Etude des mécanismes cellulaires et moléculaires de la migration des macrophages humains dans des environnements en trois dimensions

L'infiltration tissulaire des macrophages est un facteur aggravant dans de nombreuses pathologies telles que les maladies inflammatoires chroniques ou le cancer. Les macrophages qui infiltrent les tumeurs de façon continue sont appelés macrophages associés aux tumeurs (TAMs). Ils favorisent la croissance tumorale, l'angiogenèse, l'invasion tumorale et la formation de métastases. L'inhibition de l'infiltration des macrophages est donc devenue une évidence thérapeutique. Récemment, l'équipe a démontré que les macrophages utilisent le mode migratoire amiboïde (dépendant de ROCK) ou mésenchymal (dépendant des protéases) selon l'architecture de la matrice extracellulaire (MEC) en trois-dimensions (3D) qu'ils traversent. De plus, l'étude du mode migratoire mésenchymal a montré qu'il est dépendant de Hck (une tyrosine kinase spécifique des phagocytes) et de sa capacité à réorganiser les podosomes en rosettes (structures riches en actine dégradant la MEC). Mon projet de thèse s'est articulé autour de deux axes de recherche : 1) l'identification des substrats de Hck et la caractérisation de leur rôle dans l'organisation des podosomes et la migration 3D des macrophages, et 2) l'étude de la migration 3D des monocytes/macrophages primaires humains dans un modèle mimant le microenvironnement tumoral : les sphéroïdes tumoraux. Par une approche protéomique j'ai identifié des partenaires et substrats potentiels de Hck dont la Filamine A (FLNa), une protéine assurant notamment la liaison entre le cytosquelette d'actine et les intégrines. En utilisant différents outils (protéines recombinantes, anticorps, shRNA...) j'ai montré que : 1) Hck phosphoryle la FLNa in vitro, 2) la FLNa est associée aux podosomes et est nécessaire à leur organisation en rosettes sous le contrôle de Hck, 3) les podosomes des cellules déficientes en Flna ont une durée de vie plus courte, et 4) l'expression de la FLNa est nécessaire à la migration mésenchymale, mais pas à la migration amiboïde des macrophages dans une MEC en 3D. Ainsi la FLNa est impliquée dans la formation et à la stabilisation des podosomes, à leur organisation en rosettes, la migration mésenchymale des macrophages et pourrait se situer dans la voie de signalisation de Hck. En parallèle, j'ai mis au point un modèle de sphéroïdes tumoraux qui m'a permis de montrer que l'infiltration des monocytes ou des macrophages, dans ce modèle tissulaire in vitro, est dépendante de ROCK et des protéases, signature de l'utilisation des deux modes migratoires. Puis en incubant ces sphéroïdes au sein de MEC, j'ai démontré que la présence de macrophages infiltrés dans les sphéroïdes est nécessaire pour déclencher le pouvoir invasif des cellules tumorales qui émigrent des sphéroïdes en suivant les macrophages et infiltrent la MEC environnante. Les macrophages Hck-/- présentant un défaut de migration mésenchymale, sont significativement moins efficaces dans la promotion de l'invasion des cellules tumorales. Ces résultats indiquent que l'activité de migration et de remodelage de la matrice exercée par les macrophages est prépondérante dans l'invasion tumorale in vitro. Ces résultats ont permis d'établir le mode migratoire des macrophages infiltrant un modèle tissulaire in-vitro et de démontrer le mécanisme d'action des macrophages dans l'invasion tumorale. Ainsi, mes travaux de thèse ont permis de progresser dans la caractérisation des mécanismes moléculaires et cellulaires de la migration 3D des macrophages humains. En effet, j'ai pu 1) identifier une protéine nécessaire à la migration mésenchymale des macrophages, 2) mettre en évidence l'utilisation par les macrophages des modes migratoires amiboïde et mésenchymal lors de leur infiltration dans un modèle de tumeur en trois-dimensions, les sphéroïdes tumoraux et 3) montrer que le remodelage de la matrice par les macrophages, lors de leur migration, joue un rôle prépondérant dans l'invasion tumorale.Tissue infiltration of macrophages is an aggravating factor in many diseases such as chronic inflammation and cancer. Macrophages that infiltrate tumors are called tumor-associated macrophages (TAMs). They promote tumor growth, angiogenesis, invasion and metastasis. Thus, inhibition of macrophage infiltration has become a therapeutic goal. Recently, the team demonstrated that macrophages use the amoeboid (depending on ROCK) or the mesenchymal (depending on proteases) migratory mode according to the extracellular matrix (ECM) architecture in three dimensions (3D). In addition, the study of the mesenchymal migration mode showed that it is dependent on Hck (a phagocyte-specific tyrosine kinase) and its ability to reorganize podosomes (ECM-degrading actin-rich structures) into rosettes. My thesis project was organized around two axes 1) the identification of substrates of Hck and the characterization of their role in the organization of podosomes and 3D migration of macrophages, and 2) the study of the 3D migration mechanisms of primary human monocytes/ macrophages within an in vitro tumor model: tumor cell spheroids. By a proteomic approach, I have identified potential partners and substrates of Hck, including the protein Filamin A (FLNa), a protein interacting with the actin cytoskeleton and integrins. Using different tools (recombinant proteins, antibodies, shRNA ...) I showed that: 1) Hck phosphorylates FLNa in vitro, 2) FLNa is localized to podosomes and is necessary for their organization as rosettes under the control of Hck, 3) the podosomes of FLNa-deficient cells have a shorter life span, and 4) the expression of FLNa is required for mesenchymal migration, but not for amoeboid migration of macrophages in a 3D ECM. Thus, FLNa could be a substrate of Hck necessary for the formation and stabilization of podosomes and their organization as rosettes, and is required for the mesenchymal migration of macrophages. In parallel, I developed a model of tumor cell spheroids, which allowed me to show that the infiltration of monocytes or macrophages in this in vitro tissue model of tumor is dependent on ROCK and proteases, signature of the use of the two migration modes. Then, when spheroids were embedded into ECM, I demonstrated that the presence of macrophages infiltrated into the spheroids is necessary to trigger the invasiveness of tumor cells. Indeed, macrophages infiltrate first the surrounding ECM and tumor cells follow macrophages in the matrix outside of the spheroid. Hck-/- macrophages, that are defective in mesenchymal migration, are significantly less effective in promoting the invasion of tumor cells. These results indicate that the activity of migration and matrix remodeling exerted by macrophages is prominent in tumor invasion. These results have established the migratory mode of macrophages infiltrating an in vitro tumor model and a mechanism required for tumor invasiveness promoted by macrophages. Thus during my thesis, I characterized the molecular and cellular mechanisms of 3D migration of human macrophages. Indeed, I have been able to: 1) identify a protein necessary for the mesenchymal migration of macrophages, 2) highlight the use by macrophages of the amoeboid and mesenchymal migration modes during their infiltration into an in vitro tumor model in 3D and 3) show that the matrix remodeling activity of macrophages during their migration plays a critical role in tumor cell invasion

