Abstract
Investigations into the roles of mature adipocytes within the breast tumour microenvironment (TME) have increased with the acceptance that both the cellular and acellular components of the TME are intricately linked in malignant progression. Mature adipocytes at the invasive front of developing breast tumours in vivo display an altered morphology and phenotype. However, in vitro research into the interactions occurring between mature adipocytes and breast cancer cells has been limited by a lack of appropriate model systems with which to culture these different cell types. As such, the principal aim of this thesis was to develop a novel three-dimensional (3D) in vitro breast cancer cell-mature adipocyte co-culture system, to assess the interactions between these cells relating to the breast TME.
This was approached by, firstly, culturing two breast cancer cell lines representative of Luminal A (MCF7) and Triple Negative (MDA-MB-231) breast cancer molecular subtypes within GelMA hydrogels of a clinically comparable range of hydrogel stiffness to determine the parameters necessary for the successful incorporation and culture of breast cancer cells alone. Luminal A MCF7 breast cancer cells underwent EMT-like phenotypic changes when cultured in stiff (28 kPa) GelMA hydrogels for 21 days, generating a model of stiffness induced EMT. When cultured in soft (11 kPa) GelMA hydrogels both MCF7 and MDA-MB-231 breast cancer cells demonstrated no change in EMT marker expression, thus establishing the parameters for maintenance of breast cancer cell phenotype in further investigations.
Secondly, patient-derived mature adipocytes were co-cultured with breast cancer cells with results showing that mature adipocytes co-cultured with MDA-MB-231 breast cancer cells lost expression of PLIN1 at day 21 of culture. Contrastingly, MCF7 breast cancer cells displayed an EMT-like upregulation of mesenchymal markers at day 21 of co-culture with mature adipocytes. Together highlighting a breast cancer subtype specific impact of co-culture on cellular phenotype.
Lastly, this 3D co-culture system was utilised to investigate chemotherapy responsiveness. Triple Negative MDA-MB-231 breast cancer cells co-cultured with mature adipocytes were less responsive to chemotherapy treatment, with a potential mechanism modulated by Major Vault Protein (MVP) expression. Additionally, breast cancer cell co-cultures with mature adipocytes secreted higher levels of cytokines related to biological pathways involved in cell migration, invasion, survival, inflammation and treatment response.
Cumulatively, the studies described in this thesis have provided a novel 3D in vitro breast cancer cell-mature adipocyte co-culture model with which to continue exploring the diverse roles of mature adipocytes within the breast TME.