Volumetric time-lapse imaging of biophysical cell-extracellular matrix interactions for systems mechanobiology research

项目摘要 在过去的十年里，人们对癌症的认识发展迅速，特别是随着对 物理因素，如细胞外基质(ECM)硬度和细胞力，在癌症发生中的作用。 这项研究表明，细胞外基质僵硬改变不仅是肿瘤的症状，而且现在已被认为是引发 恶性肿瘤的实际发生和发展。另一个关键发现是细胞牵引力增加 随着转移潜能的增加，提示细胞牵引力可能是一种生物标记物 转移。此外，已经发现细胞群体的(2D)集体行为可以显著地 与分离的癌细胞不同，细胞在3D基质中的迁移行为明显不同 二维曲面上的偏移。尽管这推动了癌症中3D微环境的采用 机制生物学研究，目前用于量化ECM机械性能和局部细胞的成像方法 Forms仅提供2D成像，或者当它们支持3D成像时，它们不提供远程体积成像 用细胞分辨率测量集体力学行为。这项提议的中心目标是 是为PI最近开发的基于OCT的技术开发定量重建能力 细胞牵引力和ECM机械性能的体积成像小组。这些新的数量 功能将与荧光共聚焦显微镜模块集成，以演示一种新的成像 具有前所未有的能力的平台，用于生物物理细胞-细胞外基质相互作用的时间推移成像研究 3D环境。目标1将开发ECM机械的定量3D重建能力 并通过流变仪和原子力显微镜(AFM)对其进行验证。目标2将展示我们的 使用低密度细胞培养、集成细胞分辨率的3D细胞牵引力的OCT成像 毫米级体积的ECM机械性能成像。这些小说的展示， 在低密度细胞培养中的集成成像能力将在致密肿瘤中进行演示 球体细胞培养，我们将比较牵引力和主要球体边界的细胞外基质重塑 与周围的入侵链相比较。AIM 3将在我们的OCT系统中增加一个共焦荧光成像模块， 我们将演示该成像平台可以执行3D细胞牵引的延时重建 力和细胞诱导的细胞外基质力学性质在三维迁移的多细胞群体中的变化 胶原蛋白。这将使不同电池类型的时变牵引力的首次直接比较成为可能 同时在3D胶原蛋白中迁移。我们用于系统机械生物学研究的新型3D成像平台 可以导致对癌症潜在的生物物理(机械)特征的更深层次的理解，这些特征可以用于 在未来的设计和测试新的‘机械疗法’，以目标/调节的机械性能 ECM。

Project Summary The understanding of cancer has evolved rapidly over the last decade, particularly with discoveries regarding the role of physical factors, such as extracellular matrix (ECM) stiffness and cellular forces, in carcinogenesis. This research has shown that altered ECM stiffness is not just a symptom of tumors, but is now known to trigger the actual onset of and progression of malignancy. Another key finding is that cellular traction stresses increase with increasing metastatic potential, suggesting that cell traction forces could be a biomarker for the likelihood of metastasis. Additionally, it has been found that (2D) collective behavior of cell populations can be significantly different from that of isolated cancer cells, and that cell migratory behavior in 3D matrices is significantly different migration on 2D surfaces. Although this has motivated the adoption of 3D microenvironments in cancer mechanobiology research, current imaging methods to quantify ECM mechanical properties and local cellular forces only provide 2D imaging, or when they do support 3D imaging, they do not provide long-range volumetric measurements of collective mechanical behavior with cellular resolution. The central objective of this proposal is to develop quantitative reconstruction capabilities for OCT-based techniques recently developed by the PI's group for volumetric imaging of cell traction forces and ECM mechanical properties. These new quantitative capabilities will be integrated with a fluorescence confocal microscopy module, to demonstrate a novel imaging platform with unprecedented capabilities for time-lapse imaging studies of biophysical cell-ECM interactions in 3D environments. Aim 1 will develop the capabilities for quantitative 3D reconstruction of ECM mechanical properties and validate it against rheometry and atomic force microscopy (AFM). Aim 2 will demonstrate our OCT-based imaging of 3D cell traction forces using low-density cell cultures, integrating cellular resolution imaging of ECM mechanical properties over millimeter-scale volumes. The demonstration of these novel, integrated imaging capabilities in low-density cell cultures will be followed by a demonstration in dense tumor spheroid cell cultures, where we will compare traction forces and ECM remodeling at the main spheroid boundary versus surrounding invasion strands. Aim 3 will add a confocal fluorescence imaging module to our OCT system, and we will demonstrate that this imaging platform can perform time-lapse reconstruction of 3D cell traction forces and cell-induced changes in ECM mechanical properties in a multiple-cell population migrating in 3D collagen. This will enable the first direct comparison of the time-varying traction forces of different cell types simultaneously migrating in 3D collagen. Our novel 3D imaging platform for systems mechanobiology research could lead to a deeper understanding of potential biophysical (mechanical) hallmarks of cancer, that can be used in the future to design and test new `mechano-therapies' that target/modulate the mechanical properties of the ECM.