Microarrayed human bone marrow organoids for modeling blood stem cell dynamics

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D in vitro model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells. Accordingly, the mesenchymal compartment shows partial maintenance of its self-renewal and multilineage potential, while endothelial cells self-organize into an interconnected vessel-like network. Intriguingly, such an endothelial compartment enhances the recruitment of HSPCs in a chemokine ligand/receptor-dependent manner, reminiscent of HSPC homing behavior in vivo. Additionally, we also model LSC migration and nesting in BMOs, thus highlighting the potential of this system as a well accessible and scalable preclinical model for candidate drug screening and patient-specific assays

A StarDist Journey (dataset and exercises)

<p>A dataset (with images of nuclear stainings) with some step-by-step exercises to discover <a href="https://github.com/stardist/stardist">StarDist</a>, the usefulness of the tool and its limitations.</p&gt

A StarDist Journey (dataset and exercises)

<p>A dataset (with images of nuclear stainings) with some step-by-step exercises to discover <a href="https://github.com/stardist/stardist">StarDist</a>, the usefulness of the tool and its limitations.</p&gt

Downsizing FeNb11O29 anode material through ultrafast solid-state microwave-assisted synthesis for enhanced electrochemical performance

International audienceWadsley-Roth oxide FeNb11O29 powder samples are successfully prepared using a simple, cost-effective, and ultrafast microwave-assisted solid-state synthesis for the first time. While conventional solid-state route in furnace requires hours of high-temperature treatment, both monoclinic and orthorhombic polymorphs of FeNb11O29 were obtained in minutes under microwave heating. Combining such short heat treatment with submicrometric oxide precursors enables to limit particle growth during the synthesis. The electrochemical benchmark clearly shows that FeNb11O29 powder samples obtained rapidly from submicrometric oxide precursors exhibit enhanced cycling performance. For example, the monoclinic polymorph prepared in only 5 min offers a high capacity of 179 mAh g-1 (90 % retention) after 500 cycles at 2 A g-1, approximately 20 % more than with conventional synthesis protocol. Electrochemical analysis demonstrates that the extra capacity is gained at low voltage and is probably induced by an easier ionic diffusion occurring in smaller particles. This work confirms the interest of solid-state microwave heating to design of electrode materials with limited particle growth and better cycling performance

La migration des phagocytes

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Correlative multicolor 3D SIM and STORM microscopy

