Periodic Reporting for period 2 - DeadorAlive (Understanding the mechanical control of cell extrusion in collective assemblies)
Okres sprawozdawczy: 2023-06-01 do 2024-05-31
Epithelia are assemblies of multiple cells whose complex dynamic behavior relies on physical properties including jamming-unjamming mechanisms, active turbulence and active nematic principles. The homeostasis of epithelia is crucial to maintain barrier function and integrity while epithelial cells are constantly challenged by the environment. To face these challenges, epithelia are dynamics and have to deal constantly with cell renewal and extrusion, whose balance is key for epithelia homeostasis. On top of this role in tissue homeostasis, cell extrusion is a major cause of tissue shape changes and tumor progression. Extrusion mechanisms can thus lead to different cell fates namely dead or live cells but the factors selecting different cell fates are unknown. Extruding cells and their neighbors experience various mechanical stresses that lead to cell shape changes and could determine the way cells are extruded and their fate. However, these mechanical stresses and their impact on tissue organization remain to be determined. We thus hypothesize that mechanical constraints coming from the active forces generated by neighboring cells and the passive physical properties of the environment can determine the modes of cell extrusion and the fate of extruded cells. Here the project “Dead or Alive” proposes to tackle the molecular mechanisms and physical principles that determine the manner by which cells are extruded and the collective response of surrounding cells, and to evaluate their contribution in tissue homeostasis, morphogenesis and tumor progression. By combining tools from soft matter physics, cell biology and engineering, our project investigates how active and passive physical signals are overarching components of the behaviors of tissue at different temporal and spatial scales, and may further establish novel paths to understand the mechanobiology of epithelial tissues in normal and pathological conditions.
Cell extrusion necessarily plays a crucial role in epithelial homeostasis, and a better understanding of it will allow us to better assess the factors involve in this equilibrium. This equilibrium is known to be susceptible to perturbations, particularly in the context of cancer. Given how instrumental extrusion is in the dissemination of secondary tumors from the primary tumor, DeadOrAlive for the scientific community could also yield long term medical/social benefits.
We use epithelial cell lines (normal and cancerous) and intestinal organoids as model systems. Integrating active matter, mechanics, engineering and cell signaling approaches across scales from sub-cellular, cellular and multicellular levels, we aim at understanding the physical principles and the molecular mechanisms that control cell extrusion mechanisms as well as evaluating their contribution in physiological tissue development and cancer progression.
At these different levels, we aim to:
i) identify and quantify the role of cytoskeleton and nuclear-associated structures in dedicating the fate of extruded cells, i.e. live or dead;
ii) identify the feedback interactions between extruding cells and their neighbors;
iii) determine the impact of the physical cues of the environment on cell fate, extrusion modes and collective response of the neighbors;
iv) determine the robustness and stochasticity of cell extrusion processes;
v) build physical models to integrate parameter measurements.
Cell extrusion necessarily plays a crucial role in epithelial homeostasis, and a better understanding of it will allow us to better assess the factors involve in this equilibrium. This equilibrium is known to be susceptible to perturbations, particularly in the context of cancer. Given how instrumental extrusion is in the dissemination of secondary tumors from the primary tumor, DeadOrAlive for the scientific community could also yield long term medical/social benefits.
We use epithelial cell lines (normal and cancerous) and intestinal organoids as model systems. Integrating active matter, mechanics, engineering and cell signaling approaches across scales from sub-cellular, cellular and multicellular levels, we aim at understanding the physical principles and the molecular mechanisms that control cell extrusion mechanisms as well as evaluating their contribution in physiological tissue development and cancer progression.
At these different levels, we aim to:
i) identify and quantify the role of cytoskeleton and nuclear-associated structures in dedicating the fate of extruded cells, i.e. live or dead;
ii) identify the feedback interactions between extruding cells and their neighbors;
iii) determine the impact of the physical cues of the environment on cell fate, extrusion modes and collective response of the neighbors;
iv) determine the robustness and stochasticity of cell extrusion processes;
v) build physical models to integrate parameter measurements.
“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.
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.
We have obtained new results for understanding tissue homeostasis, the fate of extruded cells and how they are extruded. We provide a new framework to understand live versus dead cell extrusions. Along this line, we discover a master regulator of the cell competition outcome in the tissue as the force transmission capability of the competing cells. Since cell competition is a fundamental mechanism for maintaining tissue health, this discovery has overarching implications in most vital biological processes including morphogenesis, as well as devastating diseases such as acute inflammation and cancer. Our combined experimental and computational analyses led to a number of unexpected findings that challenge some of the established hallmarks of mechanical cell competition. Through extensive examination of normal, genetically transformed, and cancerous tissues with varying degrees of force transmission capability, we provide unequivocal evidence that none of the well-established frameworks for cell competition is able to determine the cell competition outcome. Instead, our work reveals higher force transmission capability as the universal winning strategy across all examined assays.
We obtained unanticipated results on the role of substrate stiffness in the regulation of epithelial integrity.
Further results will be obtained until the end of the project:
1/ on the regulation of intestinal homeostasis using organoids;
2/ on the rheology and mechanical properties of extruding cells;
3/ On the nuclear mechanics and cell extrusion mechanisms;
4/ on the role of substrate geometry.
We obtained unanticipated results on the role of substrate stiffness in the regulation of epithelial integrity.
Further results will be obtained until the end of the project:
1/ on the regulation of intestinal homeostasis using organoids;
2/ on the rheology and mechanical properties of extruding cells;
3/ On the nuclear mechanics and cell extrusion mechanisms;
4/ on the role of substrate geometry.