Novel Approaches for Identification of Idiopathic Pulmonary Fibrosis Therapy Targets Using Microscale Collagen Hydrogels

项目摘要/摘要 特发性肺纤维化(IPF)通过间质瘢痕形成损害呼吸，导致(1) 肺僵硬，防止吸入；(2)限制与邻近肺血管的气体交换。病人 依靠肺移植延长他们的寿命中位数超过3年后诊断为唯一的生存 用于IPF治疗的治疗药物延缓了疾病的进展，但无法阻止或逆转纤维化。这个 缺乏药物治疗策略，加上肺移植存活率低，表明 显然需要开发有效的治疗方案。不幸的是，药物发现在很大程度上 受生理上无关的动物模型和试剂的限制，体外信息最少 站台。这项提案的主要目标是展示我们识别重要途径的能力 纤维化进展，并通过探测IPF疾病空间，最终帮助治疗开发。这个 概述的方法通过系统地探测IPF的异源种群来实现生物学发现 通过两种互补途径研究纤维化细胞的功能行为。 第一个提出的方法是在胶原蛋白水凝胶包裹之前对细胞群体进行分层，以使 关于特定表面标记如何影响纤维化行为的研究。使用流分类技术，主要 特发性肺纤维化患者来源的成纤维细胞可分为高表达群体和低表达群体 受体，一种增强这些珍贵细胞的稀有性和价值的方法。然后层叠成纤维细胞 使用试剂效率高的微流控设备将胶原蛋白水凝胶包裹在微型胶原蛋白水凝胶中，减少细胞总数 每种情况所需的体积和最大限度地增加可研究的表面受体的数量 来自给定的细胞来源。低受体表达和高受体表达的影响将被量化，并使用 先前优化的肝纤维化功能的几个指标。第二种方法确定了潜在的治疗方法 根据单个纤维细胞的收缩能力，对其进行功能分类和测序。单个成纤维细胞 被封装到前面描述的微凝胶中并进行培养以允许铺展、粘连和 细胞收缩到周围的基质中。当细胞对附近的胶原纤维施力时，它们 将凝胶压紧成一个较小的球体。由于这些成纤维细胞收缩能力的异质性， 构造的大小变化很大，基于大小的排序机制被用来分隔大部分 收缩能力最弱的细胞。然后使用RNA测序直接将转录图谱与成纤维细胞进行比较 收缩能力，并确定上调的感兴趣的通路。要验证测序命中是否重要 驱动纤维化功能的机制、功能阻断抗体和药物抑制剂被用于 确认纤维化亚群的纤维化功能丧失。

Project Summary/Abstract Idiopathic pulmonary fibrosis (IPF) impairs respiration through scarring of the interstitial space, resulting in (1) a stiffened lung that prevents inhalation and (2) limited gas exchange to adjacent pulmonary vasculature. Patients rely on lung transplants to extend their life beyond the median 3-year survival post-diagnosis as the only therapeutic agents used for IPF treatment slow disease progression are unable to cease or reverse fibrosis. The deficiency of pharmaceutical treatment strategies coupled with poor lung transplant survival rates indicates an obvious need for development of effective treatment options. Unfortunately, drug discovery has largely been restricted by physiologically irrelevant animal models and by reagent exhaustive, minimally informative in vitro platforms. The main goal of this proposal is to demonstrate our ability to identify pathways of importance in fibrotic progression and, by probing the IPF disease space, ultimately aid in therapeutic development. The outlined methodology enables biological discovery by systematically probing heterogenous populations of IPF cells by investigating functional behaviors of fibrotic cells from two complementary approaches. The first proposed methodology stratifies cell populations prior to encapsulation in collagen hydrogels to enable studies on how specific surface markers effect fibrotic behavior. Using flow sorting techniques, primary fibroblasts derived from IPF patients are separated into high and low expressing populations of specific surface receptors, a method that enhances the rarity and value of these precious cells. Stratified fibroblasts are then encapsulated in miniature collagen hydrogels using reagent-efficient microfluidic devices, reducing total cell volumes required for each condition and maximizing the number of surface receptors that can be investigated from a given cell source. Effects of low and high receptor expression will then be quantified and compared using several metrics of fibrotic function previously optimized. The second approach identifies potential therapeutic targets by functionally sorting and sequencing single fibrotic cells based on their contractility. Single fibroblasts are encapsulated into the previously described microgels and cultured to allow for spreading, adhering, and contracting of the cells into the surrounding matrix. As the cells exert force on the nearby collagen fibers, they compact the gel into a smaller sphere. Due to heterogeneity in the contraction ability of these fibroblasts, constructs are greatly varied in size and size-based sorting mechanisms are employed to segregate the most and least contractile cells. RNA sequencing is then used to directly compare transcriptomic profiles to fibroblast contractility and to identify upregulated pathways of interest. To validate sequencing hits as important mechanisms driving fibrotic functions, function-blocking antibodies and pharmaceutical inhibitors are used to confirm loss in fibrotic function for fibrogenic subpopulations.