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

使用微型胶原水凝胶识别特发性肺纤维化治疗靶点的新方法

• 批准号: 9909024
• 负责人: Katherine Anne Cummins
• 金额: $ 3.94万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-02 至 2023-03-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909024
• 关键词: Animal Model; Automobile Driving; Behavior; Biological; Biological Assay; Blocking Antibodies; Cell Count; Cell Separation; Cell Size; Cell Volumes; Cell physiology; Cell surface; Cells; Characteristics; Cicatrix; Clinical Research; Collagen; Collagen Fiber; Contracts; Coupled; Data; Deposition; Development; Devices; Diagnosis; Disease; Disease Progression; Disease model; Drug Targeting; ECM receptor; Encapsulated; Expression Profiling; Extracellular Matrix; FDA approved; Fibroblasts; Fibrosis; Fluorescence-Activated Cell Sorting; Gases; Gel; Genetic; Goals; HMMR gene; Heterogeneity; Hydrogels; Impairment; In Vitro; Inhalation; Integrins; Investigation; Life; Link; Lung; Lung Capacity; Lung Transplantation; Methodology; Methods; Microfluidic Microchips; Microfluidics; Microspheres; Modeling; Molecular; Myofibroblast; Pathogenicity; Pathway interactions; Patient-Focused Outcomes; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Physiological; Population; Population Heterogeneity; Process; Production; Proteins; Pulmonary Fibrosis; Reagent; Respiration; Role; Signal Pathway; Signal Transduction; Sorting - Cell Movement; Source; Stratification; Surface; Survival Rate; System; Techniques; Therapeutic Agents; Tissues; Treatment Efficacy; base; cell behavior; collagenase; drug discovery; effective therapy; exhaustion; experimental study; idiopathic pulmonary fibrosis; improved; inhibitor/antagonist; interest; interstitial; miniaturize; nanolitre; novel; novel strategies; outcome forecast; patient stratification; physical separation; prevent; receptor; receptor expression; receptor for advanced glycation endproducts; response; single-cell RNA sequencing; targeted treatment; therapeutic development; therapeutic target; therapy outcome; transcriptome; transcriptome sequencing; transcriptomics; treatment strategy

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.

项目概要/摘要 特发性肺纤维化 (IPF) 通过间质空间疤痕损害呼吸，导致 (1) 肺部僵硬，阻止吸入；(2) 限制与邻近肺血管系统的气体交换。患者 依靠肺移植来延长他们的生命，使其超过诊断后的中位 3 年生存率 用于 IPF 治疗的治疗药物可以减缓疾病进展，但无法阻止或逆转纤维化。这 药物治疗策略的缺乏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已经被 受到生理上不相关的动物模型和体外试剂详尽、信息极少的限制 平台。该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，并通过探索 IPF 疾病空间，最终有助于治疗开发。这 概述的方法通过系统地探测 IPF 的异质群体来实现生物学发现 通过两种互补的方法研究纤维化细胞的功能行为。 第一个提出的方法是在封装在胶原水凝胶中之前对细胞群进行分层，以便能够 研究特定表面标记如何影响纤维化行为。使用流式分选技术，初级 来自 IPF 患者的成纤维细胞被分为特定表面的高表达群体和低表达群体 受体，一种提高这些珍贵细胞的稀有性和价值的方法。然后分层成纤维细胞 使用试剂高效的微流体装置封装在微型胶原水凝胶中，减少细胞总数 每种条件所需的体积以及最大化可研究的表面受体的数量 来自给定的细胞来源。然后使用低和高受体表达的影响进行量化和比较 先前优化的纤维化功能的几个指标。第二种方法确定潜在的治疗方法 通过根据单个纤维化细胞的收缩性对其进行功能性分类和测序来确定目标。单个成纤维细胞 被封装到先前描述的微凝胶中并培养以允许扩散、粘附和 细胞收缩到周围的基质中。当细胞对附近的胶原纤维施加力时，它们 将凝胶压实成更小的球体。由于这些成纤维细胞收缩能力的异质性， 构建体的大小差异很大，并且采用基于大小的排序机制来分离最 和最小收缩细胞。然后使用 RNA 测序直接比较转录组图谱与成纤维细胞 收缩性并确定感兴趣的上调途径。验证测序命中的重要性 驱动纤维化功能的机制、功能阻断抗体和药物抑制剂用于 确认纤维化亚群的纤维化功能丧失。