Tumour progression in metastatic breast cancer cell lines: engineered in vitro models

Abstract

Breast tumour progression is a multifactorial phenomenon in which the tumour cells share a dynamic relationship with the tumour micro-environment (TME). TME is a complex combination of both cellular (fibroblasts, immune cells, endothelial cells) and non-cellular components (extracellular matrix (ECM), tissue architecture, signalling molecules, interstitial fluid, etc). The TME gets altered by the surrounding tumour and its dysregulation in turn affects tumour progression. Dysregulation of non-cellular TME cues such as ECM, extracellular pH, and interstitial fluid flow are correlated to breast cancer progression but their direct impact on breast cancer phenotypes has not been examined yet. Traditional preclinical models, like 2D cell culture in vitro systems, fail to mimic such cell-TME interactions, whereas use of animal models for such studies renders it difficult to control and decouple the TME variables. Hence the aim of this thesis was to engineer 3D in vitro models that could precisely mimic these TME changes and decouple them to understand their individual as well as collective contribution to breast cancer progression. Breast cancer cells were cultured in alginate-gelatin hydrogels used to mimic normal (1-2 kPa) and tumour (6-10 kPa) breast tissue stiffness. These were further cultured in a perfusion system (500 μL/min) to mimic interstitial fluid flow with media of different pH (pH 7.4 and 6.5). The in vitro model effectively decoupled these cues and captured their distinctive effect on two breast cancer cell lines’ (MDA-MB 231 and MCF-7) proliferation, morphology, and cancer stem cell population. Overall, we observed that high stiffness and acidic pH are the main factors involved in increasing stem cell content of both the breast cancer cell lines. With metastasis sharing a major burden of breast cancer related deaths, impact of matrix stiffness on migration, invasion and bone metastasis was also probed using in vitro techniques. Bone microenvironment was mimicked by using “biohybrid” poly-ɛ-caprolactone (PCL) scaffolds that mimicked bone stiffness (45-55 MPa), porosity (~40-50%), and contained bone-ECM deposited by osteoblasts. Alginate hydrogels of different stiffness were combined with PCL scaffolds to model the “breast to bone metastasis”, where high primary tumour matrix stiffness was found to be associated with increased migration, invasion and expression of osteolytic factors (PTHrP and IL-6) in metastatic breast cancer cell line MDA-MB 231. This thesis effectively explored primary tumour TME factors and the far-reaching implication they can have on metastasis and tumour progression. Additionally, these 3D in vitro models present themselves as future platforms for disease prognosis and drug efficacy studies in presence of relevant TME cues.

Date of Award	31 Dec 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Kaye Williams (Supervisor), Ayse Latif (Supervisor) & Annalisa Tirella (Supervisor)