The role of integrins in the spatial organisation of the mammary gland.

The mammary epithelium is organised into a bilayer of inner polarised luminal epithelial (LE) lining the lumen space and outer myoepithelial (ME) cells, surrounded by a laminin-rich extracellular matrix (ECM). Although this architectural design is found in numerous internal organs, the processes involved in forming the concentric bilayer remain unknown. β1- integrin, a receptor that detects the ECM, is expressed more in ME than LE cells. We hypothesised that i) affinity to the ECM or ii) repulsion at the apical membrane drives ME cell positioning towards the ECM. I begin this thesis by validating lineage-specific β1-integrin deletion mouse models before characterising lumen formation in organoid cultures. I explore the role of ME β1-integrin-ECM signalling in polarity orientation and lumen formation to understand the differences between in vivo and in vitro data. Genetic deletion of β1-integrin in LE or ME cells did not affect cell positioning in primary culture organoids. I further tested by overlaying 2D cells with an exogenous ECM to relocate ECM affinity and disrupt the apical membrane. In WT culture, ME cells were positioned next to the top surface due to their affinity to the ECM, but β1-integrin-/- ME cells failed. β1-integrin deletion in LE cells inhibited apical membrane disruption in response to an ECM overlay and significantly reduced ME relocation. These findings support our hypotheses; however, further investigations are needed to establish the hierarchy of the two mechanisms. Using the experimental data, I developed a computational model to run predictive simulations of cell sorting under different integrin expressions. This identified integrins are required for establishing and maintaining cell positioning. Finally, I tested to what extent cell positioning and cell lineage-specific β1- integrin are essential for normal tissue function using organoid and collagen gel contraction assays. Overall, my thesis provides novel insights into the multifaceted role of integrins in the spatial organisation and function of the mammary gland.