Measurement of cellular forces in a 3D in vitro system to unravel vascular disease mechanisms

sponsorship: Mechanical forces play a fundamental role in regulating biological functions in health and disease. While force-driven cellular interactions are well-documented in the context of cancer cell invasion, their role in vascular abnormalities remains largely unexplored, partly due to the lack of suitable in vitro systems. Cerebral cavernous malformation (CCM) is characterized by leaky, tumor-like vessels in the brain, where CCM mutant endothelial cells recruit neighboring wild-type cells to form and aggravate mosaic lesions. However, the mechanisms underlying such aberrant behavior are poorly understood. In this work, we developed an in vitro system for mimicking and imaging dynamic early angiogenic sprouting into biomimetic polyethylene glycol (PEG) matrices. We made this system compatible with a novel inverse method for 3D traction force microscopy (TFM), enabling accurate quantification of cell-generated forces on the ECM. Our novel method constrains solutions by imposing force equilibrium in the hydrogel domain and was validated with realistic in-silico simulations[1]. Moreover, we made it available for the scientific community as part of TFMLAB, an easy-to-use open-source Matlab toolbox[2]. By combining our in vitro system with the analyses provided by TFMLAB, we demonstrated that mechanically hyperactive CCM-2 silenced tip cells guide wild-type cells by exerting and transmitting pulling forces, while also leaving degraded paths in the matrix as cues promoting invasion in a ROCK-dependent manner[3]. This transmission of forces is associated with a reinforcement of β1-integrin adhesions and actin cytoskeleton in the wild-type followers. Wild-type cells are also reprogrammed into stalk cells through activation of matrisome and DNA replication programs, leading to cell proliferation. By integrating biophysical computational methodologies to quantify cellular forces with advanced molecular techniques, we provide new insights in the mechanisms of vascular malformations and open avenues to study the role of cell mechanics in tissue heterogeneity and disease progression. Owing to the generic nature of our integrated methods combined with our novel findings on cell mechanics mechanisms, we believe our study can be a source of inspiration for tissue engineering and regenerative medicine. References 1. Barrasa-Fano J, Shapeti A, de Jong J, Ranga A, Sanz-Herrera JA, Van Oosterwyck H (2021) Advanced in silico validation framework for three-dimensional traction force microscopy and application to an in vitro model of sprouting angiogenesis. Acta Biomater 126:326–338 2. Barrasa-Fano J, Shapeti A, Jorge-Peñas Á, Barzegari M, Sanz-Herrera JA, Van Oosterwyck H (2021) TFMLAB: A MATLAB toolbox for 4D traction force microscopy. SoftwareX 15:100723 3. Shapeti A, Barrasa-Fano J, Rahman A, et al (2023) Force-mediated recruitment and reprogramming of healthy endothelial cells drive vascular lesion growth. bioRxiv 2023.11.27.568780