Demystifying the effects of a three-dimensional microenvironment in tissue morphogenesis

Abstract

Tissue morphogenesis and homeostasis are dependent on a complex dialogue between multiple cell types and chemical and physical cues in the surrounding microenvironment. The emergence of engineered three-dimensional (3D) tissue constructs and the development of tractable methods to recapitulate the native tissue microenvironment ex vivo has led to a deeper understanding of tissue-specific behavior. However, much remains unclear about how the microenvironment and aberrations therein directly affect tissue morphogenesis and behavior. Elucidating the role of the microenvironment in directing tissue-specific behavior will aid in the development of surrogate tissues and tractable approaches to diagnose and treat chronic-debilitating diseases such as cancer and atherosclerosis. Toward this goal, 3D organotypic models have been developed to clarify the mechanisms of epithelial morphogenesis and the subsequent maintenance of tissue homeostasis. Here we describe the application of these 3D culture models to illustrate how the microenvironment plays a critical role in regulating mammary tissue function and signaling, and discuss the rationale for applying precisely defined organotypic culture assays to study epithelial cell behavior. Experimental methods are provided to generate and manipulate 3D organotypic cultures to study the effect of matrix stiffness and matrix dimensionality on epithelial tissue morphology and signaling. We end by discussing technical limitations of currently available systems and by presenting opportunities for improvement.

Fig. 1

Biochemical and biophysical cues from the extracellular matrix regulate tissue-specific epithelial differentiation. Illustration depicting ECM regulation of tissue-specific differentiation through a progressively complex hierarchy of adhesion-regulated events functionally linked to changes in cell shape, receptor-initiated biochemical signaling, assembly of multicellular structures, and reciprocal biochemical and physical modification of the ECM microenvironment adjacent to the epithelial tissue. Undifferentiated cell (top): an undifferentiated cell interacting with a highly rigid 2D ECM substratum, such as matrix-coated tissue culture plastic, will adhere rapidly and, if given sufficient ECM ligand, will spread appreciably using multiple adhesion receptors, including integrins, and assemble mature focal adhesions. Epithelial cells grown on a rigid 2D matrix proliferate readily to form viable polarized cellular monolayers with adherens and tight junctions as well as prominent focal adhesions. Such cells exhibit robust Rho GTPase activation in response to exogenous stimuli, and require activated PI3 kinase or ERK signaling to survive. Under these conditions, epithelial cells do not assemble 3D tissue-like structures or express differentiated proteins in response to “differentiation cues.” Mechanical cues (second tier): an epithelial cell interacting with a highly compliant ECM readily adheres using multiple matrix receptors, including integrins, and assembles small immature focal complexes but fails to spread appreciably. Instead cells interacting with a compliant matrix exhibit profound reorganization of their actin cytoskeleton. MECs grown under these conditions can be induced to express lactoferrin if given the correct exogenous soluble cues. 3D ECM and biochemical cues (third tier): epithelial cells interacting with a highly compliant matrix in three dimensions adhere through multiple adhesion receptors including integrins, syndecans, and DG, and proliferate readily in response to exogenous growth factors. MECs interacting with a highly compliant 3D ECM can be induced to express abundant quantities of the differentiation protein β-casein. Multicellular organization, morphogenesis, and tissue differentiation (fourth to sixth tiers): in response to a 3D compliant ECM, ductal epithelial cells begin to interact with one another and assemble multicellular polarized structures with cell–cell junctions including adherens, scribble, and gap junctions. MECs assembled into multicellular 3D-polarized tissue-like structures begin to deposit and assemble an endogenous basally polarized basement membrane, show enhanced expression of milk protein expression such as β-casein, and exhibit enhanced long-term survival and apoptosis resistance to multiple exogenous stimuli including chemotherapeutics, immune receptor activators, and gamma irradiation. Long term culture of epithelial cells in the context of a 3D compliant ECM permits completion of tissue-like morphogenesis characterized by the assembly of an apically and basally polarized, growth-arrested tissue with a cleared central lumen and spatial restriction of various membrane associated proteins including growth factor receptors. Once a fully polarized and growth-arrested structure has formed, mammary acini can now be induced to express additional milk proteins such as WAP in response to lactogenic hormones. However, in response to an increase in matrix stiffness as occurs following chronic inflammation, injury, or tumorigenesis, or following genetic mutations and oncogene activation, tissue integrity becomes progressively compromised reversing the cell state to a less differentiated condition. In extreme cases, cells can behave analogous to undifferentiated, highly contractile single cells.

Fig. 2

Matrix stiffness modulates MEC growth and morphogenesis. Phase-contrast and confocal immunofluorescence images of 3D MEC colonies on 3D rBM-crosslinked PA gels of increasing elastic moduli (E = 150–5000 Pa) after 20 days, showing progressively disrupted colony morphology as matrix stiffness increases (top). Cell–cell adherens junctions are disrupted and luminal clearance is compromised with even a modest increase in the elastic modulus of the matrix (E = 1050 Pa central panel; β-catenin and actin). Basal polarity is perturbed (disorganized β4 integrin and absence of basally deposited laminin-5) once the matrix stiffness stiffens appreciably (E > 5000 Pa; right panel). Scale bar is 20μm. Adapted from Paszek et al. (2005).