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

研究背景：病理性基质重塑是许多人类疾病的核心，并且在克罗恩病和溃疡性结肠炎等炎症性肠道疾病（IBD）患者中，会导致肠道纤维化和瘘管形成。我们最近的工作将人类肠道类器官（HIO）与合成水凝胶相结合，开辟了在还原组织模型中研究基质重塑的可能性;然而，由于缺乏体外工具来可视化，量化和探索病理基质重塑如何有助于疾病，工作受到限制。目的和目标：本提案的目的是开发一个工具包，研究肠道HIO模型中的基质重塑。我们假设，一种多管齐下的方法，将水凝胶与动态机械性能、原子力显微镜（AFM）-力光谱学（用于绘制细胞介导的刚度变化）、多粒子跟踪微流变学（MPT）（用于监测活培养物中的局部水凝胶降解）和基于FRET的基质金属蛋白酶（MMP）活性传感器相结合，将使我们能够全面地可视化、定量、并在IBD的HIO模型中操纵基质重塑。然后，我们将展示我们的工具包的效用，通过应用它来研究如何基质重塑影响细胞经历上皮间充质转化（EMT），反之亦然，在HIO模型的gut.To完成这一点，我们将：目标1：建立一个基于HIO的IBD模型和地图间充质基质重塑使用AFM，MPT，和FRET为基础的MMP活性传感器的组合。我们将HIO封装在PEG基水凝胶中，并用细胞因子处理它们以建立IBD-在-a-dish中。然后，我们将使用AFM刚度映射，MPT监测局部水凝胶降解和基于FRET的MMP活性传感器来映射周围类器官基质重塑。该目标将在我们的IBD培养皿模型中产生HIO周围的硬度、降解和MMP活性图作为时间的函数。据我们所知，这些将是显示IBD体外模型中HIO周围间充质如何重塑的第一个测量结果。目的2：开发水凝胶，重现在基于HIO的IBD模型中观察到的动态机械调节。将三烯添加到我们现有的水凝胶中，以创建一个3D培养平台，该平台可通过水解随时间可控地软化，或在反应中二次硬化。自由基交联，我们将通过流变学来解释。这一目标将使我们能够将HIO封装在水凝胶中，其动态刚度与我们的IBD盘中模型中的周围器官空间相匹配。它还将提供第一个证据，以是否机械变化本身驱动可能驱动IBD.Objective3：演示工具包的相关性在HIO为基础的EMT.We模型将诱导EMT HIO使用细胞因子和监测矩阵重塑使用我们的工具包。然后，我们将从机制上确定病理性基质重塑是否是EMT的原因或后果，以及机械刚度本身是否有助于EMT。潜在的应用和益处：通过建立一个工具包来监测和操纵体外基质重塑，我们可以开始机械地解开有助于病理基质重塑的分子和信号通路，特别是在IBD等疾病中。目前IBD的研究工作几乎完全集中在减轻炎症上。然而，通过揭示基质重塑在疾病发病机制中的作用，有可能确定未来治疗的靶向基质成分。此外，尽管我们在此将努力集中在基于HIO的IBD模型上，但我们的工具包将适用于其他组织/疾病模型，并有可能允许其他人在以病理基质重塑为标志的其他条件下解开潜在的疾病机制，如肺/肝纤维化和癌症转移。

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.