Optogenetic manipulation of cell contraction signal network dynamics in tumors

机械力在多细胞组合和组织的发育和功能中起着重要作用。在组织中，单个细胞既可以产生机械力，也可以感知机械力，这些单个机械力之间的相互作用被认为是细胞排列自组织的必要条件。调节力产生或感知的控制机制的改变可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的组织组织异常。由于肿瘤组织的复杂性，包括肿瘤细胞、癌症相关成纤维细胞和免疫细胞等多种相关细胞类型，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细胞收缩动力学，无论是在整个单个细胞水平，还是在亚细胞水平。这种光遗传学工具是基于线粒体高度可调的、可逆的细胞收缩调节因子GEF-H1释放，以控制其胞质水平。我们的研究表明，GEF-H1的细胞质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成分。因此，通过光调节GEF-H1的细胞质浓度，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胞质GEF-H1水平，从而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，持续时间和振幅，无论是在急性扰动实验或在数天的慢性扰动。在这里，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该工具。然后，我们将把这些细胞注射到小鼠耳皮肤中，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。我们将重点关注肿瘤和成纤维细胞作为发力细胞，研究它们对肿瘤、成纤维细胞和免疫细胞的影响。这个提议的关键概念是，我们绕过复杂的内源性控制机制，通过我们新的光遗传学工具通过外部的、依赖于光的控制来施加细胞收缩动力学。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的表型反应读数相结合，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。

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.