F. Fibroblast biology in three-dimensional collagen matrices.Trends Cell Biol. 2003; 13: 264-269Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, Walpita and Hay, 2002Walpita D. Hay E. Studying actin-dependent processes in tissue culture.Nat. Rev. Mol. Cell Biol. 2002; 3: 137-141Crossref PubMed Scopus (75) Google Scholar). Studies of gene expression and mRNA splicing patterns also reveal considerable differences when cells are cultured under 2D versus 3D conditions (Birgersdotter et al., 2005Birgersdotter A. Sandberg R. Ernberg I. Gene expression perturbation in vitro–a growing case for three-dimensional (3D) culture systems.Semin. Cancer Biol. 2005; 15: 405-412Crossref PubMed Scopus (384) Google Scholar, Li et al., 2006Li C. Kato M. Shiue L. Shively J.E. Ares Jr., M. Lin R.J. Cell type and culture condition-dependent alternative splicing in human breast cancer cells revealed by splicing-sensitive microarrays.Cancer Res. 2006; 66: 1990-1999Crossref PubMed Scopus (61) Google Scholar).Table 1Key Strengths and Weaknesses of 3D ModelsAdvantages• Cell morphology and signaling are often more physiological than routine 2D cell culture• Permit rapid experimental manipulations and testing of hypotheses• Permit much better real-time and/or fixed imaging by microscopy than in animalsDisadvantages• Vary in their ability to mimic in vivo tissue conditions• Currently lack vasculature and normal transport of small molecules, host immune responses, and other cell-cell interactions• Generally mimic static or short-term conditions, whereas in vivo systems often progress Open table in a new tab Table 23D-Dependent Cell Behavior and SignalingBiological Function2D versus 3DRegulatory MechanismsCell ShapeLoss of epithelial cell polarity and altered epithelial and fibroblast shape in 2DGrowth factor receptors and pathways; cell-adhesion signals associated with cell survival and matrix plasticityGene ExpressionCells in 2D versus 3D often have different patterns of gene expressionECM, hormones, and adhesion moleculesGrowth3D matrix-dependent regulation of cell growthAdhesion and growth factor-related pathways plus survival or apoptotic genesMorphogenesis3D matrix-induced vessel sprouting and gland branchingECM, adhesion, growth factor-related pathways and apoptotic genesMotilityAltered single and collective cell motility patterns in 3D matricesECM and its regulators; adhesions and growth factor-related pathways; phospholipidsDifferentiation3D matrix-induced cell differentiationECM and growth factors; motor moleculesSee Table S2 for references and additional information. Open table in a new tab See Table S2 for references and additional information. In any 3D model system, the specific cellular and matrix microenvironment provided to cells can substantially influence experimental outcome. For example, embedding tumor cells in a 3D collagen matrix as single cells, small aggregates, or larger aggregates can result, respectively, in individual cell migration and invasion, collective cell invasion, or mixtures of invasive and necrotic cells (Friedl, 2004Friedl P. Prespecification and plasticity: shifting mechanisms of cell migration.Curr. Opin. Cell Biol. 2004; 16: 14-23Crossref PubMed Scopus (506) Google Scholar, Mueller-Klieser, 1997Mueller-Klieser W. Three-dimensional cell cultures: from molecular mechanisms to clinical applications.Am. J. Physiol. 1997; 273: C1109-C1123PubMed Google Scholar). Because 3D in vitro model systems lack the complex vascular systems that perfuse tissues in vivo, oxygenation, nutrition, and waste removal occur by simple diffusion. Consequently, as the tissue thickness of a 3D model increases, transport limitations for these molecules will become increasingly important. Although nutrient restrictions may sometimes mimic in vivo tissue and tumor microenvironments better than the uniformly rich oxygenation and nutrition provided to monolayer cells in 2D cultures, they also introduce significant and potentially confounding variables to 3D models. This is because cells at different depths from the surface can be in different nutritional states (Keith and Simon, 2007Keith B. Simon M.C. Hypoxia-inducible factors, stem cells, and cancer.Cell. 2007; 129: 465-472Abstract Full Text Full Text PDF PubMed Scopus (846) Google Scholar, Levenberg, 2005Levenberg S. Engineering blood vessels from stem cells: recent advances and applications.Curr. Opin. Biotechnol. 