Agrawal, Ayushi; (2023) Development of advanced in vitro models to investigate the role of the tumour microenvironment in the metastatic cascade. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Cancer metastasis is the leading cause of most solid tumour-related deaths. It is defined as the spread of cancer cells from their primary region of origin to a distant organ via systemic circulation. Thus, modelling the process of cancer metastasis in vitro to understand each step of the metastatic cascade has the potential to improve the translatability of anti-cancer drugs at the preclinical stage. To replicate the realistic behaviour of cancer cells in vitro, it is essential to incorporate all components of the Tumour Microenvironment (TME) - acellular TME comprising the extracellular matrix (ECM), and the cellular TME including stromal cells, and the blood vessels. In this thesis, we advance currently available in vitro models to delineate the role of the acellular and cellular TME to better understand tumour pathophysiology. We employed spheroids of lung (A549, SK-MES-1) and colorectal (HT29) origin as in vitro models mimicking solid tumours in vivo. Using microfluidics, we developed in vitro models of angiogenesis and cancer cell intravasation, two important early steps of the metastatic cascade. By incorporating hybrid matrices (fibrin, collagen, Matrigel), we recreated the complex tumour-stromal-vascular interactions in 3D, resulting in the visualisation of intravasation events. In addition to high spatiotemporal resolution imaging, this platform enables parametric analyses in a controlled environment to decipher the mechanisms in play. To demonstrate the role of the cellular TME (Normal Fibroblasts (NFs), Cancer Associated Fibroblasts (CAFs), Endothelial Cells (ECs)), we developed a novel multilayer in vitro spheroid contractility assay using traction microscopy. The assay quantifies the contractile displacements that tumour spheroids exert on the ECM through real-time tracking of fluorescent beads. We observed a negative correlation between the levels of ECM deformations and cancer invasion patterns. Additionally, we found that stromal cells modulate cancer spheroids’ ability to deform and realign the ECM through the secretion of pro-inflammatory cytokines (IL-6 and IL-8). Besides, we also compared the deformation levels and invasion patterns for non-metastatic (A549) and metastatic (SK-MES-1) lung tumour cells, validating in vivo observations. These novel in vitro assays provide mechanistic insights into the cellular and molecular players in cancer dynamics, overcoming the technical difficulties in observing real-time events in vivo. The outcomes of our in vitro models are in agreement with in vivo observations while improving currently available in vitro assays. Such models can be used to predict and discover new opportunities for enhanced therapeutic intervention during early-stage tumour dissemination.

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