Sorting and characterization of mechanically heterogeneous cell populations based on cellular contractility

项目摘要 细胞收缩性在发育和疾病中起着关键作用。最近的证据表明，即使 在来自相同来源的细胞群中，细胞收缩性存在巨大的异质性。 时空组织细胞收缩性的失调常常导致发育缺陷。在 像癌症这样的侵袭性疾病通常是高度收缩的，存在弱收缩的 亚群正受到越来越多的关注。粘附细胞以其感知和识别细胞的能力而闻名。 动态地适应当地的微环境。因此，机械表型的异质性可能是一个 这是遗传异质性或细胞可塑性和机械适应的结果。机械医学或机械- 基于靶向特定物理细胞和组织相互作用的疗法，包括异常细胞 收缩性，如癌症，纤维化和心血管疾病，以及衰老，正在出现， 有很大的潜力。虽然已知机械不均匀性和塑性有助于抵抗 针对特定分子途径的治疗，目前尚不清楚机械表型的变化是否 预测疾病的结果，或者是否由于疾病进展而发生机械适应。这个项目 提出将粘附细胞表型分选成具有不同收缩表型的亚群， 这些分类的亚群来检验初始收缩表型和异质性 确定疾病结果对备择假设，机械适应当地 微环境和表型转换有助于疾病进展， 力学不均匀性癌症转移将用作假设检验的主要生物学模型。 在目标1中，将优化基于细胞收缩性的细胞分选的工程方法。荧光- 活化细胞分选（FACS）将与工程化的高通量细胞收缩性筛选相结合 平台、自动化显微镜以及用于细胞分离的细胞的光活化和荧光标记。在Aim中 2，将分选的收缩亚群用于在体外和体内检验主要的生物学假设 与另一种假设的对比模拟癌症环境条件的工程系统 进展将被设计为表征细胞的迁移、增殖、存活和代谢。 亚群，以及它们的机械适应。这些亚群的转移潜力和 将在小鼠肿瘤中检查它们在转移级联沿着的各个阶段的机械适应性 模型该建议的创新方面是通过细胞收缩性进行分类的概念，其目标是 揭示了初始机械表型在癌症等疾病进展和发育中的作用。 本项目将使用新的工程细胞分选方法来剖析机械的各自作用， 异质性和适应性的疾病进展，从而奠定了基础，为今后的工作，以确定 关键的分子途径，以精确地为目标的机械医学的发展。

Project Summary Cellular contractility plays a critical role in both development and disease. Recent evidence suggests that even within a cell population derived from the same source, vast heterogeneity exists in terms of cellular contractility. Dysregulation of spatiotemporally organized cellular contractility often results in developmental defects. In invasive diseases like cancer which are often highly contractile, the existence of a weakly contractile subpopulation is receiving increasing attention. Adherent cells are known for their ability to sense and dynamically adapt to their local microenvironment. Hence, the heterogeneity in mechanical phenotype may be a result of genetic heterogeneity or cellular plasticity and mechanical adaptation. Mechanomedicines or mechano- based therapies that target specific physical cellular and tissue interactions, including abnormal cellular contractility, in diseases like cancer, fibrosis, and cardiovascular disease, as well as aging, are emerging and hold great potential. While mechanical heterogeneity and plasticity are known to contribute to resistance to therapies that target a specific molecular pathway, it is not clear whether a change in mechanical phenotype predicts disease outcome or if mechanical adaption happens as a result of disease progression. This project proposes to phenotypically sort adherent cells into subpopulations with distinct contractile phenotypes and use these sorted subpopulations to test the hypothesis that the initial contractile phenotype and heterogeneity determine the disease outcome against the alternative hypothesis that mechanical adaptation to the local microenvironment and phenotypical switching contribute to disease progression regardless of the initial mechanical heterogeneity. Cancer metastasis will be used as the main biological model for hypothesis testing. In Aim 1, the engineering approach for cell sorting based on cellular contractility will be optimized. Fluorescence- activated cell sorting (FACS) will be coupled with an engineered high throughput cell contractility screening platform, automated microscopy, and photoactivation and fluorescent labeling of cells for cell separation. In Aim 2, the sorted contractile subpopulations will be used to test the main biological hypothesis in vitro and in vivo against the alternative hypothesis. Engineered systems mimicking the environmental conditions in cancer progression will be designed to characterize the migration, proliferation, survival, and metabolism of the subpopulations, as well as their mechanical adaptation. The metastatic potential of these subpopulations and their mechanical adaptations at various stages along the metastatic cascade will be examined in a mouse tumor model. The innovative aspect of this proposal is the concept to sort by cellular contractility with the goal of uncovering the role of initial mechanical phenotype in the progression of diseases like cancer and in development. This project will use the novel engineered cell sorting approach to dissect the respective roles of mechanical heterogeneity and adaptability in disease progression, thus laying the foundation for future work to identify the key molecular pathways to precisely target for the development of mechanomedicine.