2005; 16: 516-523Crossref PubMed Scopus (61) Google Scholar). In addition, both the composition and stiffness of the extracellular matrix surrounding the cells have major effects on cell signaling and behavior (Cukierman et al., 2002Cukierman E. Pankov R. Yamada K.M. Cell interactions with three-dimensional matrices.Curr. Opin. Cell Biol. 2002; 14: 633-639Crossref PubMed Scopus (734) Google Scholar, Discher et al., 2005Discher D.E. Janmey P. Wang Y.L. Tissue cells feel and respond to the stiffness of their substrate.Science. 2005; 310: 1139-1143Crossref PubMed Scopus (4409) Google Scholar, Grinnell, 2003Grinnell F. Fibroblast biology in three-dimensional collagen matrices.Trends Cell Biol. 2003; 13: 264-269Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar, Paszek et al., 2005Paszek M.J. Zahir N. Johnson K.R. Lakins J.N. Rozenberg G.I. Gefen A. Reinhart-King C.A. Margulies S.S. Dembo M. Boettiger D. et al.Tensional homeostasis and the malignant phenotype.Cancer Cell. 2005; 8: 241-254Abstract Full Text Full Text PDF PubMed Scopus (2656) Google Scholar). For example, collagen gels can mimic loose or dense connective tissue depending on the concentration of collagen; such gels have been used widely in studies of fibroblast and tumor cell migration and signaling (Grinnell, 2003Grinnell F. Fibroblast biology in three-dimensional collagen matrices.Trends Cell Biol. 2003; 13: 264-269Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). For growth and differentiation of epithelial cells, however, reconstituted gels of an extract containing basement membrane components and growth factors termed Matrigel (or EHS matrix or Cultrex) are much more effective (Kleinman and Martin, 2005Kleinman H.K. Martin G.R. Matrigel: basement membrane matrix with biological activity.Semin. Cancer Biol. 2005; 15: 378-386Crossref PubMed Scopus (968) Google Scholar). 3D matrices generated by cells in vitro provide yet another class of matrix (Cukierman et al., 2001Cukierman E. Pankov R. Stevens D.R. Yamada K.M. Taking cell-matrix adhesions to the third dimension.Science. 2001; 294: 1708-1712Crossref PubMed Scopus (2321) Google Scholar). Each type of matrix can also have experimental drawbacks. For example, collagen gels lack other components of connective tissue, and they differ in the extent of covalent crosslinking. Matrigel consists of basement membrane components, but it is a 3D cell culture material rather than a mimetic of the flat basement membranes underneath cells. Finally, cell-derived matrices can have lower amounts of collagen, larger internal spaces, and less depth than mature tissue matrices. Thus, an important point is that because each tissue in vivo has a characteristic matrix microenvironment, for a given study it is crucial to select an appropriately matched 3D in vitro matrix. Although the molecular composition of the extracellular matrix is a well-known regulator of cellular responses, physical properties of the matrix in 3D models can also play surprisingly important roles. In particular, recent evidence points to direct roles for the stiffness (compliance) of the extracellular matrix in regulating multiple cellular functions (Discher et al., 2005Discher D.E. Janmey P. Wang Y.L. Tissue cells feel and respond to the stiffness of their substrate.Science. 2005; 310: 1139-1143Crossref PubMed Scopus (4409) Google Scholar, Paszek et al., 2005Paszek M.J. Zahir N. Johnson K.R. Lakins J.N. Rozenberg G.I. Gefen A. Reinhart-King C.A. Margulies S.S. Dembo M. 