Biomechanical regulation of cell extrusion and migration during metastasis

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Most adult tumours are comprised of tightly bound epithelial cells organised into continuous sheets. The extrusion of cancer cells from these sheets is an important initial step in metastasis. It was previous believed that tumour cell extrusion is driven by epithelialmesenchymal transition (EMT) whereby cancer cells lose epithelial phenotypes and adhesions to neighbouring cells. However, recent evidence has suggested a more complex behaviour, where different cancer subtypes perform varying degrees of EMT and in certain cases, EMT may not be required at all. Furthermore, tumour cells often disseminate via collective migration where group of cells migrate together with intact adhesions among neighbours. Cell collectives can then enter the bloodstream as circulating tumour cell clusters, which are "the most likely harbingers of metastases" due to their 50-100X greater metastatic potential than equal numbers of individual circulating cells.We currently do not fully understand how cell-cell and cell-ECM adhesions, intrinsic forces (cortical tension) or extrinsic biomechanical forces (extracellular environment) contribute to the a) extrusion of cells from epithelial sheets and b) individual vs. collective migration. Our lack of physiologically-relevant models capable of isolating these variables severely hampers our ability to study their contributions and interactions during tumour cell dissemination. In this work, we aim to use novel microfabricated devices to explore how cell-cell contacts, cell-cell interfacial tension and "squeeze" forces applied by neighbouring tissues drive tumour cell extrusion and detachment. Confinement of doublets into geometric shapes (2D micropatterned substrates) has a dramatic influence on intercellular boundaries, cortical tension and cell motility. Depending on the tensional level, cells displayed undulated, weak junctions and migrate faster (circular shapes) or strong junctions and less motility (triangular shapes), resembling healthy epithelial tissues. This indicates an essential role of cell cortex stiffness and intracellular mechanics to influence the ability to stick together or to migrate faster, and that these can be controlled via geometric confinement.We hypothesize that (i) the lower cortical tension seen in tumour cells increases tumour cell dissemination by weakening cohesion among neighbours and (ii) biomechanical forces and the balance between cell-cell & cell-ECM adhesions control the detachment and dissemination of cancer cells as individual vs. clusters from benign tumours.As a model of carcinoma development, we will use a panel of cells: primary keratinocytes, immortalized keratinocytes, and two sets of primary tumour and metastatic head and neck carcinoma cells from patients (available in the Braga lab; keratinocyte-derived tumours). We will design novel platforms to provide high controllability of mechanical stress. We aim to: Design next generation 3D-microwell arrays and microchannels to evaluate the influence of cell geometry and external mechanical forces on adhesive properties and migration; Define the response of cells at various stages of transformation to variations in intrinsic cortical tension and external mechanical forces; Compare the oncogenic signalling in the various geometric challenges (3D cellular microwell) and cell detachment/motility as cohorts (microchannels).Outcomes: This project will accelerate our understanding of the factors that drive tumour cell extrusion and motility, enabling us to devise novel anti-metastatic strategies to inhibit tumour cell invasion. The project will generate comprehensive knowledge of mechanical force regulation of metastasis, how different states of tumour progression respond to tensional challenges, molecular regulators and screening platforms to interfere with the process.