Deciphering Cellular Heterogeneity and Inheritability in Migration

解读迁移中的细胞异质性和遗传性

• 批准号: 10710996
• 负责人: Yu-Chih Chen
• 金额: $ 38.31万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10710996
• 关键词: Biological Markers; CXCR4 gene; Cells; Characteristics; Chemotaxis; Cues; Data; Ear; Embryonic Development; Failure; Goals; Heterogeneity; In Vitro; Individual; Inflammation; Inflammatory Response; Inherited; Liquid substance; Measurement; Microfluidics; Migration Assay; Modeling; Molecular; Movement; Mus; Nature; Neoplasm Metastasis; Organelles; Pattern; Physiological Processes; Population; Process; Reporter; Retrieval; Robotics; Signal Transduction; Skin; Speed; Stimulus; Stromal Cell-Derived Factor 1; Technology; Testing; Time; Tissues; angiogenesis; cancer cell; cell dimension; cell motility; image processing; in vivo; injury and repair; intravital microscopy; migration; molecular dynamics; multi-photon; response; single cell analysis; therapeutic target; transcription factor; treatment duration; wound healing

The overarching goal of our lab is to decipher cellular heterogeneity, dynamics, and inheritability using high- throughput and longitudinal single-cell analysis. Specifically, we will focus on chemotaxis toward CXCL12 as a model. Cell migration is an essential process in embryogenesis, angiogenesis, wound healing, inflammation, and cancer metastasis. Failure of cell migration can lead to defective inflammatory responses and poor repair of injured tissues. At the same time, fast migration of cancer cells is associated metastasis. Although many environmental cues, physiological processes, transcription factors, and organelle features have been discovered to regulate cell migration, we have limited understanding about why individual cells respond differently. Given the limitations of long-standing migration assays in tracking and selectively isolating individual cells, we developed a high-throughput single-cell migration platform that coordinates robotic liquid handling and autonomous image processing for rapidly quantifying motility of thousands of cells. Based on the observed cellular heterogeneity in our preliminary studies, we hypothesize that distinct mechanisms are used to enhance motility in different cells, including CXCL12 dependent signals as well as intrinsic motility drivers. We will isolate and profile fast-moving cell populations with single-cell molecular and functional analysis to test this hypothesis. We will inhibit individual and combination of multiple motility drivers to examine whether the movement of all cells can be stopped. We will further examine whether elevated cellular motility can be maintained over time and pinpoint key molecular features, focusing on copy number alterations and skewed expression of transcription factors. Compared to cellular characteristics driven by transient randomness, inheritable and stable alterations will be valuable biomarkers and therapeutic targets. Moreover, emerging evidence suggests the importance of molecular dynamics in signal transduction to determine cellular responses. Based on the preliminary data, we expect that sharp increase of the stimulus concentration rather than the duration of treatment is the key to induce cell movement. With the cutting-edge single-cell tracking capability, we will collect time-varying and quantitative information of thousands of cells, including cellular speed and persistence and fluorescent reporters of key migratory regulators. The dynamic cell data will reveal unique temporal patterns in fast- and slow- moving cells, which cannot be revealed with conventional one-time measurements. In addition to in vitro studies, we will track cell movement in the mouse ear skin with multiphoton intravital microscopy. We expect that without treatment, most cells move slowly yet a small number of cells move rapidly in vivo. Furthermore, the treatments that suppress in vitro cell movement will function in the same way in vivo. The proposed multi-dimensional cell migration studies will change how we understand cellular decision in migration and heterogeneity between cells. While we focus on chemotaxis toward CXCL12, we envision the technology and paradigm we establish will be widely applied to regulate heterogeneous cell populations in other contexts.

我们实验室的首要目标是利用高分子材料来破译细胞异质性、动态和遗传性。 通量和纵向单细胞分析。具体来说，我们将重点关注趋化性对CXCL12作为一种 模型细胞迁移是胚胎发生、血管生成、伤口愈合、炎症， 和癌症转移。细胞迁移的失败可导致炎症反应缺陷和细胞修复不良。 受伤的组织同时，癌细胞的快速迁移与转移有关。尽管许多 已经发现了环境线索、生理过程、转录因子和细胞器特征 为了调节细胞迁移，我们对为什么单个细胞的反应不同的理解有限。给定 长期的迁移试验在跟踪和选择性分离单个细胞方面的局限性， 开发了一种高通量单细胞迁移平台，可协调机器人液体处理， 自动图像处理，用于快速量化数千个细胞的运动性。基于观察到的 细胞异质性在我们的初步研究中，我们假设不同的机制被用来增强 在不同的细胞中的运动，包括CXCL12依赖性信号以及内在运动驱动。我们将隔离 并通过单细胞分子和功能分析来分析快速移动的细胞群，以验证这一假设。 我们将抑制单个和多个运动驱动程序的组合，以检查是否所有的运动 细胞可以被阻止。我们将进一步研究是否可以随着时间的推移保持细胞运动性的提高， 精确定位关键分子特征，重点关注拷贝数改变和转录的偏斜表达 因素与瞬时随机性驱动的细胞特征相比， 将是有价值的生物标志物和治疗靶点。此外，新出现的证据表明， 信号转导中的分子动力学，以确定细胞反应。根据初步数据，我们 预计刺激浓度的急剧增加而不是治疗时间的增加是诱导的关键 细胞运动凭借尖端的单细胞跟踪能力，我们将收集时变和定量的 数千个细胞的信息，包括细胞的速度和持久性以及关键的荧光报告基因。 迁移调节器。动态细胞数据将揭示快速和缓慢移动细胞的独特时间模式， 这不能用常规的一次性测量来揭示。除了体外研究，我们还将跟踪 用多光子活体显微镜观察小鼠耳皮肤中的细胞运动。我们认为如果不进行治疗， 大多数细胞在体内移动缓慢，而少数细胞在体内移动迅速。此外， 抑制体外细胞运动在体内也将以同样的方式发挥作用。所提出的多维单元格 迁移研究将改变我们对细胞迁移和细胞间异质性的理解。 当我们专注于对CXCL12的趋化性时，我们设想我们建立的技术和范式将是 广泛应用于调节其他环境中的异质细胞群体。