Van Hear, James Hudson; (2024) Tissue repair in colorectal cancer organoids. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Colorectal cancer (CRC) is the second leading cause of cancer deaths worldwide, with incidence projected to increase in the near future. This is due to a slow accumulation of mutations, such as APC loss and KRAS constitutive activation, resulting in patients only presenting symptoms in the advanced stages of disease. An understanding of early disease stages is essential to address the clinical challenge presented by colorectal cancer. Wound healing pathologies severely impact patient quality of life, with incidence of intestinal chronic wounding conditions, such as IBD, also set to rise in the coming years. Treatment and clinical complications associated with wound healing represent a massive socio-economic burden on health services around the world. The link between wound healing and cancer has long been known, with ample evidence of shared signalling pathways, cell and tissue behaviours. Chronic wounding can promote a pro-tumourigenic environment, and acute wounding has been shown to initiate tumourigenesis in tissues harbouring oncogenic mutations. In recent years, the importance of mechanobiology in repair and disease has been established in a wide range of animal systems. The aim of this thesis is to use mechanobiological approaches to investigate tissue repair in CRC organoids, bringing together the study of mechanobiology, cancer and tissue repair. First, I validate CRC organoids as an effective model system, optimising a set of culture conditions which aim to control inter-experiment variability and reduce the effect of hyperactive signalling from exogenous growth factor ligands in the organoid growth medium. Using multiphoton ablation and confocal imaging to simulate intestinal damage, I then characterise differences in the wound healing response between WT and KRAS-shAPC organoids in terms of cell and tissue behaviours, as well as mechanical features. WT organoids drive wound closure considerably faster than KRAS-shAPC organoids, correlating with the ability to rapidly alter cellular morphology and remodel the wound area through cell intercalations. The healing defect in KRAS-shAPC organoids also correlates with stronger apical F-actin expression, cortical microtubule localisation, and subtly increased tissue tension, all features of more rigid tissues. Next, I reduced tissue tension through pharmacological perturbation of actomyosin contractility with Rho-kinase inhibitor (Y-27632), which partially rescued the healing defect in KRAS-shAPC organoids. Modulating the external mechanical environment through organoid culture in synthetic PEG-based hydrogels also highlighted key differences in mechanosensitivity, with WT organoid formation and growth greatly impaired in stiffer hydrogel conditions, and evidence of increased deformability. KRAS-shAPC organoids, however, were only modestly affected in the stiffer hydrogel conditions. Finally, I optimised TOBis CyTOF for use with hydrogels to interrogate how the mechanical environment influences phosphoproteomic signalling in CRC organoids. By combining these approaches, I aim to understand fundamental biophysical aspects of intestinal wound healing and CRC.

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