Optimisation and validation of 3D models of progressive human lung fibrosis

特发性肺纤维化（IPF）是一种影响老年人的疾病，它涉及在形成肺气囊的细胞和在全身运输含氧血液的潜在血管之间建立僵硬的疤痕组织。这种疤痕组织阻止氧气进入血液（以及血液中的二氧化碳），并破坏肺部的气囊，导致呼吸逐渐受损，最终死亡。IPF无法治愈，现有的治疗方法只能减缓疾病的进展。在IPF中，负责产生疤痕组织的细胞，成纤维细胞，被发现在称为成纤维细胞病灶的小聚集体中，这被认为是疾病的活跃区域。先前的研究还发现了一种分子，转化生长因子β (TGFB)，它驱动成纤维细胞产生疤痕组织。动物，主要是小鼠，已被广泛用于研究IPF。用一种叫做博来霉素的药物治疗的老鼠在肺部形成了疤痕组织，就像在IPF中看到的那样。然而，重要的是，这种疤痕组织的形成在小鼠中是可逆的，而人类疾病则是永久性的，并逐渐恶化。用一种更好地反映人类IPF的不同模型代替这些小鼠是这个项目的目标。用动物研究疾病机制的另一种方法是在烧瓶中培养人类细胞，并对它们进行各种治疗，以模拟它们在患病组织中的行为。然而，与人体内的三维环境相比，烧瓶中的细胞生长在单一的二维层中。南安普顿的研究人员开发了一种3D成纤维细胞培养模型，该模型允许从IPF肺组织中分离的成纤维细胞更自然地生长。这些成纤维细胞的三维聚集体类似于用TGFB处理后硬化疤痕组织中的成纤维细胞灶。然而，尽管TGFB可能对疤痕组织的形成做出了重要贡献，但我之前的研究已经确定它不是唯一的责任。例如，致密堆积的纤维母细胞病灶缺氧也有助于瘢痕组织的形成，其他介质的存在也是如此。此外，我的研究发现了成纤维细胞病灶中的其他细胞类型，这表明目前仅使用成纤维细胞的模型过于简单，无法准确反映实际疾病。该项目的目的是开发3D纤维化模型，以更好地反映在IPF肺组织中发现的成纤维细胞焦点。我将用模拟缺氧对细胞影响的化学物质处理3D细胞培养物，并将在IPF肺液中识别的其他分子添加到3D培养物中，以更好地代表IPF中成纤维细胞灶的环境。我将使用一种叫做RNA测序的技术将3D培养模型与来自IPF患者的成纤维细胞灶进行比较。这可以量化控制细胞行为的遗传信息，从而根据这些基因特征的变化来识别细胞行为的变化。我们已经有了来自成纤维细胞病灶的RNA测序数据，所以我们可以用它来比较不同3D培养条件下的基因特征与实际疾病中的基因特征的匹配程度。我们还将与药物发现弹射器（MDC）合作，使用一种单独的技术，该技术允许对这些基因特征进行快速量化，以验证这些数据。在我们开发了这个模型之后，我们将通过添加其他细胞来进一步改进它，以反映成纤维细胞灶中存在的多种细胞类型。这种多细胞模型也将通过RNA测序进行分析，并与成纤维细胞灶进行比较。我们还将与MDC合作，确定细胞的遗传和代谢特征如何在3D模型和成纤维细胞灶上发生空间变化。如果成功，这种3D细胞培养模型将为肺纤维化研究提供一种替代动物模型的方法，并为评估新的治疗方法提供一个新的测试平台。

Idiopathic pulmonary fibrosis (IPF) is a disease affecting older adults which involves the build up of stiff scar tissue between cells that form the air sacs of the lung and the underlying blood vessels which transport oxygenated blood around the body. This scar tissue prevents the transfer of oxygen into the blood (and CO2 from the blood), and destroys the air sacs of the lung, leading to progressively impaired breathing and eventual death. IPF has no cure, and existing treatments only slow progression of the disease. In IPF, the cells responsible for producing scar tissue, fibroblasts, are found in small aggregates called fibroblastic foci which are thought to be the active areas of disease. Studies have also previously identified a molecule, transforming growth factor beta (TGFB) which drives fibroblasts to produce scar tissue.Animals, principally mice, have been extensively used to study IPF. Mice treated with a drug called bleomycin develop a build-up of scar tissue in their lungs like that seen in IPF. Importantly, however, this scar tissue build-up is reversible in mice, in contrast to human disease, where it is permanent and progressively worsens. Replacing these mice with a different model which better reflects IPF in humans is the goal of this project.One alternative to using animals to study disease mechanisms is growing human cells in flasks and subjecting them to various treatments to mimic how they behave within diseased tissue. However, cells in flasks grow in a single, 2D layer, in contrast to the 3D environment in the human body. Researchers in Southampton have developed a 3D fibroblast culture model which allows fibroblasts isolated from IPF lung tissue to grow more naturally. These 3D aggregates of fibroblasts resemble fibroblastic foci in the stiffened scar tissue they produce when treated with TGFB. However, while TGFB likely makes an important contribution to scar tissue formation, my previous research has identified that it is not solely responsible. For example, lack of oxygen in the densely packed fibroblastic foci also contributes to scar tissue formation, as does the presence of other mediators. In addition, my research has identified other cell types in fibroblastic foci, suggesting that the current model which just uses fibroblasts is too simplistic to accurately reflect the actual disease. The aim of this project is to develop the 3D fibrosis model to better reflect a fibroblastic focus found in IPF lung tissue. I will treat 3D cell cultures with chemicals which mimic the effects of lack of oxygen on the cells, and will add other molecules identified in IPF lung fluid to the 3D cultures to better represent the environment of fibroblast foci in IPF. I will compare the 3D culture model to fibroblastic foci from people with IPF using a technique called RNA sequencing. This quantifies the genetic messages that control how cells behave, allowing changes in cell behaviour to be identified based on the change in these gene signatures. We already have RNA sequencing data from fibroblastic foci, so we can use that to compare how well the gene signatures from the different 3D culture conditions match those in actual disease. We will also use a separate technology which allows fast quantification of these gene signatures to validate these data, in collaboration with the Medicines Discovery Catapult (MDC).After we have developed this model, we will seek to improve it further by adding other cells into it, reflecting the multiple cell types present in fibroblastic foci. This multicellular model will also be analysed by RNA sequencing and compared to fibroblastic foci. We will also collaborate with MDC to identify how the cells' genetic and metabolic signatures change spatially across the 3D model and fibroblast foci. If successful, this 3D cell culture model will give an alternative to animal models for the study of lung fibrosis and provide a novel testbed for evaluation of new treatments.