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

研究背景：病理基质重塑是许多人类疾病的核心，在炎症性肠病（IBD）患者中，如克罗恩病和溃疡性结肠炎，有助于肠纤维化和瘘管形成。我们最近的工作将人类肠道类器官（HIO）与合成水凝胶结合，为研究还原论组织模型中的基质重塑开辟了可能性；然而，由于缺乏体外工具来可视化，量化和探测病理基质重塑如何导致疾病，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来研究肠道HIO模型中的基质重塑。我们假设，多管齐下的方法结合水凝胶与动态力学特性，原子力显微镜(AFM)-力光谱来绘制细胞介导的刚度变化，多粒子跟踪微流变学（MPT）来监测活培养物中的局部水凝胶降解，以及基于fret的基质金属蛋白酶（MMP）活性传感器，将使我们能够全面可视化，量化和操纵IBD HIO模型中的基质重塑。然后，我们将展示我们的工具包的效用，应用它来研究基质重塑如何影响细胞进行上皮-间质转化（EMT），反之亦然，在肠HIO模型中。为了实现这一目标，我们将：目标1：建立一个基于hio的IBD模型，并利用AFM， MPT和基于fret的MMP活性传感器的组合来绘制间质基质重塑图。我们将把HIO包封在peg基水凝胶中，并用细胞因子处理，建立IBD-in-a-dish。然后，我们将使用AFM刚度映射，MPT来监测局部水凝胶降解，以及基于fret的MMP活性传感器来绘制类器官周围基质重塑。在我们的IBD-in-a-dish模型中，这个目标将产生围绕HIO的刚度、退化和MMP活性图，作为时间的函数。据我们所知，这将是首次在体外IBD模型中显示间质如何在HIO周围重塑的测量。目的2：开发水凝胶，再现hid模型中观察到的动态力学调节。我们将在现有的水凝胶中加入peg -丙烯酸酯或peg -降冰片烯，以创建一个3D培养平台，该平台可以随着时间的推移通过水解或响应二次自由基交联而可控地软化，我们将通过流变学来表征。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-in-a-dish模型中的类器官周围空间相匹配。它也将为机械变化本身是否可能驱动IBD提供第一个证据。目标3：展示工具包在基于hio的EMT模型中的相关性。我们将使用细胞因子在HIO中诱导EMT，并使用我们的工具包监测基质重塑。然后，我们将从机械上确定病理性基质重塑是EMT的原因还是结果，以及机械刚度本身是否有助于EMT。潜在的应用和好处：通过建立一个工具箱来监测和操纵体外基质重塑，我们可以开始从机械上理清导致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，可能有可能确定未来治疗的靶向基质成分。此外，尽管我们的工作重点是基于hio的IBD模型，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揭示以病理基质重塑为标志的其他疾病（如肺/肝纤维化和癌症转移）的潜在疾病机制。

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.