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，对癌症的理解迅速发展，特别是在以下方面的发现： 物理因素的作用，如细胞外基质（ECM）的硬度和细胞的力量，在致癌作用。 这项研究表明，ECM硬度的改变不仅仅是肿瘤的症状， 恶性肿瘤的实际发作和进展。另一个关键发现是细胞牵引应力增加 随着转移潜力的增加，这表明细胞牵引力可能是一种生物标志物， 转移此外，已经发现，细胞群体的（2D）集体行为可以显著地改变。 与分离的癌细胞不同，并且细胞在3D基质中的迁移行为显著不同 2D曲面上的迁移。尽管这促使了3D微环境在癌症中的采用， 机械生物学研究，目前的成像方法，以量化ECM的机械性能和局部细胞 部队仅提供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。这项建议的中心目标是 是为PI最近开发的基于OCT的技术开发定量重建能力 用于细胞牵引力和ECM机械特性的体积成像的组。这些新的量化 能力将与荧光共聚焦显微镜模块集成，以展示一种新的成像方法。 该平台具有前所未有的能力，可用于生物物理细胞-ECM相互作用的延时成像研究， 3D环境。目标1将开发ECM机械的定量3D重建能力 性能和验证它对流变仪和原子力显微镜（AFM）。目标2将展示我们的 基于OCT的3D细胞牵引力成像，使用低密度细胞培养物，整合细胞分辨率 毫米级体积上ECM机械性能的成像。这些小说的示范， 在低密度细胞培养中的综合成像能力之后，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ECM重塑在主要球体边界 与周围的入侵链进行对比Aim 3将在我们的OCT系统中添加共焦荧光成像模块， 我们将证明这个成像平台可以对3D细胞牵引进行延时重建， 在3D中迁移的多细胞群体中ECM机械特性的力和细胞诱导的变化 胶原这将使不同细胞类型的时变牵引力的第一次直接比较 同时在3D胶原蛋白中迁移。用于系统机械生物学研究的新型3D成像平台 可能导致对癌症的潜在生物物理（机械）特征的更深入理解， 在未来设计和测试新的“机械疗法”，目标/调节机械性能的 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.