A new biomimetic assay reveals the temporal role of matrix stiffening in cancer cell invasion

International audienceTumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent me-tastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity

Dynamique des cellules cancéreuses dans le coeur tumoral et rôle de la matrice extracellulaire dans l'invasion métastatique

Carcinoma development is a multistep process, in which accumulation of genetic alterations promotes the sustained proliferation and altered differentiation of epithelial cells. Metastasis is a critical step in cancer progression and the major cause of cancer-associated mortality. In colon cancer, twenty-five percent of patients already have metastasis at colorectal cancer diagnosis, and an additional 25-35% will develop metastases during disease progression. The metastatic colonization of distant organs requires the completion of a complex series of biological events. A crucial event in the metastatic cascade is the acquisition of a migratory phenotype by cancer cells. Most solid tumors, including colorectal cancer, are composed of a central differentiated core region and an invasive front forming the interface between tumor and stromal tissue. Although most studies have focused on cancer cell migration in the invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we studied the behavior of cancer cells in densely packed regions at the tumor core using a mouse model of genetically-induced aggressive intestinal carcinoma. We developed a method to image tumor explants in real time using two-photon microscopy. We found that cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local "currents" and large-scale tissue dynamics. The direction of these local currents appears to be influenced by collagen structures in the tumor core. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Our model opens new avenues for studying the dynamics of cells in the tumor core and more generally of tumor progression.Le développement de carcinomes est un processus graduel, pendant lequel l'accumulation de modifications génétiques promeut la prolifération et la différenciation aberrante des cellules épithéliales. Le processus métastatique est la cause majeure de décès dus au cancer. Dans le cancer du côlon, vingt-cinq pourcent des patients présentent déjà des métastases au diagnostic de la maladie, et 25-35% en développent à la progression de la maladie. La colonisation métastatique d'organes distants requiert la réalisation d'une série complexe d'événements. L'acquisition d'un phénotype migratoire par les cellules cancéreuses est un événement crucial dans le processus métastatique. La plupart des tumeurs solides, y compris le cancer du côlon, sont composées par un cœur tumoral différencié et par un front invasif qui forme l'interface entre la tumeur et le tissu stromal. Bien que de nombreuses études ont porté sur la migration des cellules cancéreuses dans le front invasif, les cellules du cœur tumoral peuvent également potentiellement métastaser. Nous avons étudié la migration des cellules cancéreuses dans le cœur tumoral grâce à un modèle murin de tumorigenèse intestinale génétiquement induite. Nous avons développé une technique d'imagerie pour suivre en temps réel la migration cellulaire dans des explants tumoraux en utilisant la microscopie biphotonique. Nous avons trouvé que les cellules cancéreuses dans le cœur tumoral sont remarquablement dynamiques et suivent les schémas de migration coordonnées, ce qui donne naissance à des « courants » cellulaires et à des dynamiques à large échelle. La direction de ces courants est influencée par des structures de collagène dans le cœur tumoral. Bien que les cellules s'arrêtent de migrer de façon intermittente, les pauses dans la migration ne sont pas nécessaires au moment de la division cellulaire. En conclusion, notre modèle ouvre une nouvelle voie d'étude de la dynamique des cellules cancéreuses dans le cœur tumoral et plus largement de la progression tumorale

Dynamics of cancer cells in the tumor core and role of the extracellular matrix in invasion

