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

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

Podosomes of dendritic cells facilitate antigen sampling

Dendritic cells sample the environment for antigens and play an important role in establishing the link between innate and acquired immunity. Dendritic cells contain mechanosensitive adhesive structures called podosomes that consist of an actin-rich core surrounded by integrins, adaptor proteins and actin network filaments. They facilitate cell migration via localized degradation of extracellular matrix. Here, we show that podosomes of human dendritic cells locate to spots of low physical resistance in the substrate (soft spots) where they can evolve into protrusive structures. Pathogen recognition receptors locate to these protrusive structures where they can trigger localized antigen uptake, processing and presentation to activate T-cells. Our data demonstrate a novel role in antigen sampling for the podosomes of dendritic cell

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

Extracellular vesicles secreted by triple-negative breast cancer stem cells trigger premetastatic niche remodeling and metastatic growth in the lungs

Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple-negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC , respectively) from the MDA-MB-231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL-6/IL-8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α-SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells

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

Micro Immune Response On-chip (MIRO) models the tumour-stroma interface for immunotherapy testing

Immunotherapies are beneficial for a considerable proportion of cancer patients, but ineffective in others. In vitro modelling of the complex interactions between cancer cells and their microenvironment could provide a path to understanding immune therapy sensitivity and resistance. Here we develop MIRO, a fully humanised in vitro platform to model the spatial organisation of the tumour/stroma interface and its interaction with immune cells. We find that stromal barriers are associated with immune exclusion and protect cancer cells from antibody-dependent cellular cytotoxicity, elicited by targeted therapy. We demonstrate that IL2-driven immunomodulation increases immune cell velocity and spreading to overcome stromal immunosuppression and restores anti-cancer response in refractory tumours. Collectively, our study underscores the translational value of MIRO as a powerful tool for exploring how the spatial organisation of the tumour microenvironment shapes the immune landscape and influences the responses to immunomodulating therapies

Cadherins: cellular adhesive molecules serving as signalling mediators

The single pass, transmembrane proteins of the cadherin family have been appreciated as important proteins that regulate intercellular adhesion. In addition to this critical function, cadherins contribute to important signalling events that control cellular homeostasis. Many examples exist of classical, desmosomal and atypical cadherins participating in the regulation of signalling events that control homeostatic functions in cells. Much of the work on cadherin mediated signalling focuses on classical cadherins or on specific disease states such as pemphigus vulgaris. Cadherin mediated signalling has been shown to play critical roles during development, in proliferation, apoptosis, disease pathobiology and beyond. It is becoming increasingly clear that cadherins operate through a range of molecular mechanisms. The diversity of pathways and cellular functions regulated by cadherins suggests that we have only scratched the surface in terms of the roles that these versatile proteins play in signalling and cellular function.Modalities of cadherin mediated signalling. Cadherins have been shown to participate in many diverse signalling pathways and mechanisms. Not only is the range of functional outcomes that cadherins regulate wide, the number of different types of mechanisms that cadherins participate in to mediate their signalling is also impressive. Many times cadherins themselves act as scaffolds for important signalling events and the ability of the cadherin to participate in these signalling mechanisms is often dependent upon their participation in cadherin mediated adhesion (A). Other mechanisms requiring cadherin mediated adhesion include cadherin protein association with other transmembrane signalling proteins and receptors (B). There are several reports of cadherin mediated signalling that requires the formation of stable cadherin protein fragments via regulated proteolytic cleavage (C and D). Most of these mechanisms require cadherin extracellular fragment generation and often both stimulate receptor signalling pathways and interfere with cadherin mediated adhesion (C). There are, however, multiple studies describing cadherin intracellular protein fragment generation (D). These mechanisms often entail binding of the intracellular fragment to a cytoplasmic signalling partner, nuclear re‐localization, and regulation of gene transcription.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/145515/1/tjp13128.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/145515/2/tjp13128_am.pd