Understanding cell migration through microscale in vitro models

描述（由申请人提供）：细胞迁移是一个复杂的多步骤过程，是多种疾病发病过程的核心。虽然中性粒细胞是先天免疫反应的重要组成部分，但它们的不适当募集是慢性炎症性疾病（包括哮喘、关节炎和炎症性肠病）的核心；也有助于其他疾病的发病机制，如心血管疾病、肿瘤进展和阿尔茨海默病。趋化性，细胞在化学梯度内的运动，是中性粒细胞募集的基本过程。尽管它很重要，但目前的工具限制了理解调节细胞迁移的分子机制的进展。特别是，目前的方法主要局限于二维环境，需要相对大量的抽血，并使用可疑的生理相关性刺激。我们的目标是使用并进一步开发克服这些挑战的微尺度方法和微尺度体外模型，增强我们在涉及先天免疫系统疾病的背景下识别调节细胞极化和定向运动的信号通路的能力-特别是在严重先天性中性粒细胞减少症（SCN）的背景下，Hax1信号在中性粒细胞趋化中的作用。目前，趋化性研究非常耗费时间和人力，限制了可以探索的实验条件的数量。我们的方法从根本上改变了研究人员进行趋化性研究的方式。用同样的时间/精力可以检验更多的实验条件。该应用程序的一个关键优势在于使用新颖（但简单）的微流体装置，产生明确和稳定的化学梯度，不需要层流来形成或维持梯度，可以适应高通量筛选。在目标1中，我们将开发改进的3D分析，为细胞迁移提供更生理相关的背景，并开发允许使用小体积手指棒样本的方法。在目标2中，我们将开发基于微流体的伤口试验，通过细胞源梯度来分析中性粒细胞募集，以确定在多细胞环境中调节细胞极化和定向细胞迁移的因素。在目标3中，我们建议研究Hax1/HS1/G113信号如何在更复杂的3D和细胞源梯度中调节中性粒细胞的运动和募集，以模拟体内条件。使用这些系统，我们将剖析Hax1如何调节梯度传感和Hax1缺陷中性粒细胞样PLB987细胞的定向细胞迁移。未来的应用将包括使用2D和3D/细胞源微流控系统分析中性粒细胞减少症患者的中性粒细胞运动，以了解疾病发病机制并确定新的治疗靶点。

DESCRIPTION (provided by applicant): Cell migration is a complicated multistep process that is central to the pathogenesis of diverse disease processes. Although neutrophils are an essential part of the innate immune response, their inappropriate recruitment is central to chronic inflammatory disorders including asthma, arthritis and inflammatory bowel disease; and also contributes to the pathogenesis of other diseases such as cardiovascular disease, tumor progression and Alzheimer's disease. Chemotaxis, the movement of cells within a chemical gradient, is the fundamental process underlying neutrophil recruitment. Despite its importance, current tools limit progress towards understanding the molecular mechanisms that regulate cell migration. In particular, current methods are largely limited to 2D environments, require relatively large blood draws and use stimuli of questionable physiological relevance. Our goal is to use and further develop microscale methods and microscale in vitro models that overcome these challenges, enhancing our ability to identify signaling pathways that regulate cell polarization and directed motility in the context of disorders involving the innate immune system - specifically the role of Hax1 signaling in neutrophil chemotaxis in the context of severe congential neutropenia (SCN). Currently, chemotaxis studies are very time and labor intensive limiting the number of experimental conditions that can be explored. Our approach fundamentally changes the way a researcher can approach chemotaxis studies. Many more experimental conditions can be examined with the same time/effort. A key strength of this application lies in the use of novel (but simple) microfluidic devices that generate defined and stable chemical gradients that do not require laminar flow for gradient formation or maintenance and can be adapted to high throughput screening. In Aim 1, we will develop improved 3D assays that provide a more physiologically relevant context for cell migration and develop methods that allow the use of small volume finger stick samples. In Aim 2, we will develop microfluidic-based wound assays to analyze neutrophil recruitment through cell sourced gradients to determine factors that regulate cell polarization and directed cell migration in multicelluar environments In Aim 3, we propose to study how Hax1/HS1/G113 signaling modulates neutrophil motility and recruitment in more complex 3D and cell sourced gradients to mimic in vivo conditions. Using these systems, we will dissect how Hax1 regulates gradient sensing and directed cell migration with Hax1-deficient neutrophil-like PLB987 cells. Future applications will include analysis of neutrophil motility from patients presenting with neutropenia using 2D and 3D/cell sourced microfluidic systems to understand disease pathogenesis and identify novel therapeutic targets. PUBLIC HEALTH RELEVANCE: We anticipate that the research proposed in this grant will not only help to elucidate the basic mechanisms that regulate cell motility, but will also provide insight into the development of novel therapeutic approaches to treat inflammation in general. These studies will therefore have implications for treating many different disorders where cell motility is central to disease pathogenesis, including arthritis, cardiovascular disease and cancer.