Development of a biophysical toolkit to monitor and manipulate matrix remodelling in organoid based models of human disease

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Research context: Pathological matrix remodelling is central in many human diseases, and in patients with inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, contributes to intestinal fibrosis and fistula formation. Our recent work combining human intestinal organoids (HIO) with synthetic hydrogels opened the possibility of studying matrix remodelling in reductionist tissue models; however, work is limited by a lack of in vitro tools to visualise, quantify and probe how pathological matrix remodelling contributes to disease. Aims and objectives: The aim of this proposal is to develop a toolkit to study matrix remodelling in HIO models of the gut. We hypothesise that a multi-pronged approach combining hydrogels with dynamic mechanical properties, atomic force microscopy (AFM)-force spectroscopy to map cell-mediated changes in stiffness, multiple particle tracking microrheology (MPT) to monitor local hydrogel degradation in live cultures, and a FRET-based sensor of matrix metalloproteinase (MMP) activity will allow us to comprehensively visualise, quantify, and manipulate matrix remodelling in HIO models of IBD. We will then showcase our toolkit's utility by applying it to study how matrix remodelling impacts cells undergoing epithelial-mesenchymal transition (EMT) and vice versa in an HIO model of the gut.To accomplish this, we will:Objective 1: Establish an HIO-based model of IBD and map mesenchymal matrix remodelling using a combination of AFM, MPT, and a FRET-based sensor of MMP activity. We will encapsulate HIO within PEG-based hydrogels and treat them with cytokines to establish IBD-in-a-dish. We will then map peri-organoid matrix remodelling using AFM stiffness mapping, MPT to monitor local hydrogel degradation, and a FRET-based sensor of MMP activity. This objective will produce stiffness, degradation, and MMP activity maps around HIO as a function of time in our IBD-in-a-dish model. To our knowledge, these will be the first measurements that show how the mesenchyme remodels around HIO in an in vitro model of IBD.Objective 2: Develop hydrogels that recapitulate the dynamic mechanical modulation observed in HIO-based models of IBD.We will incorporate either PEG-acrylate or PEG-norbornene into our existing hydrogels to create a 3D culture platform that controllably softens over time by hydrolysis or stiffens in response secondary radical cross-linking, which we will characterise by rheology. This objective will allow us to encapsulate HIO in hydrogels whose dynamic stiffness matches that of the peri-organoid space in our IBD-in-a-dish model. It will also provide the first evidence as to whether mechanical changes themselves drive might drive IBD.Objective 3: Demonstrate the relevance of the toolkit in an HIO-based model of EMT.We will induce EMT in HIO using cytokines and monitor matrix remodelling using our toolkit. We will then mechanistically determine whether pathological matrix remodelling is a cause or consequence of EMT, and if mechanical stiffness itself contributes to EMT. Potential applications and benefits: By establishing a toolkit to monitor and manipulate matrix remodelling in vitro, we can begin to mechanistically untangle the molecular and signalling pathways that contribute to pathological matrix remodelling, particularly in diseases such as IBD. Current research efforts in IBD are focussed almost exclusively on alleviating inflammation. However, by uncovering a role for matrix remodelling in disease pathogenesis, it may be possible to identify matrix components that could be targeted for future therapies. Moreover, although we focus our efforts here on HIO-based models of IBD, our toolkit will be applicable to other tissue/disease models and has the potential to allow others to unravel underlying disease mechanisms in other conditions marked by pathological matrix remodelling such as lung/liver fibrosis and cancer metastasis.