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18336
21/09/2018

Engineered micro-devices for the isolation of circulating tumor cells in clinical routine

A.K.JIMENEZ ZENTENO

ELIA

Doctorat : 21 Septembre 2018, 190p., Président: J.GRISOLIA, Rapporteurs: E.DELAMARCHE, J.R.GREER, Examinateurs: C.PICART, S.VERBRIDGE, U.DEMIRCI, Directeurs de thèse: C.VIEU, A.CERF , N° 18336

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Résumé

Les cellules tumorales circulantes (CTCs) sont la principale voie de dissémination du cancer dans le corps humain au travers de la circulation sanguine. Ces cellules ont la capacité de se détacher de la tumeur primaire, de rejoindre la circulation sanguine et de survivre dans cet environnement. Une sous-population spécifique de ces cellules a la capacité de coloniser de nouveaux tissus et de former des métastases. L'importance de ces cellules rares dans la circulation sanguine a été intensément étudiée au cours des dernières décennies, et il a été constaté que les informations phénotypiques et génomiques qu'elles contiennent pourraient être corrélées avec celles obtenues à partir d'une biopsie tissulaire. De plus, le nombre et l'incidence des CTC chez les patients métastatiques pourraient être utilisés comme indicateurs pronostics. Ainsi, leur isolement à partir d'échantillons sanguins et leur analyse a été proposé en remplacement des biopsies conventionnelles, comme une alternative moins invasive et permettant un échantillonnage plus répété. In fine, la détection et l'analyse des CTC en routine clinique pourraient être utilisées pour le suivi en temps réel des thérapies et de leur efficacité pour améliorer la prise en charge des patients, un pas de plus vers une médecine de précision. Dans ce projet de thèse, nous avons développé de nouveaux micro-dispositifs pour la capture, sous flux, de cellules cancéreuses à partir de sang complet humain. Nous avons exploité les propriétés physiques des CTC, plus grandes et moins déformables que les cellules sanguines normales, pour discriminer ces cellules rares (<1 cellule par mL aux premiers stades de la maladie). Des micro-dispositifs ont été conçus tels des tamis à trois dimensions pour filtrer sélectivement les cellules cancéreuses tout en préservant l'intégrité et la viabilité des cellules. De plus, les dispositifs ont été conçus pour permettre l'accès au matériel biologique isolé et effectuer ainsi une identification des cellules in situ, e.g. par immunocytochimie, mais aussi potentiellement pour servir de plateforme pour une analyse fonctionnelle de ces cellules. Nous avons proposé deux approches totalement compatibles avec la routine clinique. La première consiste en un guide équipé de microdispositifs, conçu pour être introduit directement dans la circulation sanguine au travers d'un cathéter médical et effectuer la capture des cellules cancéreuses in vivo. La deuxième approche vise à réaliser l'isolement des CTCs en utilisant des microdispositifs intégrés à des plateformes ex vivo compatibles avec les consommables médicaux de prélèvement sanguin. Les deux développements technologiques ont été validés en utilisant des cellules issues de lignée cancéreuse en suspension dans un milieu de culture cellulaire ou dans du sang complet Notre approche in vivo a été optimisée sur la base d'observations réalisées à l'aide d'un banc fluidique mimant une veine artificielle et a été testée avec succès in vivo dans un modèle animal. Ce prototype a démontré sa robustesse et sa capacité à capturer des cellules cancéreuses à des concentrations proches des concentrations en situation métastatique. Une évaluation de la sensibilité a été réalisée de la même manière pour l'approche ex vivo, démontrant des performances de capture analogues. Nous croyons que ces technologies pourraient permettre des prélèvements de CTC de manière répétée et fiable pour le pronostic et le suivi thérapeutique des patients métastatiques.