Le développement de carcinomes est un processus graduel, pendant lequel l'accumulation de modifications génétiques promeut la prolifération et la différenciation aberrante des cellules épithéliales. Le processus métastatique est la cause majeure de décès dus au cancer. Dans le cancer du côlon, vingt-cinq pourcent des patients présentent déjà des métastases au diagnostic de la maladie, et 25-35% en développent à la progression de la maladie. La colonisation métastatique d'organes distants requiert la réalisation d'une série complexe d'événements. L'acquisition d'un phénotype migratoire par les cellules cancéreuses est un événement crucial dans le processus métastatique. La plupart des tumeurs solides, y compris le cancer du côlon, sont composées par un cœur tumoral différencié et par un front invasif qui forme l'interface entre la tumeur et le tissu stromal. Bien que de nombreuses études ont porté sur la migration des cellules cancéreuses dans le front invasif, les cellules du cœur tumoral peuvent également potentiellement métastaser. Nous avons étudié la migration des cellules cancéreuses dans le cœur tumoral grâce à un modèle murin de tumorigenèse intestinale génétiquement induite. Nous avons développé une technique d'imagerie pour suivre en temps réel la migration cellulaire dans des explants tumoraux en utilisant la microscopie biphotonique. Nous avons trouvé que les cellules cancéreuses dans le cœur tumoral sont remarquablement dynamiques et suivent les schémas de migration coordonnées, ce qui donne naissance à des « courants » cellulaires et à des dynamiques à large échelle. La direction de ces courants est influencée par des structures de collagène dans le cœur tumoral. Bien que les cellules s'arrêtent de migrer de façon intermittente, les pauses dans la migration ne sont pas nécessaires au moment de la division cellulaire. En conclusion, notre modèle ouvre une nouvelle voie d'étude de la dynamique des cellules cancéreuses dans le cœur tumoral et plus largement de la progression tumorale.Carcinoma development is a multistep process, in which accumulation of genetic alterations promotes the sustained proliferation and altered differentiation of epithelial cells. Metastasis is a critical step in cancer progression and the major cause of cancer-associated mortality. In colon cancer, twenty-five percent of patients already have metastasis at colorectal cancer diagnosis, and an additional 25-35% will develop metastases during disease progression. The metastatic colonization of distant organs requires the completion of a complex series of biological events. A crucial event in the metastatic cascade is the acquisition of a migratory phenotype by cancer cells. Most solid tumors, including colorectal cancer, are composed of a central differentiated core region and an invasive front forming the interface between tumor and stromal tissue. Although most studies have focused on cancer cell migration in the invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we studied the behavior of cancer cells in densely packed regions at the tumor core using a mouse model of genetically-induced aggressive intestinal carcinoma. We developed a method to image tumor explants in real time using two-photon microscopy. We found that cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local "currents" and large-scale tissue dynamics. The direction of these local currents appears to be influenced by collagen structures in the tumor core. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Our model opens new avenues for studying the dynamics of cells in the tumor core and more generally of tumor progression

Cell polarity and extrusion: how to polarize extrusion and extrude misspolarized cells?

International audienceThe barrier function of epithelia is one of the cornerstones of the body plan organisation of metazoans. It relies on the polarity of epithelial cells which organises along the apico-basal axis the mechanical properties, signalling as well as transport. This barrier function is however constantly challenged by the fast turnover of epithelia occurring during morphogenesis or adult tissue homeostasis. Yet, the sealing property of thetissue can be maintained thanks to cell extrusion: a series of remodelling steps involving the dying cell and its neighbours leading to seamless cell expulsion. Alternatively, the tissue architecture can also be challenged by local damages or the emergence of mutant cells that may alter its organisation. This includes mutants of the polarity complexes which can generate neoplastic overgrowths or be eliminated by cell competition when surrounded by wild type cells. In this review, we will provide an overview of the regulation of cell extrusion in various tissues focusing on the relationship between cell polarity, cell organisation and the direction of cell expulsion. We will then describe how local perturbations of polarity can also trigger cell elimination either by apoptosis or by cell exclusion, focusing specifically on how polarity defects can be directly causal to cell elimination. Overall, we propose a general framework connecting the influence of polarity on cell extrusion and its contribution to aberrant cell elimination

Cell Migration in Tissues: Explant Culture and Live Imaging

International audienc

Tensile Forces Originating from Cancer Spheroids Facilitate Tumor Invasion

International audienceThe mechanical properties of tumors and the tumor environment provide important information for the progression and characterization of cancer. Tumors are surrounded by an extracellular matrix (ECM) dominated by collagen I. The geometrical and mechanical properties of the ECM play an important role for the initial step in the formation of metastasis, presented by the migration of malignant cells towards new settlements as well as the vascular and lymphatic system. The extent of this cell invasion into the ECM is a key medical marker for cancer prognosis. In vivo studies reveal an increased stiffness and different architecture of tumor tissue when compared to its healthy counterparts. The observed parallel collagen organization on the tumor border and radial arrangement at the invasion zone has raised the question about the mechanisms organizing these structures. Here we study the effect of contractile forces originated from model tumor spheroids embedded in a biomimetic collagen I matrix. We show that contractile forces act immediately after seeding and deform the ECM, thus leading to tensile radial forces within the matrix. Relaxation of this tension via cutting the collagen does reduce invasion, showing a mechanical relation between the tensile state of the ECM and invasion. In turn, these results suggest that tensile forces in the ECM facilitate invasion. Furthermore, simultaneous contraction of the ECM and tumor growth leads to the condensation and reorientation of the collagen at the spheroid’s surface. We propose a tension-based model to explain the collagen organization and the onset of invasion by forces originating from the tumor

A new biomimetic assay reveals the temporal role of matrix stiffening in cancer cell invasion

