Alford, Deborah Jane; (1997) Morphogenesis of human mammary epithelial cells in vitro. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

The human mammary gland in vivo is made up of branching epithelial ducts embedded in a supporting stroma. One of the earliest changes seen in the development of breast cancers is the disruption of normal tissue architecture, thereby leading to the loss of the organisation of the branching ducts. The objective of this study was to set up an in vitro system whereby the molecules involved in branching morphogenesis of a non tumorigenic human mammary epithelial cell line could be analysed. These molecules would be potential targets for alteration in tumorigenesis, and also have a potential role in mammary gland development in vivo. This thesis describes the in vitro system used, analysis of the roles of fibroblast secreted factors and integrins in the branching morphogenesis process, and the effect of overexpression of oncogenes on the ability of cells to form organised morphogenetic structures. The in vitro system involves culturing a human mammary epithelial cell line (HB2) in 3-dimensions within a collagen type I (or fibrin) matrix. The cells grow to form compact, organised, spherical cysts, or can be induced to perform branching morphogenesis when cultured with conditioned medium from fibroblasts. We have found that there are at least two distinct soluble factors that can induce branching morphogenesis; hepatocyte growth factor (HGF), and an unidentified and potentially novel factor secreted by human foreskin fibroblasts (HFF). Preliminary characterisation of this factor has shown that it is a heat stable protein with a size of between 35 and 45 kDa. In the analysis of the role of integrins in branching morphogenesis, we have analysed both the expression of integrins at the RNA level throughout the morphogenetic time course, and the effect of specific monoclonal antibodies (mAbs) to integrin subunits. Use of specific integrin mAbs that inhibit cell- matrix interaction, has shown that integrin mediated maintenance of cell- matrix interaction is vital both for the growth of HB2 cells and for maintaining cell-cell interactions in the branching structures. In both cases the integrins involved were found to differ depending on the matrix in which the branching morphogenesis occurred, with the a2?1 important in the collagen system and the [alpha]v[beta]1 in fibrin. Both HGF and HFF conditioned medium are shown to stimulate the motility of HB2 cells on collagen, and it is hypothesised that the stimulation of cell motility is a necessary event in the branching morphogenesis process, and that the [alpha]2[beta]1 integrin has a specific role in branching morphogenesis by way of regulating this motility. Evidence for this is provided by showing that mAbs to the [alpha]2 or [beta]1 integrin subunits that reduce HGF induced motility also inhibit branching morphogenesis induced by HGF. Overexpression of the v-Ha-ras oncogene in HB2 cells also led to the disruption of cell-cell interactions in 3-dimensional morphogenetic structures. Overexpression of ras induced a reduced level of expression of the [alpha]2 integrin subunit and a concurrent reduction in adhesion of the cells to collagen type I. Further evidence that the [alpha]2[beta]1 integrin-collagen interaction is involved in regulating cell-cell interaction in 3-dimensional structures was provided by showing that the HB2 ras colony phenotype could be significantly reverted with a mAb to the [beta]1 integrin subunit which increases the integrin mediated adhesion of cells to collagen.

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