Addressing the Mechanical Interaction Between Cancer-Associated Fibroblasts and Cancer Cells by Laser Ablation.

Cells within a tumor interact by generating, transmitting, and sensing mechanical forces. Among all the cells of the tumor microenvironment, cancer-associated fibroblasts (CAFs) are a paradigmatic example of mechanical communication. In different steps of tumor progression, CAFs pull and push on cancer cells, regulating cancer cell migration, invasion, compartmentalization, and signaling. There is thus an increasing need to experimentally address mechanical interactions within a tumor. A common technique to measure these interactions is laser ablation. Cutting a tissue region with a high-power laser triggers a sudden tissue displacement whose direction and magnitude reveal the local mechanical stresses. In this chapter, we provide a detailed protocol to perform laser ablations in vitro and ex vivo. First, we describe how to prepare cocultures of primary CAFs and cancer cells and tumor explants. Then, we explain how to perform laser ablations in these two systems and how to analyze the induced tissue displacements using particle image velocimetry (PIV). Overall, we provide a workflow to perform, analyze, and interpret laser ablations to explore tumor mechanical interactions.

Mechanical forces across compartments coordinate cell shape and fate transitions to generate tissue architecture.

Morphogenesis and cell state transitions must be coordinated in time and space to produce a functional tissue. An excellent paradigm to understand the coupling of these processes is mammalian hair follicle development, which is initiated by the formation of an epithelial invagination-termed placode-that coincides with the emergence of a designated hair follicle stem cell population. The mechanisms directing the deformation of the epithelium, cell state transitions and physical compartmentalization of the placode are unknown. Here we identify a key role for coordinated mechanical forces stemming from contractile, proliferative and proteolytic activities across the epithelial and mesenchymal compartments in generating the placode structure. A ring of fibroblast cells gradually wraps around the placode cells to generate centripetal contractile forces, which, in collaboration with polarized epithelial myosin activity, promote elongation and local tissue thickening. These mechanical stresses further enhance compartmentalization of Sox9 expression to promote stem cell positioning. Subsequently, proteolytic remodelling locally softens the basement membrane to facilitate a release of pressure on the placode, enabling localized cell divisions, tissue fluidification and epithelial invagination into the underlying mesenchyme. Together, our experiments and modelling identify dynamic cell shape transformations and tissue-scale mechanical cooperation as key factors for orchestrating organ formation.