The role of collagen reorganization on mammary epithelial morphogenesis in a 3D culture model
Introduction
The breast is a branched tubulo-alveolar gland with ducts formed by an epithelium supported by a stroma that contains an extracellular matrix (ECM), fibroblasts, immune cells, adipocytes, and blood and lymphatic vessels. Some of the ECM components include type I collagen, laminins, type IV collagen, fibronectin, and other macromolecules [1], [2], [3]. Stromal–epithelial interactions mediate mammary gland organogenesis during normal and neoplastic development [4], [5], [6].
In order to dissect the mechanisms that mediate stromal–epithelial interactions, defined three-dimensional (3D) models were developed [7], [8], [9], [10], [11]. The introduction of reconstituted basement membrane (rBM), a complex mixture of macromolecules secreted by a mouse chondrosarcoma [12], has facilitated the study of interactions between breast epithelial cells and BM components. Primary epithelial cells and established epithelial cell lines cultured in rBM mostly form acini, that together with ducts are the most common structures in a resting breast [7], [13], [14]. However, since most breast cancers are of ductal origin [15], experimental surrogate 3D models should include ductal as well as alveolar structures.
Collagen type I is the most abundant of the ECM components in normal and malignant breast [16]. Increased mammographic density, due in part to the abundance of collagen type I, has been shown to increase breast cancer risk in women [17]. In addition, increased collagen density has been associated with breast cancer initiation and progression in animal studies [18], [19]. However, in 3D cultures, the role of collagen in epithelial morphogenesis is still being debated. Primary cultures of mouse mammary epithelial and myoepithelial cells grown in floating collagen gels formed structures resembling the alveoli present in vivo [20]. The human breast cell lines T47D and MCF10A formed acini and ducts when cultured in floating collagen gels, but only formed undifferentiated cellular sheets in attached gels [21], [22]. In contrast, the human breast epithelial cell line HB2 formed only acini in collagen type I gels; however, when HB2 cells were co-cultured with various fibroblast cell lines or fibroblast-conditioned media branching tubular structures developed [23]. Thus, it is clear that the type of epithelial structure formed is influenced by various factors, including the epithelial cell types, presence of fibroblasts, and the biochemical and physical properties of the gel in which they are embedded.
Collagen gels have also been used to explore the role of the focal adhesion kinase pathway (FAK) on epithelial morphogenesis through transduction of mechanical stimuli. It has been suggested that acini and ducts occurred only in matrices compliant enough for the cells to contract them [22]. Disruption of the intracellular Rho kinase signal downstream of the integrin receptor has been shown to inhibit tubulogenesis and block collagen contraction [22]. This suggests that cell-adhesion receptors actively participate in collagen reorganization [22], [24]. Collagen fibers bundle into large diameter fascicles through lateral alignment; this process is crucial in determining strain-transfer properties in 3D collagen-based matrices [25], [26]. However, the role of collagen restructuring in epithelial tubulogenesis and branching, and the associated biomechanical interactions remain unknown.
In this study, we characterized the matrix composition and organization that allow the formation of mammary structures resembling those found in vivo. We used floating, attached and partially detached collagen gel models. Our results indicate that epithelial morphogenesis in 3D collagen gels is spatio-temporally regulated and that collagen reorganization may play a role in epithelial tubulogenesis and branching.
Section snippets
Chemicals and cell culture reagents
Hydrocortisone, cholera toxin, insulin, methyl salicylate, and carmine were purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), DMEM/F12, and penicillin–streptomycin solution were obtained from Gibco/Invitrogen (Carlsbad, CA). Equine serum and fetal bovine serum (FBS) were purchased from HyClone (Logan, UT). Bovine type I collagen was purchased from Organogenesis (Canton, MA). Epidermal growth factor (EGF) and formalin were obtained from Fisher Scientific
Floating gels
At day 1, the gel area covering the entire well was approximately 4 cm2. The onset of gel contraction in MCF10A mono-cultures was observed on the third day; the gel area was reduced up to 85% of the initial value within the first 10 days of culture (Fig. 1A). Collagen gels containing only RMF began contracting after one week in culture and progressed at a slower rate than that observed in MCF10A mono-cultures. When MCF10A cells were co-cultured with RMF, collagen contraction began on the second
Discussion
Morphogenesis is mediated by the reciprocal interactions between epithelial cells and the surrounding stroma [30]. Surrogate models to study these stroma–epithelium interactions include 3D cultures in which mammary epithelial cells grown in floating collagen gels generated organoids of alveolar or tubular shape [20], [21], [31]. However, little is known about the influence of local matrix reorganization within those gels on determining the epithelial morphology. Here, we report the
Conclusion
The most remarkable finding of our study has been the plasticity of the model as revealed by the local and temporal changes observed both in the distribution of epithelial structures, their phenotype, and in collagen fiber organization. These dynamic changes became apparent by the systematic observation of the whole-mounted gels. This plasticity suggests a dynamic process initiated by the cell-mediated collagen organization that results in reciprocal interaction between the newly organized and
Acknowledgments
This work was supported in part by grants from the Parsemus Foundation, Philip Morris International, the Babylon Breast Cancer Coalition and the Great Neck Breast Cancer Coalition. The excellent technical expertise of Cheryl Schaeberle, Michael Askenase and Andrew Tharp is greatly appreciated.
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