Mecanobiology of cancerous cells on topographically and chemically well controlled surfaces

Le travail présenté dans cette thèse est le résultat d'une collaboration fructueuse entre la chimie, la physique et la biologie. En effet, des matériaux avec des propriétés physico-chimiques très contrôlées ont été mis à profit dans le but de caractériser des fonctions cellulaires complexes. Nous présentons tout d'abord la création d'un outil permettant l'étude de la mécanotransduction cellulaire. L'originalité de cet outil est basé sur son activation par étirement permettant de lier réversiblement les cellules à la surface. Nous avons ensuite étudié des comportements de cellules souches et cancéreuses en réponse à des microtopographies sous forme de piliers. Cette approche a permis de définir un comportement cancéreux caractérisé par une déformation prononcée des corps et noyaux cellulaires. Nous montrons aussi que l'utilisation de cette surface couverte de micro-piliers permet de décrire la mécano-biologie de cellules cancéreuses. En effet, ce substrat à topographie contrôlée a permis de montrer que la chimie et la rigidité du substrat n'ont que peu d'incidence sur la déformation des cellules cancéreuses, alors que les éléments du cytosquelette sont primordiaux et que sans eux, la déformation n'est pas possible. Nous avons ensuite inhibé une à une des protéines de l'enveloppe et de la lamina nucléaire afin d'évaluer leur implication dans ces mécanismes de déformation. En parallèle, un séquençage total des ARN (Acides RiboNucléiques) de cellules déformées et non déformées a été réalisé dans le but de visualiser d'éventuelles modifications dans l'expression génique. Ces déformations des cellules cancéreuses entre les micro-piliers ont été comparées à celles que subissent les cellules lors de la traversée de membranes poreuses (Chambres de Boyden). Ces comparaisons nous ont permis d'identifier que plusieurs mécanismes peuvent aboutir à la déformation de cellules cancéreuses et en particulier de leurs noyaux. Nous montrons dans une dernière partie que la mitose cellulaire s'effectue sur les surfaces microstructurées. Nous décrivons une ségrégation des chromosomes qui semble être non parallèle. Toutefois, ces divisions atypiques ne causent pas davantage d'accidents mitotiques.The work shown in this thesis is the outcome of a successful collaboration between chemistry, physics and biology. Indeed, materials with well controlled parameters have been used in order to characterize complex cellular functions. We first introduce the creation of one tool which allow the study of cells mechanotransduction. The originality of this tool is based on its activation by stretching which allow a reversible adhesion of cells to the surface.Then, we studied the behavior of stem cells and cancerous cells on micropillared surfaces. This approach allowed us to describe a cancerous behavior of cells characterized by strong deformations of cells bodies and nuclei. We also showed that the use of such micropillared surfaces allowed us to describe cancerous cells mecanobiology. Indeed, this substrate with a well controlled topography allowed us to show that substrates chemistry and stiffness have only little effects on cancerous cells deformation while cytoskeleton components are necessary. More specifically, the deformation is impossible without the cytoskeleton. We also inhibited the nuclear envelope proteins and nuclear lamina proteins in order to evaluate their involvement in cells deformation mechanism. In the same time, a total RNA (RiboNucleic Acids) sequencing of deformed and non deformed cells have been done in order to identify an eventual modification in gene expression.These deformations of cancerous cells between micropillars have been compared to the deformation of cells during the transmigration through porous membranes (Boyden chambers). These comparisons allowed us to identify several mechanisms which lead to cells deformation and more specifically to nuclei deformation.We showed in a last part that cells can divide on micropillared surfaces. We described a non parallel like segregation of chromosomes. However, these unusual mitosis didn't lead to supernumerary troubles in cell division

Mécano-biologie de cellules cancéreuses sur surfaces à topographie et chimie contrôlées

