Local light-activation of the Src oncoprotein in an epithelial monolayer promotes collective extrusion

International audienceTransformed isolated cells are usually extruded from normal epithelia and subsequently eliminated. However, multicellular tumors outcompete healthy cells, highlighting the importance of collective effects. Here, we investigate this situation in vitro by controlling in space and time the activity of the Src oncoprotein within a normal Madin-Darby Canine Kidney (MDCK) epithelial cell monolayer. Using an optogenetics approach with cells expressing a synthetic light-sensitive version of Src (optoSrc), we reversibly trigger the oncogenic activity by exposing monolayers to well-defined light patterns. We show that small populations of activated optoSrc cells embedded in the non-transformed monolayer collectively extrude as a tridimensional aggregate and remain alive, while the surrounding normal cells migrate towards the exposed area. This phenomenon requires an interface between normal and transformed cells and is partially reversible. Traction forces show that Src-activated cells either actively extrude or are pushed out by the surrounding cells in a non-autonomous way

Compétition entre populations de cellules normales et transformées

During the development of a tumour in a tissue, the cancer cells are surrounded by healthy cells. The interactions between these two cell types, transformed and normal, play an important role in the tumour stability, but remain to this day poorly understood. The aim of this thesis was to establish in vitro assays to study the interactionsbetween populations of normal and transformed cells. We benefited from a light-sensitive cell line, constructed by Olivier Destaing (IAB, Grenoble). When they areexposed to blue light, these cells overactivate the protein Src, which is known to be overexpressed in many cancers. Otherwise, they keep a normal phenotype. Using these cells, called “OptoSrc”, in combination with an optical setup, we are able to create mosaic tissues in which the pattern of mutated cells is determined by the blue illumination pattern. Our system has several advantages: a selective control in time and space of the group of transformed cells, and a gradual and reversible activation of the oncoprotein. We have shown that when we illuminate a circular islet of cells from a monolayer of OptoSrc cells, the activated cells were collectively extruded, resulting in a cohesive three-dimensional aggregate on top of the monolayer. We can control the size and appearance time of this spheroid by tuning, respectively, the area and frequency ofillumination. Besides, this collective extrusion is reversible when the blue light stimulation is stopped. Finally, we have shown that the formation of this three- dimensional aggregate coincides with the loss of E-cadherin at the membrane, as well as the apparition of vimentin, for the illuminated OptoSrc cells. Our results suggest thata group of cells overexpressing the protein Src, in a monolayer of normal cells, undergoes a partial epithelial-tomesenchymal transition.Lors du développement d’une tumeur au sein d’un tissu, les cellules cancéreuses se retrouvent entourées par les cellules saines. Les interactions entre ces deux types cellulaires, transformé et normal, jouent un rôle important dans le devenir de la tumeur, mais restent `a ce jour mal comprises. L’objectif de cette thèse a été de mettre en place des systèmes in vitro qui permettent d’étudier les interactions entre une population de cellules normales et une population de cellules transformées. Nous avons tiré profit d’une lignée de cellules épithéliales sensibles à la lumière, élaborée par Olivier Destaing(IAB, Grenoble). Lorsqu’elles sont exposées à la lumière bleue, ces cellules suractivent la protéine Src, connue pour être surexprimé dans de nombreux cancers. Sinon, elles gardent un phénotype normal. L’utilisation de ces cellules, appelées “OptoSrc”, combinée `a un dispositif optique, permet de créer des tissus mosaïques dans lesquels le motif des cellules mutées est déterminé par le motif d’illumination bleue. Notre système présente plusieurs avantages : le contrôle dans le temps et dans l’espace du motif de cellules transformées, mais aussi l’activation graduelle et réversible de l’oncoprotéine. Nous avons montré qu’en illuminant dans le bleu un îlot circulaire de cellules au sein d’une monocouche OptoSrc, les cellules activées s’extrudent collectivement, donnant naissance `a un agrégat tri-dimensionnel cohésif surplombant la monocouche. Nous pouvons contrôler la taille et le temps d’apparition de ce sphéroïde en ajustant respectivement l’aire éclairée et la fréquence d’illumination. De plus, ce phénomène d’extrusion collective est réversible lorsque le stimulus de lumière bleue s’arrête. Finalement, nous avons montré que la formation de cet agrégats s’accompagne d’une diminution des E-cadhérines à la membrane, et de l’apparition de la vimentine, pour les cellules éclairées. Nos résultats suggèrent qu’un groupe de cellules surexprimant la protéine Src, au sein d’une monocouche de cellules normales, subit une transition epithéliale-mesenchymateuse partielle

Competition between normal and transformed cell populations.

