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Coordination And Migration of Cells during 3D Vasculogenesis

Final Report Summary - CAMVAS (Coordination And Migration of Cells during 3D Vasculogenesis)

The project CAMVAS aimed at integrated understanding of mechanobiological interactions during the 3D morphogenesis of microvascular networks, such as newly formed capillaries during organogenesis, or spreading of cancer. In its execution, the project has focused mainly on the mechanical interaction of proteins of the extracellular matrix (ECM), and the cellular building blocks of microvasculature networks, i.e. the endothelial cells. The main project’s methodologies have been developed in conjunction with novel experimental bioengineering approaches. One of the challenges has been the extraction of mechanical measurements from 3D microfluidics. Microvascular network have been used, being self-assembled in vitro from endothelial cells embedded in a scaffold of ECM-like material often co-cultured with stromal cells (such as fibroblasts) for increased stability. Experimental access of mechanical quantities, forces and deformations, has been possible dynamically in this 3D evolving biological self-assembly, by integrating high-resolution confocal fluorescence microscopy and existing algorithms on 3D image correlation.

Results provided insights on the 3D process of matrix remodeling of the surrounding scaffold by endothelial cells. We have analyzed the forces and deformations that the self-assembling endothelium engages dynamically. We have imaged living cell cultures during the morphogenesis process and measured significant assembly, unfolding and remodeling, of fundamental proteins for cell anchorage with the ECM, such as fibronectin. Combining fluorescent labeling of fibrous gel scaffolds and live imaging, we have quantified mechanical deformations of the 3D essential scaffolding and its temporal evolution. Huge remodeling of fibrin fiber arrangement, through cell-generated forces causing recruitment, unbinding and re-binding has led to a new theoretical description for these matrices, based on mechanical plasticity (Fig. 1).
We have demonstrated that plasticity, i.e. non-elastic, permanent re-arrangement of the material, is a suitable material model for the early solid mechanics of the ECM caused by forces at the level of cell protruding processes in 3D (filopodia). This has required an integrated computational approach (continuum-discrete) together with the image analyses and mechanical measurements.

The project paves the way for novel techniques for mechanobiological assessment of tissue changes during morphogenesis and cancer, and for the in vitro creation of scalable tissues with vasculature, as in organ on-a-chip and organoids applications. The project has generated international collaborations for the use of novel microscopy techniques at high resolution (such as Brillouin microscopy, optical tweezers, and FRET), which can be preliminary efforts towards the mechanical characterization of stiffness changes in developing tissues models, or disease models, such as cancer and fibrosis. We have quantified the complex mechanical state of cells during the self-assembling of microvasculature (Fig. 2) with unprecedented precision and in real time. This has high potential in the field, as mechanical interactions are starting to be understood, and maintenance of a tissue, highly dependent on spatiotemporal changes of stuffness of the surrounding ECM scaffolding, are paramount in all mechanobiology-focused studies.

The research can also have a potential impact for cancer research. Indeed, the matrix remodeling, and the forces that play in the endothelium of small capillaries, can become a key target in metastatic process. The research has been carried out alongside with state-of-the-art microfluidics technologies at the host laboratories. One possible application that would highly benefit from the mechanical measurements is the extravasation on-a-chip study, as depicted in Fig. 3. We have adapted 3D traction force microscopy to these microfluidics chips in live application, one of this being the in vitro modeling of tumor cells transmigrating the endothelium to reach the ECM, a relevant step in the metastatic process.