Optogenetic manipulation of cell contraction signal network dynamics in tumors

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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.