High Content Screening of Multicellular Invasion with 3D Traction Force Microscopy

项目概要： 恶性肿瘤通过协调的细胞增殖过程扩散单个细胞和多细胞簇。 细胞分离和运动性。这些机械细胞-细胞和细胞-基质相互作用可以变化 在不同的细胞中是不均匀的，并且也随时间而塑性地变化。然而，细胞产生的力是 通常从蛋白质表达和细胞形态间接推断。此外，经典的迁移测定法 解决单细胞动力学的能力有限，并利用2D单层培养， 与肿瘤微环境的关系。因此，迫切需要精确测量细胞的生长。 仿生3D微环境中的迁移和力学。 我们的长期目标是开发技术，使肿瘤侵袭的生物力学分析离体， 筛选对靶向抑制剂的反应。技术挑战包括：1）跟踪导致 侵入表型，2）解决细胞产生的力，3）利用3D培养模型和4）扩大规模， 更高的通量测定。为了应对这些挑战，本提案的目标是对单个细胞进行分析， 迁移，形态学和力学在多细胞集群嵌入3D微环境。 我们的方法集成了两种互补的精密测量技术。刘：王先生以前 展示了对集体和个人迁移的自动化和全面的单细胞跟踪。Co-I： Franck展示了3D牵引力显微镜（TFM）和3D平均变形度量（MDM）， 细胞生成的基质变形。在IMAT R21的支持下，我们将以 模型系统的基础上诱导上皮间充质转化（EMT）的三维多细胞集群。目的 1将开发定量分析和指标，用于分析表型异质性和可塑性， 向入侵过渡。一旦建立，AIM 2将在96年内扩大这种高内容筛选方法的规模。 井板。我们将首先实现计算效率高的算法，以纠正阶段中的不一致性。 使用图形处理单元（GPU）进行定位。接下来，我们将筛选靶向抑制剂组， 迁移和EMT在不同浓度下测量单细胞迁移和力学是如何 心烦意乱我们设想这项技术将揭示癌细胞如何异常生长的新的基本见解。 作为一个复杂的系统相互作用。此外，这种整合方法可以应用于离体患者样品， 一种早期的、无标记的预后指标，用于预测药物反应和临床前治疗筛选。

PROJECT SUMMARY: Malignant carcinomas disseminate individual cells and multicellular clusters through coordinated processes of cellular detachment and motility. These mechanical cell-cell and cell-matrix interactions can vary heterogeneously across different cells and also vary plastically over time. However, cell-generated forces are often inferred indirectly from protein expression and cell morphology. Moreover, classical migration assays have limited capabilities for resolving single cell dynamics and utilize 2D monolayer culture with questionable relevance for the tumor microenvironment. Thus, there is a critical need for precision measurements of cell migration and mechanics in biomimetic 3D microenvironments. Our long-term goal is to develop technologies that enable biomechanical profiling of tumor invasion ex vivo and screen the response to targeted inhibitors. Technical challenges include 1) tracking rare events that result in invasive phenotypes, 2) resolving cell-generated forces, 3) utilizing 3D culture models and 4) scale-up for higher throughput assays. To address these challenges, the objective of this proposal is to profile single cell migration, morphology and mechanics in multicellular clusters embedded within 3D microenvironments. Our approach integrates two complementary techniques for precision measurement. PI: Wong has previously demonstrated automated and comprehensive single cell tracking of collective and individual migration. Co-I: Franck has demonstrated 3D traction force microscopy (TFM) and 3D mean deformation metrics (MDM) for cell-generated matrix deformations. With the support of this IMAT R21, we will develop this approach in a model system based on inducing the epithelial-mesenchymal transition (EMT) in 3D multicellular clusters. AIM 1 will develop quantitative analyses and metrics for profiling phenotypic heterogeneity and plasticity during the transition to invasion. Once established, AIM 2 will scale up this approach for high-content screening in a 96 well plate. We will first implement computationally efficient algorithms to correct for inconsistencies in stage positioning using graphics processing units (GPUs). Next, we will screen panels of targeted inhibitors against migration and EMT at varying concentrations to measure how single cell migration and mechanics are perturbed. We envision this technology will reveal new fundamental insights into how cancer cells aberrantly interact as a complex system. Moreover, this integrated approach can be applied to patient samples ex vivo as an early, label-free prognostic indicator, to predict drug response and for preclinical therapeutic screens.