An ultra-sensitive micro sensor for biophysical studies of single cells cultured in 3D extracellular matrix

在过去三十年中获得的大量实验证据现已证实，活细胞是可收缩的。它们在3D组织或2D基底上产生力。力和基质刚度对细胞的生长、运动和分化等一系列行为有着深远的影响。最近，人们认识到细胞不仅对来自其3D基质的这些提示作出反应，而且还重塑基质，从而影响随后的提示。癌细胞利用肿瘤内的这种互惠来支持它们的转移之旅。力学在决定这种相互作用中的确切作用仍然难以捉摸。例如，细胞如何将机械信号转化为生化过程，反之亦然。哪些过程与重塑基质有关？探究这些基本问题需要实验平台来评估三维矩阵中的细胞力，并量化决定细胞-矩阵动力学的关键参数。在过去的三十年里，测量二维单细胞力的方法已经取得了进展。目前，还没有方法可以在3D中测量细胞力。百年纪念高中（Champaign）戏剧老师将协助高中生制作一部代表癌症发展的戏剧，其中角色将代表癌症、成纤维细胞和免疫细胞。这些场景将模拟癌症的不同阶段。通过少数民族本科生参与研究和网络传播，研究将与教育相结合。PI有长期的外展活动记录，包括为学生和公众制作多个视频系列。这个项目的目标就是缩小这个差距。该公司将设计、开发和测试一种超灵敏（~10nN/um）的微力传感器，该传感器可以量化3D矩阵中的单细胞力、动力学和细胞外基质重塑。该传感器将用于量化成纤维细胞产生的力和基质重塑，并探索导致转移进展的癌症和基质细胞之间的力依赖串扰。传感器由两个网格组成，一个连接到软弹簧，另一个连接到刚性支架。液滴的液体细胞基质（如胶原蛋白）混合物被分配到网格上。由于毛细作用，它填充网格，并在网格之间形成独立的组织桥接。根据要解决的问题，组织包含一个或离散数量的细胞。细胞与基质形成粘附并产生力，这是由弹簧感知的。该传感器还可以通过拉伸矩阵来获得额外的细胞矩阵刚度。该传感器将用于研究三维胶原中单个成纤维细胞的受力、动力学和基质重塑。最后，我们将探讨三维基质中癌细胞与癌相关成纤维细胞之间的相互作用以及作用力在相互作用中的作用。此次活动的内容是，以高中生为对象制作代表癌症发展的电视剧，剧中人物将分别代表癌症、成纤维细胞、免疫细胞等。这些场景将模拟癌症的不同阶段。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Considerable experimental evidence acquired during the last three decades has now established that living cells are contractile. They generate force on 3D tissues or 2D substrates. Forces and matrix stiffness have a profound influence on a wide range of cell behavior such as growth, motility, and differentiation. More recently, it is appreciated that cells not only respond to these cues from their 3D matrix, but also remodel the matrix and hence influence the subsequent cues. Cancer cells exploit such reciprocity within the tumor to support their metastatic journey. The precise role of mechanics in determining this reciprocity remains elusive. For example, how cells transduce mechanical cues into biochemical processes and vice versa. Which processes are involved in remodeling the matrix? Probing these fundamental questions need experimental platforms that allow to evaluate cell-forces in a 3D matrix and quantify the key parameters that determine the cell-matrix dynamics. Methods to measure single cell forces on 2D have been advanced over the last three decades. Currently, there is no method available to measure cell forces in 3D. Centennial High School (Champaign) drama teacher will assist in developing a drama with high school students representing cancer development where characters will represent cancer, fibroblast and immune cells. The scenes will mimic various stages of cancer. Research will be integrated with education through involvement of undergraduate students from minorities in research, and web dissemination. The PI has a long record of outreach activities, including multiple videos series development for students and public. The goal of this project is to close this gap. It will design, develop and test an ultra-sensitive (~10nN/um) micro force sensor that allows to quantify single cell force, dynamics, and extra-cellular matrix remodeling in 3D matrix. The sensor will be applied to quantify force generation and matrix remodeling by fibroblasts, and to explore force-dependent cross talk between cancer and stromal cells leading to metastatic progression. The sensor consists of two grids, one connected to a soft spring and the other to a rigid support. A droplet of liquid cell-matrix (e.g., collagen) mixture is dispensed on the grids. It fills the grids due to capillarity and forms a free-standing tissue bridging between them. The tissue contains one or a discrete number of cells depending on the questions to be addressed. The cell(s) forms adhesion with the matrix and generates force, which is sensed by the spring. The extra cellular matrix stiffness can also be obtained by the sensor by stretching the matrix. The sensor will be used to study single fibroblasts in 3D collagen for their force, dynamics and matrix remodeling. Finally, the interactions between cancer cells and cancer associated fibroblasts in 3D matrix and the role of forces on the interaction will be explored. The outreach activities will include developing a drama with high school students representing cancer development where characters will represent cancer, fibroblast and immune cells. The scenes will mimic various stages of cancer.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.