Within the last decade, super-resolution methods that surpass the diffraction limit of light microscopy have provided invaluable insight into a variety of biological questions. Each of these approaches has inherent advantages and limitations, such that their combination is a powerful means to transform them into versatile tools for the life sciences. Here, we report the development of a combined SIM and STORM setup that maintains the optimal resolution of both methods and which is coupled to image registration to localize biological structures in 3D using multicolor labeling. We utilized this workflow to determine the localization of Bld12p/CrSAS-6 in purified basal bodies of Chlamydomonas reinhardtii with utmost precision, demonstrating its usefulness for accurate molecular mapping in 3D. (C) 2014 Optical Society of Americ

Biotin-NeutrAvidin Mediated Immobilization of Polymer Micro- and Nanoparticles on T Lymphocytes

Cells are powerful carriers that can help to improve the delivery of nanomedicines. One approach to use cells as carriers is to immobilize the nanoparticulate cargo on the cell surface. While a plethora of chemical conjugation strategies are available to bind nanoparticles to cell surfaces, only relatively little is known about the effects of particle size and cell type on the surface immobilization of nanoparticles. This study investigates the biotin-NeutrAvidin mediated immobilization of model polymer nanoparticles with sizes ranging from 40 nm to 1 mu m on two different T cell lines, viz., human Jurkat cells as well as mouse SJL/PLP7 T cells, which are of potential interest for drug delivery across the blood-brain barrier. The nanoparticle cell surface immobilization and the particle surface concentration and distribution were analyzed by flow cytometry and confocal microscopy. The functional properties of nanoparticle-modified SJL/PLP7 T cells were assessed in an ICAM-1 binding assay as well as in a two-chamber setup in which the migration of the particle-modified T cells across an in vitro model of the blood-brain barrier was studied. The results of these experiments highlight the effects of particle size and cell line on the surface immobilization of nanoparticles on living cells

Cellular Uptake and Intracellular Trafficking of Poly(N-(2-Hydroxypropyl) Methacrylamide)

Cellular uptake and intracellular trafficking of polymer conjugates or polymer nanoparticles is typically monitored using fluorescence-based techniques such as confocal microscopy. While these methods have provided a wealth of insight into the internalization and trafficking of polymers and polymer nanoparticles, they require fluorescent labeling of the polymer or polymer nanoparticle. Because in biological media fluorescent dyes may degrade, be cleaved from the polymer or particle, or even change uptake and trafficking pathways, there is an interest in fluorescent label-free methods to study the interactions between cells and polymer nanomedicines. This article presents a first proof-of-concept that demonstrates the feasibility of NanoSIMS to monitor the intracellular localization of polymer conjugates. For the experiments reported here, poly(N-(2-hydroxypropyl) methacrylamide)) (PHPMA) was selected as a prototypical polymer–drug conjugate. This PHPMA polymer contained a 19F-label at the α-terminus, which was introduced in order to allow NanoSIMS analysis. Prior to the NanoSIMS experiments, the uptake and intracellular trafficking of the polymer was established using confocal microscopy and flow cytometry. These experiments not only provided detailed insight into the kinetics of these processes but were also important to select time points for the NanoSIMS analysis. For the NanoSIMS experiments, HeLa cells were investigated that had been exposed to the PHPMA polymer for a period of 4 or 15 h, which was known to lead to predominant lysosomal accumulation of the polymer. NanoSIMS analysis of resin-embedded and microtomed samples of the cells revealed a punctuated fluorine signal, which was found to colocalize with the sulfur signal that was attributed to the lysosomal compartments. The localization of the polymer in the endolysosomal compartments was confirmed by TEM analysis on the same cell samples. The results of this study illustrate the potential of NanoSIMS to study the uptake and intracellular trafficking of polymer nanomedicines

DeconvolutionLab2: An open-source software for deconvolution microscopy

International audienceImages in fluorescence microscopy are inherently blurred due to the limit of diffraction of light. The purpose of deconvolution microscopy is to compensate numerically for this degradation. Deconvolution is widely used to restore fine details of 3D biological samples. Unfortunately, dealing with deconvolution tools is not straightforward. Among others, end users have to select the appropriate algorithm, calibration and parametrization, while potentially facing demanding computational tasks. To make deconvolution more accessible, we have developed a practical platform for deconvolution microscopy called DeconvolutionLab. Freely distributed, DeconvolutionLab hosts standard algorithms for 3D micro-scopy deconvolution and drives them through a user-oriented interface. In this paper, we take advantage of the release of DeconvolutionLab2 to provide a complete description of the software package and its built-in deconvolution algorithms. We examine several standard algorithms used in deconvolution microscopy, notably: Regularized inverse filter, Tikhonov regularization, Landweber, Tikhonov–Miller, Richardson–Lucy, and fast iterative shrinkage-thresholding. We evaluate these methods over large 3D microscopy images using simulated datasets and real experimental images. We distinguish the algorithms in terms of image quality, performance, usability and computational requirements. Our presentation is completed with a discussion of recent trends in deconvolution, inspired by the results of the Grand Challenge on deconvolution microscopy that was recently organized