“Dead or Alive” aims at understanding the role of cell extrusion in the regulation of biological tissues by integrating mechanics, engineering and cell signaling approaches across scales from sub-cellular, cellular and multicellular levels.
We first focus on the fate of extruding cells, dead or alive, during extrusion. We show that altering force transmission between cells affects the fate of extruding cells by altering mechanical stress patterns. Combining experiments with cells exhibiting various levels of E-cadherin-based junctions and physical modeling of three-dimensional cell monolayers in collaboration with Doostmohammadi’s team (Denmark), we demonstrate how modified force-transmission across adherens junctions leads to a substantial increase in live cell eliminations and promote a shift in the mode of extrusion from the apical to the basal side. We employed a comprehensive approach combining in vitro cell culture models, patient-derived xenografts and agent-based models to unveil the role of E-cadherin based junctions in the establishment of extrusion programs. We find that the absence of E-cadherin in epithelial cells and their increased cell contractility promote live cell extrusion while promoting cell invasion into the ECM when cultured on thick collagen gels or in 3D gels. We use theoretical modeling to identify the mechanical pathway that promotes various extrusion mechanisms depending on intercellular forces. In silico and experimental results reveal that cells expressing E-cadherin are subjected to higher and more persistent compressive stresses from their neighbors than cells without E-cadherin. Live cell extrusions are associated with non-apoptotic blebbing as a pro-survival mechanism further confirmed by gene expression characterization and protein analysis. Our work demonstrates that different modes of cell extrusion processes are attributed to alterations in the transmission of mechanical forces leading to genetic and protein level changes (Balasubramaniam et al. under revision, Nature Physics). In parallel, we developed various cell lines to study the role of nuclear shape fluctuations on cell extrusion. We observed nuclear shape changes in epithelial cells in correlation with downregulation of cell-cell tension. We are currently studying the interplay between cell-cell adhesion and the emergence of nuclear folds.
During the reporting period, we then study the impact of cell extrusion on neighboring cells. We are currently studying the impact of cell density and jamming-unjamming transition on cell extrusion and delamination. Our efforts led us to investigate a new area dealing with the assembly of skin tissues. In this context, the multilayering of epithelial tissues leads to basal cell delamination while differentiating to generate suprabasal layers. We observed that cell delamination occurs during a jammed state of the epithelia. We are investigating the transition between jammed and unjammed states as well as the mechanics underlying cell delamination. In the unjammed state, we determine and characterize a new mode of collective motion of a quasi-two-dimensional epithelial layer where cells move coherently as a rigid body with little rearrangements with respect to each other. This behavior is reminiscent of the one of active solids. Over time, we observe a decrease of cellular velocities, leading to a jammed state that corresponds to the starting phase of cell delamination and multilayering (Shen et al. Manuscript in preparation).
Finally, we developed optogenetic tools in epithelial cell lines to study the role of cell contractility on extrusions. We observe that high tensional state favors the elimination of cells through RhoA activity. Using such methods, we characterize the spatial distribution of extruding cells within epithelia. We developed theoretical approaches based on phase-field models in collaboration with Doostmohammadi’s team (Denmark) and continuum models with Voituriez’s team (Paris).
Finally, a part of the project focuses on collective cell extrusion. Our efforts led us to investigate the role of extrusion in mixed cultures within the context of cell competition. Cell competition is a tissue surveillance mechanism important in development, infection pathology and tumorigenesis. In these processes, cells with reduced fitness compared to their surrounding are eliminated and outcompeted. We investigate cell competition in mixed cell populations using extensive cellular models (Patient-derived xenografts, various epithelial cell lines) to reveal a previously unknown master mechanism for determining winners and losers of cell competition. Then, we unravel the functional basis of this novel mechanism by combining our experimental approach together with in silico modelling that gives access to the details of three-dimensional mechanics of cell competition. Our work reveals higher force transmission capability as the universal winning strategy across all examined assays. This unanticipated behavior can be understood by the emergence of high mechanical fluctuations at the tissue interface. We establish that these high stress fluctuations can be more effectively accommodated by cell collectives with stronger force transmission capability, preventing them from being eliminated. These findings have far-reaching implications in the way we understand tissue dynamics during morphogenesis, homeostasis and disease (Schonit et al. In revision)
We also developed approaches based on substrate stiffness and topography to study cell extrusions. Our study on substrate stiffness led us to discover a new mechanism which is a loss of epithelial monolayer integrity through spontaneous hole formation when grown on soft substrates. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from tensile on soft to compressive on stiff substrates. We modeled the epithelium as an active nematic liquid crystal. Our results confirmed the active nematic nature of the epithelium. The mechanical constraints resulting from the emergence of misalignments in the organisation of epithelial cells, known as topological defects, in a similar way to the defects observed in liquid crystals, lead to large isotropic stress fluctuations that initiate hole opening events (Sonam et al. Nature Physics 2023). Our results show that substrate stiffness provides feedback on monolayer mechanical state and that topological defects can trigger stochastic mechanical failure by creating regions of high tension. We also developed curved surfaces based on microfabrication processes to study collective cell behavior and extrusion processes (collaboration with National University of Singapore). Substrates are composed of various geometries including tubes (Glentis et al. Science Advances 2022), spheres (Wu et al. to be submitted) and domes.
The results have been published or submitted to various journals. In addition, the participants, including researchers, doctoral students and post-doctoral fellows, have presented their work at a number of national and international conferences and workshops.