Lors du développement d’une tumeur au sein d’un tissu, les cellules cancéreuses se retrouvent entourées par les cellules saines. Les interactions entre ces deux types cellulaires, transformé et normal, jouent un rôle important dans le devenir de la tumeur, mais restent à ce jour mal comprises. L’objectif de cette thèse a été de mettre en place des systèmes in vitro qui permettent d’étudier les interactions entre une population de cellules normales et une population de cellules transformées.Nous avons tiré profit d’une lignée de cellules épithéliales sensibles à la lumière, élaborée par Olivier Destaing (IAB, Grenoble). Lorsqu’elles sont exposées à la lumière bleue, ces cellules suractivent la protéine Src, connue pour être surexprimée dans de nombreux cancers. Sinon, elles gardent un phénotype normal. L’utilisation de ces cellules, appelées « OptoSrc », combinée à un dispositif optique, permet de créer des tissus mosaïques dans lesquels le motif des cellules mutées est déterminé par le motif d’illumination bleue. Notre système présente plusieurs avantages : le contrôle dans le temps et dans l’espace du motif de cellules transformées, mais aussi l’activation graduelle et réversible de l’oncoprotéine.Nous avons montré qu’en illuminant dans le bleu un îlot circulaire de cellules au sein d’une monocouche OptoSrc, les cellules activées s’extrudent collectivement, donnant naissance à un agrégat tri-dimensionnel cohésif surplombant la monocouche. Nous pouvons contrôler la taille et le temps d’apparition de ce sphéroïde en ajustant respectivement l’aire éclairée et la fréquence d’illumination. De plus, ce phénomène d’extrusion collective est réversible lorsque le stimulus de lumière bleue s’arrête. Finalement, nous avons montré que la formation de cet agrégat s’accompagne d’une diminution des E-cadhérines à la membrane, et de l’apparition de la vimentine, pour les cellules éclairées. Nos résultats suggèrent qu'un groupe de cellules surexprimant la protéine Src, au sein d’une monocouche de cellules normales, subit une transition epithéliale- mesenchymateuse partielle.During the development of a tumour in a tissue, the cancer cells are surrounded by healthy cells. The interactions between these two cell types, transformed and normal, play an important role in the tumour stability, but remain to this day poorly understood. The aim of this thesis was to establish in vitro assays to study the interactions between populations of normal and transformed cells.We benefited from a light-sensitive cell line, constructed by Olivier Destaing (IAB, Grenoble). When they are exposed to blue light, these cells overactivate the protein Src, which is known to be overexpressed in many cancers. Otherwise, they keep a normal phenotype. Using these cells, called “OptoSrc”, in combination with an optical setup, we are able to create mosaic tissues in which the pattern of mutated cells is determined by the blue illumination pattern. Our system has several advantages: a selective control in time and space of the group of transformed cells, and a gradual and reversible activation of the oncoprotein.We have shown that when we illuminate a circular islet of cells from a monolayer of OptoSrc cells, the activated cells were collectively extruded, resulting in a cohesive three-dimensional aggregate on top of the monolayer. We can control the size and appearance time of this spheroid by tuning, respectively, the area and frequency of illumination. Besides, this collective extrusion is reversible when the blue light stimulation is stopped. Finally, we have shown that the formation of this three-dimensional aggregate coincides with the loss of E-cadherin at the membrane, as well as the apparition of vimentin, for the illuminated OptoSrc cells. Our results suggest that a group of cells overexpressing the protein Src, in a monolayer of normal cells, undergoes a partial epithelial-to-mesenchymal transition

Collective stresses drive competition between monolayers of normal and Ras-transformed cells

23 pages, 12 figuresInternational audienceWe study the competition for space between two cell lines that differ only in the expression of the Ras oncogene. The two cell populations are initially separated and set to migrate antagonistically towards an in-between stripe of free substrate. After contact, their interface moves towards the population of normal cells. We interpret the velocity and traction force data taken before and after contact thanks to a hydrodynamic description of collectively migrating cohesive cell sheets. The kinematics of cells, before and after contact, allows us to estimate the relative material parameters for both cell lines. As predicted by the model, the transformed cell population with larger collective stresses pushes the wild type cell population

Collective stresses drive competition between monolayers of normal and Ras-transformed cells

23 pages, 12 figuresInternational audienceWe study the competition for space between two cell lines that differ only in the expression of the Ras oncogene. The two cell populations are initially separated and set to migrate antagonistically towards an in-between stripe of free substrate. After contact, their interface moves towards the population of normal cells. We interpret the velocity and traction force data taken before and after contact thanks to a hydrodynamic description of collectively migrating cohesive cell sheets. The kinematics of cells, before and after contact, allows us to estimate the relative material parameters for both cell lines. As predicted by the model, the transformed cell population with larger collective stresses pushes the wild type cell population

Controlling Confinement and Topology to Study Collective Cell Behaviors

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