Etude des propriétés biophysiques et mécano-sensorielles des podosomes

Les podosomes sont des microstructures du cytosquelette cellulaire constituées d'un cœur dense d'actine filamenteuse entouré à leurs bases de protéines qui établissent un lien étroit avec la matrice extracellulaire à la face ventrale des cellules. Contrairement aux adhérences cellulaires " classiques ", ces structures sont très dynamiques et ont également la particularité de pouvoir localement dégrader la matrice extracellulaire. Les podosomes sont spécifiques à quelques types cellulaires pour la plupart très mobiles, principalement issus du lignage myéloïde dont les macrophages, cellules clés de l'immunité innée. Si de nombreuses études ont été menées dans le but de caractériser l'architecture et la régulation de la formation des podosomes, de nombreuses questions demeurent sur la fonction exacte de ces structures. En effet, si le caractère très dynamique des podosomes semble participer à la perception de l'environnement par les cellules, les études sur les processus mécano-sensoriels de ces structures sont encore très sporadiques. Dans ce contexte, l'objectif de ma thèse s'est alors inscrit dans une démarche exploratoire pour tenter d'approfondir les caractéristiques mécano-sensorielles et biophysiques des podosomes des macrophages humains dans un contexte d'étude in vitro par l'utilisation combinée d'approches complémentaires comme (i) la Microscopie à Force Atomique, (ii) la microstructuration de protéines de matrices par impression par micro-contact (iii) la fabrication de matrices à rigidités modulables (iv) la mise en œuvre de substrats très fins déformables suspendus. L'ensemble de mes travaux de thèse a permis l'étude des podosomes à l'échelle nanométrique dans des cellules vivantes, ce qui a permis de révéler (i) que la hauteur des podosomes est constante quelle que soit la physico-chimie du substrat, (ii) que ces structures sont capables d'exercer sur le substrat une contrainte normale oscillante et périodique dépendante de la contraction des complexes acto-myosine et de la polymérisation d'actine, et (iii) que ces structures présentent une dynamique de rigidité périodique étroitement liée à la dynamique des contraintes que ces structures génèrent sur le substrat et qui est également dépendante de la rigidité de la matrice.Podosomes are particular sub-cellular F-actin rich structures, composed of a dense actin-core surrounded at it base by numerous proteins that establish a close contact with the extracellular matrix on the ventral face of the cell. Unlike "classical" adhesive structures, podosomes are very dynamic and are able to locally degrade the extracellular matrix. These structures are specifically found in very motile cells, which mainly belong to the myeloid cell lineage, including macrophages, which are key cells of the innate immunity. Despite numerous studies that aimed to decipher podosome architecture and signaling pathways that regulates their formation, several questions remains about the exact role of these structures. Indeed, if the dynamic behavior of podosome seems to participate in the probing activity of the cells for analyzing their surrounding environment, studies of podosome mechanosensory processes is still sporadic. In this context, the purpose of my PhD was an exploratory research in order to decipher mechanosensory and biophysical properties of podosomes in human macrophages in vitro. Thus several complementary approaches were combined such as: (i) Atomic Force Microscopy, (i) micro-contact printing of matrix proteins, (iii) fabrication of matrices with various stiffness, and (iv) the use of a thin compliant suspended substrate. Finally my work enabled to explore podosomes at a nanometer-scale level in living cells and shaded light on several aspects as: (i) the height of podosomes is constant independently of the physicochemical properties of the substrate, (ii) theses structures are able to exert a normal, oscillating and periodic strain on the substrate related to acto-myosin complexes contraction and actin-polymerization activity, (iv) these structures have periodic stiffness oscillations closely related to the periodic strain they exert on the substrate, which are modulated by the stiffness of the substrate

Collective cell durotaxis emerges from long-range intercellular force transmission

The ability of cells to follow gradients of extracellular matrix stiffness—durotaxis—has been implicated in development, fibrosis, and cancer. Here, we found multicellular clusters that exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, required the action of myosin motors, and originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stick-slip dynamics of cell-matrix adhesions was integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single-cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion.Peer ReviewedPostprint (author's final draft

Collective cell durotaxis emerges from long-range intercellular force transmission

The ability of cells to follow gradients of extracellular matrix stiffness-durotaxis-has been implicated in development, fibrosis, and cancer. Here, we found multicellular clusters that exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, required the action of myosin motors, and originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stick-slip dynamics of cell-matrix adhesions was integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single-cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion

