Correlating mechanical and genetic data at high-throughput and single cell levels to investigate metastasis

在高通量和单细胞水平上关联机械和遗传数据以研究转移

• 批准号: 10421775
• 负责人: Katherine M Young
• 金额: $ 2.06万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10421775
• 关键词: Affect; Atomic Force Microscopy; Automobile Driving; Bioinformatics; Biological Assay; CRISPR/Cas technology; Cancer cell line; Cell Separation; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Collaborations; Cytoskeletal Proteins; Data; Disease; Gene Combinations; Gene Expression; Genes; Genetic; Genotype; Goals; Guide RNA; Human Genome; Invaded; Knock-out; Knowledge; Libraries; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Measurement; Mechanics; Mesenchymal; Microfluidics; Molecular; Molecular Genetics; Morphology; Neoplasm Metastasis; Phenotype; Play; Population; Positioning Attribute; Primary Neoplasm; Process; Proteins; Research; Research Personnel; Review Literature; Role; Signal Transduction; Signaling Protein; Site; Small Interfering RNA; Sorting - Cell Movement; Structural Protein; Systems Biology; Technology; Time; Tissues; Validation; Viscosity; base; biophysical properties; cancer cell; cell motility; epithelial to mesenchymal transition; experimental study; gene discovery; genome-wide; genome-wide analysis; interest; knockout gene; mechanical properties; metastatic process; migration; neoplastic cell; new therapeutic target; novel; novel diagnostics; screening; transcription factor; tumor; whole genome

Metastasis is the cause of 90% of cancer-related deaths, a statistic that has changed little over the past 50 years. During that time, cancer researchers have recognized that changes in cells’ mechanical phenotypes dictate their ability to generate force, invade through tissues and migrate throughout the body. Several studies have implicated that as a cell’s metastatic potential increases, cell stiffness decreases. More generally, the relationship between disease state and cell mechanics suggests that changes in cell stiffness are correlated with phenotypes of invasiveness, migration, epithelial-to-mesenchymal transition (EMT), and metastasis and are controlled through various cell-signaling networks. Yet while certain genes that affect cell mechanics have been studied, a genome-wide study of genes and gene networks that modulate cell biophysical properties has not been attempted. The use of genome-wide CRISPR knockout (GeCKO) pooled screens has allowed researchers to start exploring the connection between a cell’s genotype and various phenotypes. To understand gene networks that control cell mechanics and their role in metastatic potential, we will need to uncover the genetic molecular mechanisms that allow cells to change their mechanical properties to successfully form a metastatic tumor. The long-term goal of this research is to understand the molecular and mechanical mechanisms driving metastasis that will lead to the discovery of new diagnostics and therapeutic targets to find and stop key processes of metastatic cells. To reach this goal, we will leverage a novel microfluidics approach for cell sorting based upon biophysical properties for the high-throughput discovery of genes linked to cell mechanics and metastasis. We will use this approach to determine how cellular mechanics are regulated within the context of networks of cytoskeletal and structural proteins in addition to various transcription factors and signaling proteins associated with increased metastatic potential. I will investigate this intersection with the following aims: 1) Identify genes related to mechanical changes in cancer cells through GeCKO high- throughput mechanical screen and 2) Validate phenotypic and mechanotypic importance of genes of interest. We hypothesize that there is a link between cell softening and mesenchymal, migratory phenotypes that is controlled by the expression of a network of genes of interest. The proposed studies will represent the first attempt to evaluate the entire genome for its role in directing cell mechanics to understand the connection between cancer cell genotype, phenotype and mechanotype both across the whole genome and on the single cell level. Being able to collect information about the combination of gene expression data and cell stiffness measurements in a high throughput fashion and an in-depth exploration of genes related to mechanics and metastatic potential will lead to great advancement of our understanding of the metastatic cascade with potential application in many other fields where cell mechanics play a role in disease state.

转移是90%的癌症相关死亡的原因，这一统计数据在过去几乎没有变化 50年在此期间，癌症研究人员已经认识到，细胞机械表型的变化 决定了它们产生力量、侵入组织并在全身迁移的能力。几项研究 暗示随着细胞转移潜能的增加，细胞硬度降低。更一般地 疾病状态和细胞力学之间的关系表明，细胞刚度的变化是相关的， 具有侵袭性、迁移、上皮-间质转化（EMT）和转移的表型， 通过各种小区信令网络进行控制。然而，尽管某些影响细胞力学的基因 已经研究过，对调节细胞生物物理特性的基因和基因网络的全基因组研究已经 没有尝试过。全基因组CRISPR敲除（GeCKO）合并筛选的使用允许 研究人员开始探索细胞的基因型和各种表型之间的联系。到 了解控制细胞力学的基因网络及其在转移潜能中的作用，我们将需要 揭示遗传分子机制，使细胞改变其机械性能， 成功形成转移性肿瘤。 这项研究的长期目标是了解分子和机械机制驱动 转移，这将导致发现新的诊断和治疗目标，以找到和阻止关键 转移细胞的过程。为了实现这一目标，我们将利用一种新的微流体方法， 基于生物物理特性的分选，用于高通量发现与细胞力学相关的基因 和转移。我们将使用这种方法来确定细胞力学是如何调节内 除了各种转录因子外，细胞骨架和结构蛋白网络的背景， 与转移潜能增加相关的信号蛋白。我会调查这个交叉路口 以下目的：1）通过GeCKO高通量筛选癌细胞机械变化相关基因， 通量机械筛选和2）确定感兴趣基因的表型和机械型重要性。 我们假设细胞软化和间质迁移表型之间存在联系 这是由感兴趣的基因网络的表达控制的。拟议的研究将代表 首次尝试评估整个基因组在指导细胞力学中的作用，以了解 癌细胞基因型、表型和机械型之间的关系， 细胞水平。能够收集有关基因表达数据和细胞硬度组合的信息 以高通量的方式进行测量，并深入探索与力学和生物力学相关的基因， 转移潜力将导致我们对转移级联的理解有很大的进步， 在细胞力学在疾病状态中起作用的许多其他领域中的潜在应用。

项目成果

Scaling microfluidic throughput with flow-balanced manifolds to simply control devices with multiple inlets and outlets.

使用流量平衡歧管扩展微流体吞吐量，以简单地控制具有多个入口和出口的设备。

• DOI: 10.1063/5.0080510
• 发表时间: 2022
• 期刊: Biomicrofluidics
• 影响因子: 3.2
• 作者: Young,KatherineM;Shankles,PeterG;Chen,Theresa;Ahkee,Kelly;Bules,Sydney;Sulchek,Todd
• 通讯作者: Sulchek,Todd