Development of a Novel In Vitro Bioengineered Human Breast Ductal Mucosal Model to Investigate the Invasive Properties of Breast Cancer During its Development.

Abstract

Breast cancer is one of the mostly commonly diagnosed cancers globally, where it poses a significant healthcare burden in both developed and developing countries. Over recent years, it has become increasingly apparent that the tumour microenvironment is a major driver of the adoption of specific migratory phenotypes in breast cancer. However, in vitro models investigating breast cancer invasion often do not recapitulate this important aspect of breast cancer biology, thereby reducing their physiological relevance and predictive power.

This project aimed to develop novel three-dimensional (3D) migration assays, based on available Alvetex® technologies, that account for the tumour microenvironment. Their effectiveness at recapitulating in vivo behaviours was then compared against conventional 2D invasion assays and the literature. Through the use of three immortalised breast cancer cell lines: MCF-10A, MCF-7, and MDA-MB-231’s, the three main stages of ductal breast cancer were able to be simulated, namely: Healthy tissue, Ductal Carcinoma In Situ (DCIS), and Invasive Ductal Carcinoma (IDC), respectively.

Initially the impact of a 3D geometric space on breast cancer invasion characteristics was investigated using Alvetex® Strata. Despite the increase in in vivo-like characteristics of each cell line in this platform, the physiological relevance of these models was limited due to the lack of presence of Extracellular Matrix (ECM) constituents and stromal cells. Using Co-culture techniques, optimised in the Przyborski lab, Human Neonatal Dermal Fibroblasts (HDFn’s) were cultured in Alvetex® Scaffold with the immortalised cell lines to create and optimise a complex 3D breast cancer invasion model. The physiological relevance of these models was then assessed using immunostaining and histological analysis to confirm the presence of in vivo characteristics and reproducibility of this platform. This led to the creation of a novel reproducible 3D invasion assay for breast cancer that accounts for a physiologically relevant mammary microenvironment. The modular nature of this model was then explored, testing its compatibility with primary mammary fibroblast and epithelial cells to further increase physiological relevance, while also exploring the potential for patient personalised Alvetex® models.

Although physiological relevance is important in invasion models, so is the compatibility of a platform with anti-migratory compounds, as treatments of in vitro models with known inhibitors is a cornerstone for increasing our understanding of invasive processes, as well as identifying novel compounds. Thus, each model platform (2D, Alvetex® Strata, Alvetex® Scaffold) was treated with a known migration inhibitor, Caffeic Acid Phenyl-Ethyl Ester (CAPE), to demonstrate their compatibility with these pipelines.

Together, the data presented in this thesis demonstrates the ability of this novel 3D Co-culture system to recapitulate the migratory behaviour of breast cancer cells in its distinct developmental stages; in a platform that is compatible with both drug treatment protocols and the use of primary cell lines.