Optogenetic manipulation of cell contraction signal network dynamics in tumors

机械力在多细胞组件和组织的发育和功能中起着重要的作用。在组织内，单个细胞既可以产生机械力，也可以感知机械力，这些单独的机械力之间的相互作用被认为是细胞排列的自组织所必需的。调节力量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，并导致肿瘤形成或纤维化等病理生理过程中异常的组织结构。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、肿瘤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用标准方法研究。在这里，我们建议应用我们实验室最近开发的一种新的光遗传学工具来控制细胞收缩动力学，无论是在整个单个细胞的水平上，还是在亚细胞水平上。这一光遗传工具基于高度可调的、可逆的细胞收缩调节因子全环基金-H1从线粒体释放，以控制其胞浆水平。我们的研究表明，胞浆中环境基金-H1的浓度是通过包括正反馈调节和负反馈调节的信号网络来控制亚细胞收缩脉冲的关键成分。因此，通过用光调节细胞内环境基金-H1的浓度，我们能够在亚细胞水平上控制单个细胞的收缩动力学。此外，我们还可以快速和可逆地控制细胞内的环境基金-H1水平，以在整个细胞水平上触发单个收缩脉冲。这使得能够详细控制细胞收缩动力学，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中还是在几天的慢性扰动中。在这里，我们建议应用这一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。我们将首先在低转移黑色素瘤细胞和癌症相关成纤维细胞的体外模型中建立这一工具。然后，我们将把这些细胞注射到小鼠的耳朵皮肤中，以研究亚细胞或整个细胞的收缩动力学如何影响与肿瘤相关的细胞行为。我们将重点关注肿瘤和成纤维细胞作为产生力量的细胞，并研究它们对肿瘤、成纤维细胞和免疫细胞的影响。在这个建议中的关键概念是，我们通过我们的新型光遗传工具，通过外部、光依赖的控制来施加细胞收缩动力学，从而绕过复杂的内源性控制机制。因此，我们希望通过选择性地实施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的表型反应读数结合起来，揭示因果关系。因此，这些见解将澄清我们对肿瘤进展中复杂的基于力的细胞相互作用的看法。

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.