Abstract

Circulating tumor cells (CTCs) are believed to represent the main pathway of cancer dissemination in the human body through the circulatory system. These cells have the ability to detach from the primary tumor, enter into the bloodstream, and survive in this environment. A specific subpopulation of these cells possesses the capacity of colonizing new tissues and forming metastases. The relevance of these rare cells in the bloodstream has been intensively investigated during the last decades, finding that phenotypic and genomic information they carry could be correlated with that of solid biopsies. Moreover, the number and incidence of CTCs in metastatic patients could be used as an indicator for prognosis. Thus, their isolation from blood samples and analysis has been proposed as a surrogate to solid biopsies, having the added value of being a less invasive procedure and allow a more repeated measure. In fine, the routine analysis of CTCs in clinical practice could be used for the real-time monitoring of therapies and the adaptation of treatment in order to improve the outcome of patients, a step forward towards so-called precision medicine. In this PhD project, we have developed novel micro-devices for the capture, in flow conditions, of tumor-derived cells from human whole blood. CTCs being larger and less deformable than normal blood cells, we exploited theses physical traits to discriminate them. Sieve-like micro-devices were engineered to selectively sort out tumor-derived cells having as a priority the preservation of cell integrity and viability. In addition, devices were designed to allow direct access to the isolated biological material and thus perform in situ cell identification, such as immunocytochemistry, but also to potentially serve as a platform for functional analysis. We proposed two approaches compatible with clinical routine. The first approach consists in a customized guiding-strip equipped with integrated microfilters, designed to be introduced directly within the bloodstream through a conventional medical catheter to perform the capture of tumor-derived cells in vivo. The second approach aims to perform CTC isolation ex vivo through the integration of microfilters into a platform compatible with blood collection medical sets. Both technological developments were validated using a cancerous cell line suspended in either cell culture medium or whole blood. Our in vivo approach was optimized using a customized fluidic bench mimicking an artificial human vein and was tested successfully in an animal model. This prototype demonstrated its robustness and capability to capture tumor-derived cells in concentrations within the range found in metastatic patients. This sensitivity appraisal was carried out for the ex vivo approach, demonstrating analogous capture performances. We believe that these technologies could enable repeated and reliable CTC detection for prognosis and monitoring of treatment efficiency in metastatic patients.

Mots-Clés / Keywords
Liquid-biopsy; CTCs; Cancer-monitoring; Microfabrication; Microfiltration; 3D-microdevices; Biopsie liquide; Surveillance du cancer; Microdispositifs 3D;

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18283
01/08/2018

Direct laser fabrication of meso-scale 2D and 3D architectures with micrometric feature resolution

A.ACCARDO, R.COURSON, R.RIESCO ALVAREZ, V.RAIMBAULT, L.MALAQUIN

ELIA, TEAM, MEMS, MICA

Revue Scientifique : Additive Manufacturing, Vol.22, pp.440-446, Août 2018 , N° 18283

Lien : https://hal.laas.fr/hal-01875364

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Abstract

The realization of 2D and 3D meso-scale architectures is an area of research involving a wide range of disciplines ranging from materials science, microelectronics, phononics, microfluidics to biomedicine requiring millimeter to centimeter-sized objects embedding micrometric features. In the recent years, several technologies have been employed to provide optimal features in terms of object size flexibility, printing resolution, large materials library and fabrication speed. In this work, we report a fully customizable single-photon absorption 3D fabrication methodology based on direct laser fabrication. To validate this approach and highlight the versatility of the setup, we have fabricated a comprehensive ensemble of 2D and 3D designs with potential applications in biomimetics, 3D scaffolding and microfluidics. The high degree of tunability of the reported fabrication system allows tailoring the laser power, slicing and fabrication speed for each single area of the design. These unique features enable a rapid prototyping of millimeter to centimeter-sized objects involving 3D architectures with true freestanding subunits and micrometric feature reproducibility. The presented strategy fills indeed the current technological gap related to the development of meso-scale architectures required in multidisciplinary fields of research.

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18347
01/06/2018

The Role of Glycans in Bacterial Adhesion to Mucosal Surfaces: How Can Single-Molecule Techniques Advance Our Understanding?