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Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties.Biomaterials. 2007; 28: 671-679Crossref PubMed Scopus (278) Google Scholar, Ingber, 2006Ingber D.E. Cellular mechanotransduction: putting all the pieces together again.FASEB J. 2006; 20: 811-827Crossref PubMed Scopus (1112) Google Scholar, Vogel and Sheetz, 2006Vogel V. Sheetz M. Local force and geometry sensing regulate cell functions.Nat. Rev. Mol. Cell Biol. 2006; 7: 265-275Crossref PubMed Scopus (1642) Google Scholar). Biologically, cells need to sense and respond appropriately to their local microenvironment. The stiffness of microenvironments is variable; examples include loose versus dense connective tissue, soft versus hard tissues (such as bones and teeth), and early versus late stages of wound healing. The stiffness of a matrix and its susceptibility to remodeling by cellular contractile processes, matrix secretion, and enzymatic degradation can affect the distribution of cell surface integrin receptors and the types of cell adhesions and cytoskeletal structures formed (Cukierman et al., 2001Cukierman E. Pankov R. Stevens D.R. Yamada K.M. Taking cell-matrix adhesions to the third dimension.Science. 2001; 294: 1708-1712Crossref PubMed Scopus (2321) Google Scholar, Katz et al., 2000Katz B.Z. Zamir E. Bershadsky A. Kam Z. Yamada K.M. Geiger B. Physical state of the extracellular matrix regulates the structure and molecular composition of cell-matrix adhesions.Mol. Biol. Cell. 2000; 11: 1047-1060Crossref PubMed Scopus (345) Google Scholar, Walpita and Hay, 2002Walpita D. Hay E. Studying actin-dependent processes in tissue culture.Nat. Rev. Mol. Cell Biol. 2002; 3: 137-141Crossref PubMed Scopus (75) Google Scholar). Matrix stiffness also alters intracellular signaling via Rho kinase and Rac (Pankov et al., 2005Pankov R. Endo Y. Even-Ram S. Araki M. Clark K. Cukierman E. Matsumoto K. Yamada K.M. A Rac switch regulates random versus directionally persistent cell migration.J. Cell Biol. 2005; 170: 793-802Crossref PubMed Scopus (356) Google Scholar, Paszek et al., 2005Paszek M.J. Zahir N. Johnson K.R. Lakins J.N. Rozenberg G.I. Gefen A. Reinhart-King C.A. Margulies S.S. Dembo M. Boettiger D. et al.Tensional homeostasis and the malignant phenotype.Cancer Cell. 2005; 8: 241-254Abstract Full Text Full Text PDF PubMed Scopus (2656) Google Scholar, Wozniak et al., 2003Wozniak M.A. Desai R. Solski P.A. Der C.J. Keely P.J. ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix.J. Cell Biol. 2003; 163: 583-595Crossref PubMed Scopus (396) Google Scholar). Stiffness can also enhance cell proliferation, in some cases promoting neoplasia (Paszek et al., 2005Paszek M.J. Zahir N. Johnson K.R. Lakins J.N. Rozenberg G.I. Gefen A. Reinhart-King C.A. Margulies S.S. Dembo M. Boettiger D. et al.Tensional homeostasis and the malignant phenotype.Cancer Cell. 2005; 8: 241-254Abstract Full Text Full Text PDF PubMed Scopus (2656) Google Scholar, Pelham and Wang, 1997Pelham Jr., R.J. Wang Y. Cell locomotion and focal adhesions are regulated by substrate flexibility.Proc. Natl. Acad. Sci. USA. 1997; 94: 13661-13665Crossref PubMed Scopus (2319) Google Scholar). Different in vitro 3D models provide a range of matrix stiffness that can mimic the range found in specific tissues in living organisms. Wide differences in stiffness exist between soft adipose tissue and the tightly woven basement membrane (such as encountered by mammary epithelial cells), as well as between loose matrices used by cells for migration during embryogenesis, dense connective tissue in skin, and precalcified osteoid versus rigid mature bone (Discher et al., 2005Discher D.E. Janmey P. Wang Y.L. Tissue cells feel and respond to the stiffness of their substrate.Science. 