The work shown in this thesis is the outcome of a successful collaboration between chemistry, physics and biology. Indeed, materials with well controlled parameters have been used in order to characterize complex cellular functions. We first introduce the creation of one tool which allow the study of cells mechanotransduction. The originality of this tool is based on its activation by stretching which allow a reversible adhesion of cells to the surface.Then, we studied the behavior of stem cells and cancerous cells on micropillared surfaces. This approach allowed us to describe a cancerous behavior of cells characterized by strong deformations of cells bodies and nuclei. We also showed that the use of such micropillared surfaces allowed us to describe cancerous cells mecanobiology. Indeed, this substrate with a well controlled topography allowed us to show that substrates chemistry and stiffness have only little effects on cancerous cells deformation while cytoskeleton components are necessary. More specifically, the deformation is impossible without the cytoskeleton. We also inhibited the nuclear envelope proteins and nuclear lamina proteins in order to evaluate their involvement in cells deformation mechanism. In the same time, a total RNA (RiboNucleic Acids) sequencing of deformed and non deformed cells have been done in order to identify an eventual modification in gene expression.These deformations of cancerous cells between micropillars have been compared to the deformation of cells during the transmigration through porous membranes (Boyden chambers). These comparisons allowed us to identify several mechanisms which lead to cells deformation and more specifically to nuclei deformation.We showed in a last part that cells can divide on micropillared surfaces. We described a non parallel like segregation of chromosomes. However, these unusual mitosis didn't lead to supernumerary troubles in cell division.Le travail présenté dans cette thèse est le résultat d'une collaboration fructueuse entre la chimie, la physique et la biologie. En effet, des matériaux avec des propriétés physico-chimiques très contrôlées ont été mis à profit dans le but de caractériser des fonctions cellulaires complexes. Nous présentons tout d'abord la création d'un outil permettant l'étude de la mécanotransduction cellulaire. L'originalité de cet outil est basé sur son activation par étirement permettant de lier réversiblement les cellules à la surface. Nous avons ensuite étudié des comportements de cellules souches et cancéreuses en réponse à des microtopographies sous forme de piliers. Cette approche a permis de définir un comportement cancéreux caractérisé par une déformation prononcée des corps et noyaux cellulaires. Nous montrons aussi que l'utilisation de cette surface couverte de micro-piliers permet de décrire la mécano-biologie de cellules cancéreuses. En effet, ce substrat à topographie contrôlée a permis de montrer que la chimie et la rigidité du substrat n'ont que peu d'incidence sur la déformation des cellules cancéreuses, alors que les éléments du cytosquelette sont primordiaux et que sans eux, la déformation n'est pas possible. Nous avons ensuite inhibé une à une des protéines de l'enveloppe et de la lamina nucléaire afin d'évaluer leur implication dans ces mécanismes de déformation. En parallèle, un séquençage total des ARN (Acides RiboNucléiques) de cellules déformées et non déformées a été réalisé dans le but de visualiser d'éventuelles modifications dans l'expression génique. Ces déformations des cellules cancéreuses entre les micro-piliers ont été comparées à celles que subissent les cellules lors de la traversée de membranes poreuses (Chambres de Boyden). Ces comparaisons nous ont permis d'identifier que plusieurs mécanismes peuvent aboutir à la déformation de cellules cancéreuses et en particulier de leurs noyaux. Nous montrons dans une dernière partie que la mitose cellulaire s'effectue sur les surfaces microstructurées. Nous décrivons une ségrégation des chromosomes qui semble être non parallèle. Toutefois, ces divisions atypiques ne causent pas davantage d'accidents mitotiques

Nucleo-mechanosensing crosstalk between 3-D pillar topography and cyto-nucleoskeleton

Nucleo-mechanosensing crosstalk between 3-D pillar topography and cyto-nucleoskeleto

Antiplatelet therapy guided by CYP2C19 point-of-care pharmacogenetics plus multidimensional treatment decisions

Aim: Implementation of CYP2C19 point-of-care (POC) pharmacogenetic (PGx) testing with personalized treatment recommendations. Methods: POC CYP2C19 genotyping plus expert evaluation of risk factors for ischemic and bleeding events. Results: 167 patients underwent PGx testing, 54 (32.3%) were CYP2C19 loss of function carriers, and POC versus standard PGx analysis results for *2 and *3 variants matched in 100%. Antiplatelet therapy was adjusted in 44 patients (26.3%), but always required consideration of patient-specific factors. Conclusion: CYP2C19 POC-PGx is reliable and offers clinically relevant advantages for immediate evidence-based adaptations of antiplatelet therapy, whereas in less acute cases conventional PGx testing can also have advantages. Antiplatelet therapy has become more complex, and implementation of PGx-based personalized antiplatelet therapy requires complementary expert knowledge.ISSN:1462-2416ISSN:1744-804

Interactions between bio-systems & 3D µ-structured surfaces

Intervention également présentée au colloque Mifobio 2016 (30 septembre - 6 octobre 2016)Interactions between bio-systems & 3D µ-structured surface

Nuclear plasticity of cancerous cells on micropillared surfaces: interest for understanding mechanotransduction of metastatic cells

