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

项目概要 在过去十年中，人们对癌症的认识迅速发展，特别是关于癌症的发现 物理因素（例如细胞外基质 (ECM) 硬度和细胞力）在致癌过程中的作用。 这项研究表明，ECM 硬度的改变不仅仅是肿瘤的症状，而且现在已知会引发 恶性肿瘤的实际发生和进展。另一个重要发现是细胞牵引应力增加 随着转移潜力的增加，表明细胞牵引力可能是转移可能性的生物标志物 转移。此外，还发现细胞群的（2D）集体行为可以显着 与分离的癌细胞不同，并且 3D 矩阵中的细胞迁移行为显着不同 二维表面上的迁移。尽管这促使在癌症中采用 3D 微环境 机械生物学研究、当前量化 ECM 机械特性和局部细胞的成像方法 力仅提供 2D 成像，或者当它们支持 3D 成像时，它们不提供远程体积测量 以细胞分辨率测量集体机械行为。本提案的中心目标 旨在为 PI 最近开发的基于 OCT 的技术开发定量重建功能 小组用于细胞牵引力和 ECM 机械性能的体积成像。这些新的定量 功能将与荧光共焦显微镜模块集成，以展示新颖的成像 具有前所未有的能力的平台，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 机械定量 3D 重建的能力 特性并根据流变测定法和原子力显微镜 (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物进行基于 OCT 的 3D 细胞牵引力成像，整合细胞分辨率 毫米级体积上的 ECM 机械性能成像。这些小说的示范， 低密度细胞培养物中的集成成像能力随后将在致密肿瘤中进行演示 球体细胞培养，我们将比较主球体边界处的牵引力和 ECM 重塑 与周围的入侵链相比。 Aim 3 将为我们的 OCT 系统添加共焦荧光成像模块， 我们将证明该成像平台可以对 3D 细胞牵引进行延时重建 3D 迁移的多细胞群中 ECM 机械性能的力和细胞诱导的变化 胶原蛋白。这将能够首次直接比较不同细胞类型随时间变化的牵引力 同时在 3D 胶原蛋白中迁移。我们用于系统力学生物学研究的新型 3D 成像平台 可以使人们更深入地了解癌症的潜在生物物理（机械）特征，可以使用 未来设计和测试新的“机械疗法”，其目标/调节机械性能 细胞外基质。

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.