2005; 310: 1139-1143Crossref PubMed Scopus (4409) Google Scholar, Engler et al., 2006Engler A.J. Sen S. Sweeney H.L. Discher D.E. Matrix elasticity directs stem cell lineage specification.Cell. 2006; 126: 677-689Abstract Full Text Full Text PDF PubMed Scopus (9275) Google Scholar, Paszek et al., 2005Paszek M.J. Zahir N. Johnson K.R. Lakins J.N. Rozenberg G.I. Gefen A. Reinhart-King C.A. Margulies S.S. Dembo M. Boettiger D. et al.Tensional homeostasis and the malignant phenotype.Cancer Cell. 2005; 8: 241-254Abstract Full Text Full Text PDF PubMed Scopus (2656) Google Scholar). Pathological processes such as fibrosis or microenvironmental changes within and around developing tumors can also alter tissue stiffness and cellular responses (Engler et al., 2004Engler A.J. Griffin M.A. Sen S. Bonnemann C.G. Sweeney H.L. Discher D.E. Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments.J. Cell Biol. 2004; 166: 877-887Crossref PubMed Scopus (1251) Google Scholar). For example, dense, nonpliable desmoplastic tissue is associated with some carcinomas (Paszek et al., 2005Paszek M.J. Zahir N. Johnson K.R. Lakins J.N. Rozenberg G.I. Gefen A. Reinhart-King C.A. Margulies S.S. Dembo M. Boettiger D. et al.Tensional homeostasis and the malignant phenotype.Cancer Cell. 2005; 8: 241-254Abstract Full Text Full Text PDF PubMed Scopus (2656) Google Scholar) and sites predisposed for secondary metastases (Kaplan et al., 2005Kaplan R.N. Riba R.D. Zacharoulis S. Bramley A.H. Vincent L. Costa C. MacDonald D.D. Jin D.K. Shido K. Kerns S.A. et al.VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.Nature. 2005; 438: 820-827Crossref PubMed Scopus (2267) Google Scholar). Another critical in vivo property provided by 3D models is appropriate cell polarity. Polarity in vivo depends both on the cell type and the cellular microenvironment (Figure 2). Epithelial cells are often polarized, with apical and basal surfaces that are important for tissue organization and directional secretion of products. Their basal surfaces rest on thin, flat basement membranes comprised of collagen IV, laminin, and many other matrix proteins. In many tissues, particularly secretory organs, epithelial cells are organized into spherical 3D structures surrounding a lumen to function as acini of glands, alveoli (lung), or glomeruli (kidney). Tissue organization is lost when these cells are explanted onto flat 2D tissue culture substrates, and this organization and differentiated function can be restored or maintained when the cells are placed into 3D culture conditions (Griffith and Swartz, 2006Griffith L.G. Swartz M.A. Capturing complex 3D tissue physiology in vitro.Nat. Rev. Mol. Cell Biol. 2006; 7: 211-224Crossref PubMed Scopus (1676) Google Scholar, Nelson and Bissell, 2006Nelson C.M. Bissell M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer.Annu. Rev. Cell Dev. Biol. 2006; 22: 287-309Crossref PubMed Scopus (789) Google Scholar). Cells explanted into routine tissue cultures often flatten and lose differentiation markers. When placed back in appropriate 3D culture conditions, epithelial cells generally regain apical-basal polarity, and glandular cells form a lumen into which differentiated products are secreted. The inset image shows the morphology of human salivary gland cells reaggregated in vitro. In contrast, mesenchymal derivatives in 3D (lower right) regain a fibroblastic spindle shape and lose their artificial dorsal-ventral polarity. Epithelial cells often rest on a relatively thin 2D basement membrane facing a lumen. They can sometimes undergo an epithelial-mesenchymal transition to become migratory in a 3D stroma. Because basement membranes are thin and basically 2D, epithelial cells in vivo resting on a flat basement membrane can be considered to be adhering to a 2D substrate (Figure 2). We predict, therefore, that it should eventually be possible to elicit normal, differentiated epithelial cell function on appropriately designed 2D surfaces in vitro, e.g., with a composition and stiffness mimicking basement membranes when combined with soluble stromal factors. In direct contrast, cells such as fibroblasts lack this highly polar apical-basal organization in vivo. However, when placed onto 2D culture substrates, these cells acquire an upper (dorsal) and lower (ventral) surface, and prominent cell adhesions to the substrate form on the ventral surface. Fibroblasts lose this artificial dorsal-ventral polarity when placed back into a mesenchymal 3D matrix, and they regain their in vivo morphology (Amatangelo et al., 2005Amatangelo M.D. Bassi D.E. Klein-Szanto A.J. Cukierman E. Stroma-derived three-dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts.Am. J. Pathol. 