Intervention aussi présentée par Karine Anselme à la Journée MEV de l’Institut Jean Lamour le 13 septembre 2016   Affiliations : a. Institut de Sciences des Matériaux de Mulhouse, Université de Strasbourg, Université de Haute-Alsace, CNRS UMR7361, 68057 Mulhouse, France, b. Department of Microsystems Engineering (IMTEK), Albert-Ludwigs-Universität Freiburg, 79110 Freiburg, Germany, c. DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology, 76131 Karlsruhe, Germany, d. Laboratoire Physico-Chimie Curie, Institut Curie, CNRS UMR168, 75248 Paris, France, e. INSERM UMR-S1113, 3 Avenue Molière, 67200 Strasbourg, FranceSeveral years ago, we have shown that strong deformations of cancerous cell bodies can be obtained when osteosarcoma cells are cultured on micropillared substrates and that these alterations also affect the nucleus shape. These strong deformations of cells and of their organelles which don't affect viability and proliferation draw an analogy with the metastatic process. That is why the understanding of cells mechanics and of the related inner forces in this model is essential. Here, we analyse the nuclear deformation on micropillared surfaces of cancer cells. As the deformability of cells is strongly connected with their cytoskeleton dynamics, we combine live imaging and AFM elasticity measurements with treatment with inhibitors of cytoskeleton and LINC complex molecules to decipher the involvement of each cytoskeleton component in the nucleus deformation. In parallel the kinetics of deformation of cancer cells on micropillars is analysed in relation with the chromatin condensation. Our findings confirm that actomyosin microfilaments associated with intermediate filaments and LINC molecules play a major role in this deformation. Microtubules are not always involved depending of the cell type. We also demonstrate that pulling down forces imparted by the cytoskeleton are necessary to promote deformation by changing the chemistry on top or in between interpillar space of pillars by spin coating cell adhesive (PnBA) and repellent polymers (PDMAA)

Biomimetic Cryptic Site Surfaces for Reversible Chemo- and Cyto-Mechanoresponsive Substrates

International audienceChemo-mechanotransduction, the way by which mechanical forces are transformed into chemical signals, plays a fundamental role in many biological processes. The first step of mechanotransduction often relies on exposure, under stretching, of cryptic sites buried in adhesion proteins. Likewise, here we report the first example of synthetic surfaces allowing for specific and fully reversible adhesion of proteins or cells promoted by mechanical action. Silicone sheets are first plasma treated and then functionalized by grafting sequentially under stretching poly(ethylene glycol) (PEG) chains and biotin or arginine-glycine-aspartic acid (RGD) peptides. At unstretched position, these ligands are not accessible for their receptors. Under a mechanical deformation, the surface becomes specifically interactive to streptavidin, biotin antibodies, or adherent for cells, the interactions both for proteins and cells being fully reversible by stretching/unstretching, revealing a reversible exposure process of the ligands. By varying the degree of stretching, the amount of interacting proteins can be varied continuously

Actomyosin, vimentin and LINC complex pull on osteosarcoma nuclei to deform on micropillar topography

International audienceCell deformation occurs in many critical biological processes, including cell extravasation during immune response and cancer metastasis. These cells deform the nucleus, its largest and stiffest organelle, while passing through narrow constrictions in vivo and the underlying mechanisms still remain elusive. It is unclear which biochemical actors are responsible and whether the nucleus is pushed or pulled (or both) during deformation. Herein we use an easily-tunable poly-L-lactic acid micropillar topography, mimicking in vivo constrictions to determine th

Tuning InAs Nanowire Density for HEK293 Cell Viability, Adhesion, and Morphology: Perspectives for Nanowire-Based Biosensors

Arrays of nanowires (NWs) are currently being established as vehicles for molecule delivery and electrical- and fluorescence-based platforms in the development of biosensors. It is conceivable that NW-based biosensors can be optimized through increased understanding of how the nanotopography influences the interfaced biological material. Using state-of-the-art homogenous NW arrays allow for a systematic investigation of how the broad range of NW densities used by the community influences cells. Here it is demonstrated that indium arsenide NW arrays provide a cell-promoting surface, which affects both cell division and focal adhesion up-regulation. Furthermore, a systematic variation in NW spacing affects both the detailed cell morphology and adhesion properties, where the latter can be predicted based on changes in free-energy states using the proposed theoretical model. As the NW density influences cellular parameters, such as cell size and adhesion tightness, it will be important to take NW density into consideration in the continued development of NW-based platforms for cellular applications, such as molecule delivery and electrical measurements

Tuning InAs Nanowire Density for HEK293 Cell Viability, Adhesion, and Morphology: Perspectives for Nanowire-Based Biosensors

Arrays of nanowires (NWs) are currently being established as vehicles for molecule delivery and electrical- and fluorescence-based platforms in the development of biosensors. It is conceivable that NW-based biosensors can be optimized through increased understanding of how the nanotopography influences the interfaced biological material. Using state-of-the-art homogenous NW arrays allow for a systematic investigation of how the broad range of NW densities used by the community influences cells. Here it is demonstrated that indium arsenide NW arrays provide a cell-promoting surface, which affects both cell division and focal adhesion up-regulation. Furthermore, a systematic variation in NW spacing affects both the detailed cell morphology and adhesion properties, where the latter can be predicted based on changes in free-energy states using the proposed theoretical model. As the NW density influences cellular parameters, such as cell size and adhesion tightness, it will be important to take NW density into consideration in the continued development of NW-based platforms for cellular applications, such as molecule delivery and electrical measurements