Aberrant DNA methylation in non-small cell lung cancer-associated fibroblasts

Epigenetic changes through altered DNA methylation have been implicated in critical aspects of tumor progression, and have been extensively studied in a variety of cancer types. In contrast, our current knowledge of the aberrant genomic DNA methylation in tumor-associated fibroblasts (TAFs) or other stromal cells that act as critical coconspirators of tumor progression is very scarce. To address this gap of knowledge, we conducted genome-wide DNA methylation profiling on lung TAFs and paired control fibroblasts (CFs) from non-small cell lung cancer patients using the HumanMethylation450 microarray. We found widespread DNA hypomethylation concomitant with focal gain of DNA methylation in TAFs compared to CFs. The aberrant DNA methylation landscape of TAFs had a global impact on gene expression and a selective impact on the TGF-β pathway. The latter included promoter hypermethylation-associated SMAD3 silencing, which was associated with hyperresponsiveness to exogenous TGF-β1 in terms of contractility and extracellular matrix deposition. In turn, activation of CFs with exogenous TGF-β1 partially mimicked the epigenetic alterations observed in TAFs, suggesting that TGF-β1 may be necessary but not sufficient to elicit such alterations. Moreover, integrated pathway-enrichment analyses of the DNA methylation alterations revealed that a fraction of TAFs may be bone marrow-derived fibrocytes. Finally, survival analyses using DNA methylation and gene expression datasets identified aberrant DNA methylation on the EDARADD promoter sequence as a prognostic factor in non-small cell lung cancer patients. Our findings shed light on the unique origin and molecular alterations underlying the aberrant phenotype of lung TAFs, and identify a stromal biomarker with potential clinical relevance

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells

Colorectal cancer (CRC) tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing single cell RNA-sequencing (scRNA-seq) expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth

Collective cell durotaxis emerges from long-range intercellular force transmission

The ability of cells to follow gradients of extracellular matrix stiffness—durotaxis—has been implicated in development, fibrosis, and cancer. Here, we found multicellular clusters that exhibited durotaxis even if isolated constituent cells did not. This emergent mode of directed collective cell migration applied to a variety of epithelial cell types, required the action of myosin motors, and originated from supracellular transmission of contractile physical forces. To explain the observed phenomenology, we developed a generalized clutch model in which local stick-slip dynamics of cell-matrix adhesions was integrated to the tissue level through cell-cell junctions. Collective durotaxis is far more efficient than single-cell durotaxis; it thus emerges as a robust mechanism to direct cell migration during development, wound healing, and collective cancer cell invasion

A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion

Cancer-associated fibroblasts (CAFs) promote tumour invasion and metastasis. We show that CAFs exert a physical force on cancer cells that enables their collective invasion. Force transmission is mediated by a heterophilic adhesion involving N-cadherin at the CAF membrane and E-cadherin at the cancer cell membrane. This adhesion is mechanically active; when subjected to force it triggers β-catenin recruitment and adhesion reinforcement dependent on α-catenin/vinculin interaction. Impairment of E-cadherin/N-cadherin adhesion abrogates the ability of CAFs to guide collective cell migration and blocks cancer cell invasion. N-cadherin also mediates repolarization of the CAFs away from the cancer cells. In parallel, nectins and afadin are recruited to the cancer cell/CAF interface and CAF repolarization is afadin dependent. Heterotypic junctions between CAFs and cancer cells are observed in patient-derived material. Together, our findings show that a mechanically active heterophilic adhesion between CAFs and cancer cells enables cooperative tumour invasion

Protrusion force microscopy reveals oscillatory force generation and mechanosensing activity of human macrophage podosomes

International audiencePodosomes are adhesion structures formed in monocyte-derived cells. They are F-actin-rich columns perpendicular to the substrate surrounded by a ring of integrins. Here, to measure podosome protrusive forces, we designed an innovative experimental setup named protrusion force microscopy (PFM), which consists in measuring by atomic force microscopy the deformation induced by living cells onto a compliant Formvar sheet. By quantifying the heights of protrusions made by podosomes onto Formvar sheets, we estimate that a single podosome generates a protrusion force that increases with the stiffness of the substratum, which is a hallmark of mechanosensing activity. We show that the protrusive force generated at podosomes oscillates with a constant period and requires combined actomyosin contraction and actin polymerization. Finally, we elaborate a model to explain the mechanical and oscillatory activities of podosomes. Thus, PFM shows that podosomes are mechanosensing cell structures exerting a protrusive force