Marchese, SD; (2013) Understanding the Impact of Estrogens in Mammary Gland Formation using an in vitro Three-Dimensional Model. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Increased ERα expression correlates with increased breast cancer risk, suggesting that the ER and estrogens play a role in cancer initiation and progression. Nonetheless, a clear understanding of the involvement of the ER in breast epithelium formation, carcinogenesis and cancer progression is lacking. In addition to this, the role of other estrogen-responsive receptors, such GPER-1 remains unknown. The mammary gland is comprised of milk ducts terminating in secretory units termed acini. These are comprised of a layer of epithelial cells surrounding a hollow lumen. These cells are in turn surrounded by a layer of myoepithelial cells attached to an underlying basement membrane. 3D cultures of immortalised breast cells recapitulate many of the features of acini in vivo, such as spherical growth-arrested acini with a layer of epithelial cells attached to an underlying basement membrane, surrounding a hollow lumen. We aimed to use 3D cultures of ERα, ERβ and GPER competent MCF-12A cells to assess the effects of estrogenic compounds on acini formation. MCF-12A cells were cultured in Matrigel with etOH (solvent control), E2, bisphenol A (BPA) or n-propylparaben. To assess the role of the ER or GPER-1, cells were also pre-treated with the ER and GPER -1 antagonists, as well as inhibitors of the MAPK and PI3K signalling cascades. Immunocytochemical staining using antibodies against activated caspase-3 and laminin-V was performed. Image acquisition was achieved by laser confocal microscopy. Control acini were encapsulated by a basement membrane with central cells either cleared or undergoing apoptosis. In comparison, cells treated with E2, BPA or propyparaben possessed filled lumen with little or no sign of apoptotic central cells and were larger and disorganised. Co-incubation with ER or GPER-1 antagonists, or PI3K and MAPK inhibitors reverted some of the effects of the test compounds resulting in growth-arrested, spherical acini with evidence of luminal clearing. We have successfully established an in vitro 3D model using immortalised ER competent cells to study the effects of estrogens on mammary gland formation. We have demonstrated that estrogenic compounds disrupt acini by inducing luminal filling and preventing growth arrest. Moreover, these effects seem to be mediated by both estrogen responsive receptors ER and GPER-1.

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