International audienceTumor initiation and growth is associated with significant changes in the surrounding tissue. During carcinoma progression, a global stiffening of the extracellular matrix is observed and is interpreted as a signature of aggressive invasive tumors. However, it is still unknown whether this increase in matrix rigidity promotes invasion and whether this effect is constant along the course of invasion. Here we have developed a biomimetic in vitro assay that enabled us to address the question of the importance of tissue rigidity in the chronology of tumor invasion. Using low concentrations of the sugar threose, we can effectively stiffen reconstituted collagen I matrices and control the stiffening in time with no direct effect on residing cells. Our findings demonstrate that, depending on the timing of its stiffening, the extracellular matrix could either inhibit or promote cancer cell invasion and subsequent metastasis: while matrix stiffening after the onset of invasion promotes cancer cell migration and tumor spreading, stiff matrices encapsulate the tumor at an early stage and prevent cancer cell invasion. Our study suggests that adding a temporal dimension in in vitro models to analyze biological processes in four dimensions is necessary to fully capture their complexity

Cancer cells in the tumor core exhibit spatially coordinated migration patterns

International audienceIn early stages of metastasis, cancer cells exit the primary tumor and enter the vasculature. Although most studies have focused on the tumor invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we imaged tumor explants from spontaneously-forming tumors in real time using long-term two-photon microscopy. Cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local "currents" and large-scale tissue dynamics. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Use of pharmacological inhibitors indicates that migration patterns in tumors are actively driven by the actin cytoskeleton. Under these conditions, we also observed a relationship between migration speed and correlation length, suggesting that cells in tumors are near a jamming transition. Our study provides new insight into the dynamics of cancer cells in the tumor core, opening new avenues of research in understanding the migratory properties of cancer cells and later metastasis

CT26 invasion after cutting the collagen gel close to the spheroid.

<p><b>(A)</b> Collagen (z projection, 4x objective): red TAMRA collagen was polymerized and cut with scalpel on one side. Left images show the invasion of GFP-positive cells (green) at 0, 24 and 72 hours post-seeding. The dashed line indicates an edge of the cut collagen and the arrow shows the direction of collagen contraction. Right: Fluorescent images of angular unwinded spheroid where each horizontal line corresponds to the radial fluorescence intensity profile. The y-axis of these images corresponds to the different angles. Arrows show the area of invasion on the side of the cut. The black dotted line makes the left limit of the area used to calculate the average fluorescence as shown in B. <b>(B)</b> Average fluorescent intensity of cells extending over the initial spheroid surface as a function of angle Θ. This is used to estimate the outgrowth of cells from the original spheroid size. The different colors represent the average intensity after 0 (red), 24 (green) and 72 hours (violet) of invasion. The shading of the graph shows the side facing the cut (light blue), the opposing side facing the inside of the gel (light red) and the two sides perpendicular to the cut (white area). The dotted line represents the outgrowth in spheroids embedded in uncut collagen at the corresponding times. <b>(C)</b> Invasion area quantification on the side of the cut (after 0 hours) and average invasion area on uncut side. Invasion area was quantified as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156442#pone.0156442.g003" target="_blank">Fig 3</a> (mean ± standard error, n = 54 of 6 independent experiments). <b>(D)</b> Sketch of tension-dependent invasion model.</p

Invasion of cancer cells CT26 into collagen type I.

<p><b>(A)</b> Image sequence of CT26-GFP (green) cell invasion in TAMRA-labeled collagen type I (red). Cells initiate invasion (white arrow) after approximately 9 hours (onset of invasion). Scale bar: 50 μm. <b>(B)</b> Kymographs of collagen and cells from the image sequence (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156442#pone.0156442.s011" target="_blank">S2 Movie</a>). Images were taken every one hour for 24 hours. Scale 50 μm and 3 hours. <b>(C)</b> Magnification of the boxed region showing movement of the collagen fibers towards the spheroid. The kymograph illustrates the two antagonizing movements of compression due to spheroid growth (close to spheroid, blue arrow and blue line), and the collagen contraction in the invasion zone (stripes toward the spheroid, yellow arrow and yellow line). <b>(D)</b> Zoom in confocal images of CT26-GFP multicellular cancer cell spheroid with cells invading (green) into collagen type I network (red) showing the collagen organization: parallel fibers (white arrow) and radial fibers (yellow arrow). Scale 20 μm. <b>(E)</b> Collagen fiber orientation i) Color-coded orientation map. ii) Quantitative orientation measurement (table—angles) on selected ROIs (circles). Spheroid surface orientation: 45°, orientation normal to surface: -45° <b>(F)</b> Collagen signatures found in a single NICD/p53^-/- mouse intestinal tumor are imaged using intravital two-photon microscopy. i) TACS-1, curly collagen structure; ii) TACS-2, straight and aligned collagen, parallel to the tumor edge and iii) TACS-3, collagen aligned perpendicularly to the tumor edge. Epithelial cancer cells (nuclear GFP, green), collagen (SHG, magenta). Arrowheads point to distinct collagen organization. Scale bars, 50μm.</p