2005; 167: 475-488Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, Cukierman et al., 2001Cukierman E. Pankov R. Stevens D.R. Yamada K.M. Taking cell-matrix adhesions to the third dimension.Science. 2001; 294: 1708-1712Crossref PubMed Scopus (2321) Google Scholar, Grinnell, 2003Grinnell F. Fibroblast biology in three-dimensional collagen matrices.Trends Cell Biol. 2003; 13: 264-269Abstract Full Text Full Text PDF PubMed Scopus (636) Google Scholar). Three-dimensionality per se—independent of matrix composition—can physiologically reprogram fibroblasts. There are striking differences in morphology, proliferation, and directionality of migration between cells cultured on a 3D matrix versus cells cultured on a matrix of identical biochemical composition that has been flattened to provide a 2D surface (Cukierman et al., 2001Cukierman E. Pankov R. Stevens D.R. Yamada K.M. Taking cell-matrix adhesions to the third dimension.Science. 2001; 294: 1708-1712Crossref PubMed Scopus (2321) Google Scholar, Pankov et al., 2005Pankov R. Endo Y. Even-Ram S. Araki M. Clark K. Cukierman E. Matsumoto K. Yamada K.M. A Rac switch regulates random versus directionally persistent cell migration.J. Cell Biol. 2005; 170: 793-802Crossref PubMed Scopus (356) Google Scholar, Zaman et al., 2006Zaman M.H. Trapani L.M. Sieminski A.L. Mackellar D. Gong H. Kamm R.D. Wells A. Lauffenburger D.A. Matsudaira P. Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.Proc. Natl. Acad. Sci. USA. 2006; 103: 10889-10894Crossref PubMed Scopus (834) Google Scholar). One explanation for this may involve cellular detection of matrix contact with both ventral and dorsal surfaces. Simply bringing a collagenous substrate into contact with both cell surfaces restores more normal 3D morphology in fibroblasts (Beningo et al., 2004Beningo K.A. Dembo M. Wang Y.-l. Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors.Proc. Natl. Acad. Sci. USA. 2004; 101: 18024-18029Crossref PubMed Scopus (217) Google Scholar); however, whether other cell functions such as signaling and proliferation are similarly regulated remains to be examined. Taken together, studies of epithelial cells and fibroblasts indicate that an important feature of 3D models is their ability to mimic normal tissue organization to induce appropriate polarity of each cell type (Figure 2). Nevertheless, polarity and phenotype are not always fixed; at specific stages of embryonic development and in some cancers, epithelial cells can undergo an epithelial-to-mesenchymal transition involving the loss of cell-cell adhesions and polarity, accompanied by activation of cell migration (Hay, 2005Hay E.D. The mesenchymal cell, its role in the embryo, and the remarkable signaling mechanisms that create it.Dev. Dyn. 2005; 233: 706-720Crossref PubMed Scopus (486) Google Scholar, Thiery and Sleeman, 2006Thiery J.P. Sleeman J.P. Complex networks orchestrate epithelial-mesenchymal transitions.Nat. Rev. Mol. Cell Biol. 2006; 7: 131-142Crossref PubMed Scopus (3060) Google Scholar). Morphogenesis—the development of form in the embryo—has recently been analyzed extensively in a variety of 3D model systems, particularly organ cultures and cell line models. A major goal in embryology has been to understand the mechanisms and regulation of branching morphogenesis (Figure 3), a process essential for the formation of glands and organs, including lungs, kidneys, salivary and mammary glands, prostate, and the vasculature (Affolter et al., 200
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