Guillaume Duclos

Emergence of collective modes and tri-dimensional structures from epithelial confinement

Many in vivo processes, including morphogenesis or tumour maturation, involve small populations of cells within a spatially restricted region. However, the basic mechanisms underlying the dynamics of confined cell assemblies remain largely to be deciphered and would greatly benefit from well-controlled in vitro experiments. Here we show that confluent epithelial cells cultured on finite population-sized domains, exhibit collective low-frequency radial displacement modes as well as stochastic global rotation reversals. A simple mathematical model, in which cells are described as persistent random walkers that adapt their motion to that of their neighbours, captures the essential characteristics of these breathing oscillations. As these epithelia mature, a tri-dimensional peripheral cell cord develops at the domain edge by differential extrusion, as a result of the additional degrees of freedom of the border cells. These results demonstrate that epithelial confinement alone can induce morphogenesis-like processes including spontaneous collective pulsations and transition from 2D to 3D.

Minimization of thermodynamic costs in cancer cell invasion

Metastasis, the truly lethal aspect of cancer, occurs when metastatic cancer cells in a tumor break through the basement membrane and penetrate the extracellular matrix. We show that MDA-MB-231 metastatic breast cancer cells cooperatively invade a 3D collagen matrix while following a glucose gradient. The invasion front of the cells is a dynamic one, with different cells assuming the lead on a time scale of 70 h. The front cell leadership is dynamic presumably because of metabolic costs associated with a long-range strain field that precedes the invading cell front, which we have imaged using confocal imaging and marker beads imbedded in the collagen matrix. We suggest this could be a quantitative assay for an invasive phenotype tracking a glucose gradient and show that the invading cells act in a cooperative manner by exchanging leaders in the invading front.

Mechanical cell competition kills cells via induction of lethal p53 levels

Cell competition is a quality control mechanism that eliminates unfit cells. How cells compete is poorly understood, but it is generally accepted that molecular exchange between cells signals elimination of unfit cells. Here we report an orthogonal mechanism of cell competition, whereby cells compete through mechanical insults. We show that MDCK cells silenced for the polarity gene scribble (scribKD) are hypersensitive to compaction, that interaction with wild-type cells causes their compaction and that crowding is sufficient for scribKD cell elimination. Importantly, we show that elevation of the tumour suppressor p53 is necessary and sufficient for crowding hypersensitivity. Compaction, via activation of Rho-associated kinase (ROCK) and the stress kinase p38, leads to further p53 elevation, causing cell death. Thus, in addition to molecules, cells use mechanical means to compete. Given the involvement of p53, compaction hypersensitivity may be widespread among damaged cells and offers an additional route to eliminate unfit cells.

Architecture and migration of an epithelium on a cylindrical wire

Significance Cell sheets often organize in tubular structures, for example, in the kidney. Also, cells from epithelial tumors are known to wrap around vessels or muscle fibers as they migrate collectively. By plating cells on thin glass wires, we mimic these physiological conditions in vitro and show that high curvature favors cell detachment at the front edge. This switch from collective to individual migration may reproduce features often observed in cancer invasion. High curvature also induces a circumferential organization of the actin cytoskeleton reminiscent of in vivo embryonic morphogenesis situations where tissues develop on a cylindrical template. Finally, monolayer migration is halted at submicron radii, and the tissue reconfigures into hollow cysts at its leading tip. In a wide range of epithelial tissues such as kidney tubules or breast acini, cells organize into bidimensional monolayers experiencing an out-of-plane curvature. Cancer cells can also migrate collectively from epithelial tumors by wrapping around vessels or muscle fibers. However, in vitro experiments dealing with epithelia are mostly performed on flat substrates, neglecting this out-of-plane component. In this paper, we study the development and migration of epithelial tissues on glass wires of well-defined radii varying from less than 1 µm up to 85 µm. To uncouple the effect of out-of-plane curvature from the lateral confinement experienced by the cells in these geometries, we compare our results to experiments performed on narrow adhesive tracks. Because of lateral confinement, the velocity of collective migration increases for radii smaller than typically 20 µm. The monolayer dynamics is then controlled by front-edge protrusions. Conversely, high curvature is identified as the inducer of frequent cell detachments at the front edge, a phenotype reminiscent of the Epithelial−Mesenchymal Transition. High curvature also induces a circumferential alignment of the actin cytoskeleton, stabilized by multiple focal adhesions. This organization of the cytoskeleton is reminiscent of in vivo situations such as the development of the trachea of the Drosophila embryo. Finally, submicron radii halt the monolayer, which then reconfigures into hollow cysts.

RalB regulates contractility-driven cancer dissemination upon TGFβ stimulation via the RhoGEF GEF-H1

RalA and RalB proteins are key mediators of oncogenic Ras signaling in human oncogenesis. Herein we investigated the mechanistic contribution of Ral proteins to invasion of lung cancer A549 cells after induction of epithelial-mesenchymal transition (EMT) with TGFβ. We show that TGFβ-induced EMT promotes dissemination of A549 cells in a 2/3D assay, independently of proteolysis, by activating the Rho/ROCK pathway which generates actomyosin-dependent contractility forces that actively remodel the extracellular matrix, as assessed by Traction Force microscopy. RalB, but not RalA, is required for matrix deformation and cell dissemination acting via the RhoGEF GEF-H1, which associates with the Exocyst complex, a major Ral effector. Indeed, uncoupling of the Exocyst subunit Sec5 from GEF-H1 impairs RhoA activation, generation of traction forces and cell dissemination. These results provide a novel molecular mechanism underlying the control of cell invasion by RalB via a cross-talk with the Rho pathway.