Optogenetic manipulation of cell contraction signal network dynamics in tumors

肿瘤细胞收缩信号网络动力学的光遗传学操作

• 批准号: 426018514
• 负责人: Professorin Dr. Perihan Nalbant, Ph.D.
• 金额: --
• 依托单位: Zentrum für Medizinische Biotechnologie (ZMB)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/426018514?language=en
• 关键词: Optogenetic; manipulation; cell; contraction; signal

Mechanical forces play important roles in the development and function of multicellular assemblies and tissues. Within tissues, individual cells can both produce and sense mechanical forces, and the interplay between these individual mechanical agents is thought to be essential for the self-organization of cellular arrangements. Alterations in control mechanisms that regulate force production or sensing can lead to failure in this interplay and result in aberrant tissue organization in pathophysiological processes like tumor formation or fibrosis.Due to the complexity of tumor tissues, that contain several relevant cell types including tumor cells, cancer-associated fibroblasts and immune cells, the interplay between individual cells is very difficult to study via standard methods. Here we propose to apply a novel optogenetic tool that we recently developed in our labs to control cell contraction dynamics either at the level of whole individual cells, or at the subcellular level. This optogenetic tool is based on highly tunable, reversible release of the cell contraction regulator GEF-H1 from mitochondria to control its cytosolic levels. Our studies revealed that the cytosolic concentration of GEF-H1 is a critical component that controls subcellular contraction pulses via a signal network that includes both positive and negative feedback regulation. Thus, by tuning the cytosolic concentration of GEF-H1 with light, we were able to control the contraction dynamics at the subcellular level in individual cells. In addition, we can also control cytosolic GEF-H1 levels rapidly and reversibly to trigger individual contraction pulses at the level of entire cells. This enables detailed control of cell contraction dynamics, including pulse count, frequency, duration and amplitude, both in acute perturbation experiments or in chronic perturbations over several days.Here, we propose to apply this optogenetic tool to study the role of cell contraction dynamics in tumor progression. We will first establish this tool in models of low-metastatic melanoma cells and cancer-associated fibroblasts in vitro. We will then inject these cells into the mouse ear skin to study how subcellular or whole cell contraction dynamics affect tumor-related cell behaviors. We will focus on tumor and fibroblast cells as force producing cells and study their effect on tumor, fibroblasts and immune cells.The key concept in this proposal is that we bypass complex endogenous control mechanisms by imposing cell contraction dynamics through external, light-dependent control via our novel optogenetic tool. We thus expect to uncover cause and effect relationships by selective enforcement of cell contraction signals from either tumor or fibroblast cells and combining these perturbation input signals with phenotypic response readouts in the tumor associated cell types. These insights will therefore clarify our view on the complex force-based cellular interplay in tumor progression.

机械力在多细胞集合体和组织的发育和功能中起着重要作用。在组织内，单个细胞可以产生和感受机械力，这些单个机械因子之间的相互作用被认为是细胞排列自组织的关键。调节力的产生或感知的控制机制的改变可能导致这种相互作用的失败，并导致病理生理过程中的异常组织结构，如肿瘤形成或纤维化。由于肿瘤组织的复杂性，包含几种相关的细胞类型，包括肿瘤细胞，癌症相关的成纤维细胞和免疫细胞，单个细胞之间的相互作用很难通过标准方法进行研究。在这里，我们建议应用我们最近在实验室开发的一种新的光遗传学工具来控制整个单个细胞水平或亚细胞水平的细胞收缩动力学。这种光遗传学工具基于细胞收缩调节剂GEF-H1从线粒体的高度可调、可逆释放，以控制其胞质水平。我们的研究表明，GEF-H1的胞浆浓度是通过包括正反馈和负反馈调节的信号网络控制亚细胞收缩脉冲的关键组分。因此，通过用光调节GEF-H1的胞质浓度，我们能够在单个细胞的亚细胞水平上控制收缩动力学。此外，我们还可以快速可逆地控制胞质GEF-H1水平，以在整个细胞水平上触发单个收缩脉冲。这使得细胞收缩动力学的详细控制，包括脉冲计数，频率，持续时间和幅度，无论是在急性扰动实验或慢性扰动超过days.Here，我们建议应用这种光遗传学工具来研究细胞收缩动力学在肿瘤进展中的作用。我们将首先在体外低转移性黑色素瘤细胞和癌症相关成纤维细胞模型中建立这种工具。然后，我们将这些细胞注射到小鼠耳皮肤中，以研究亚细胞或全细胞收缩动力学如何影响肿瘤相关细胞的行为。我们将专注于肿瘤和成纤维细胞作为力产生细胞，并研究它们对肿瘤，成纤维细胞和免疫细胞的影响。该提案的关键概念是我们通过外部施加细胞收缩动力学绕过复杂的内源性控制机制，通过我们的新型光遗传学工具，光依赖性控制。因此，我们期望通过选择性地执行来自肿瘤或成纤维细胞的细胞收缩信号并将这些扰动输入信号与肿瘤相关细胞类型中的表型响应读数相结合来揭示因果关系。因此，这些见解将澄清我们对肿瘤进展中基于复杂力的细胞相互作用的看法。