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.

我们实验室的总体目标是使用高级的细胞异质性，动力学和遗传性破译。 吞吐量和纵向单细胞分析。具体而言，我们将重点介绍针对CXCL12的趋化性 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。细胞迁移的失败会导致炎症反应有缺陷，修复不良 受伤的组织。同时，癌细胞的快速迁移是相关的转移。虽然很多 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，我们对单个细胞为何反应不同的理解有限。给出 长期迁移测定在跟踪和选择性隔离单个细胞中的局限性，我们 开发了一个高通量单细胞迁移平台，该平台协调机器人液体处理和 自主图像处理，以快速量化数千个细胞的运动性。基于观察到的 在我们的初步研究中，我们假设使用不同的机制来增强细胞异质性 不同细胞的运动性，包括CXCL12依赖信号以及内在的运动驱动因素。我们将孤立 并具有单细胞分子和功能分析的快速移动细胞群，以检验该假设。 我们将抑制个人和多个运动驱动因素的组合，以检查所有人的运动是否运动 可以停止细胞。我们将进一步研究是否可以随着时间的推移维持升高的细胞运动和 精确的关键分子特征，重点关注拷贝数变化和转录的偏斜表达 因素。与瞬时随机性驱动的细胞特征相比，可遗传和稳定的改变 将是有价值的生物标志物和治疗靶标。此外，新兴证据表明 信号转导中的分子动力学确定细胞反应。根据初步数据，我们 期望刺激浓度急剧增加，而不是治疗持续时间是诱导的关键 细胞运动。具有尖端的单细胞跟踪能力，我们将收集时变和定量 数千个细胞的信息，包括细胞速度和持久性以及钥匙的荧光记者 迁移监管机构。动态细胞数据将揭示在快速和慢速移动细胞中的独特时间模式， 传统的一次性测量无法揭示。除了体外研究外，我们还将跟踪 小鼠耳朵皮肤中的细胞运动，带有多光子浸泡显微镜。我们希望没有治疗， 大多数细胞移动缓慢，但少数细胞在体内迅速移动。此外， 抑制体外细胞运动在体内将以相同的方式起作用。提出的多维细胞 迁移研究将改变我们了解细胞迁移和细胞之间异质性的细胞决策的方式。 尽管我们专注于趋化CXCL12，但我们设想我们确定的技术和范式将是 在其他情况下广泛应用于调节异质细胞群体。