C.FORMOSA, M.CASTELAIN, H.MARTIN YKEN, K.DUNKER, E.DAGUE, M.SLETMOEN

LISBP, NTNU, Trondheim, ELIA

Revue Scientifique : Microorganisms, Vol.6, N°2, Juin 2018 , N° 18347

Lien : https://hal.laas.fr/hal-01907666

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Abstract

Bacterial adhesion is currently the subject of increased interest from the research community, leading to fast progress in our understanding of this complex phenomenon. Resent research within this field has documented the important roles played by glycans for bacterial surface adhesion, either through interaction with lectins or with other glycans. In parallel with this increased interest for and understanding of bacterial adhesion, there has been a growth in the sophistication and use of sensitive force probes for single-molecule and single cell studies. In this review, we highlight how the sensitive force probes atomic force microscopy (AFM) and optical tweezers (OT) have contributed to clarifying the mechanisms underlying bacterial adhesion to glycosylated surfaces in general and mucosal surfaces in particular. We also describe research areas where these techniques have not yet been applied, but where their capabilities appear appropriate to advance our understanding.

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18179
23/05/2018

Simple Synthetic Molecular Hydrogels from Self- Assembling Alkylgalactonamides as Scaffold for 3D Neuronal Cell Growth

A.CHALARD, L.VAYSSE, P.JOSEPH, L.MALAQUIN, S.ASSIE-SOULEILLE, B.LONETTI, J.C.SOL, I.LOUBINOUX, J.FITREMANN

MILE, INSERM, ELIA, I2C, IMRCP

Revue Scientifique : ACS applied materials & interfaces, Vol.10, N°20, pp.17004-17017, Mai 2018 , N° 18179

Lien : https://hal.laas.fr/hal-01810656

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Abstract

In this work, we demonstrated that the hydrogel obtained from a very simple and single synthetic molecule, N-heptyl-galactonamide was a suitable scaffold for the growth of neuronal cells in 3D. We evidenced by confocal microscopy the presence of the cells into the gel up to a depth of around 200 µm, demonstrating that the latter was permissive to cell growth and enabled a true 3D colonization and organization. It also supported successfully the differentiation of adult human neuronal stem cells (hNSCs) into both glial and neuronal cells and the development of a really dense neurofilament network. So the gel appears to be a good candidate for neural tissue regeneration. In contrast with other molecular gels described for cell culture, the molecule can be obtained at the gram scale by a one-step reaction. The resulting gel is very soft, a quality in accordance with the aim of growing neuronal cells, that requires low modulus substrates similar to the brain. But because of its fragility, specific procedures had to be implemented for its preparation and for cell labeling and confocal microscopy observations. Notably, the implementation of a controlled slow cooling of the gel solution was needed to get a very soft but nevertheless cohesive gel. In these conditions, very wide straight and long micrometric fibers were formed, held together by a second network of flexible narrower nanometric fibers. The two kinds of fibers guided the neurite and glial cell growth in a different way. We also underlined the importance of a tiny difference in the molecular structure on the gel performances: parent molecules, differing by a one-carbon increment in the alkyl chain length, N-hexyl-galactonamide and N-octyl-galactonamide, were not as good as N-heptyl-galactonamide. Their differences were analysed in terms of gel fibers morphology, mechanical properties, solubility, chain parity and cell growth.

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18104
13/03/2018

Bone degradation machinery of osteoclasts: An HIV-1 target that contributes to bone loss

B.RAYNAUD-MESSINA, L.BRACQ, M.DUPONT, S.SOURIANT, S.M.USMANI, A.PROAG, K.PINGRIS, V.SOLDAN, C.THIBAULT, F.CAPILLA, T.AL SAATI, I.GENNERO, P.JURDIC, P.JOLICOEUR, J.L.DAVIGNON, T.R.MEMPEL, S.BENICHOU, I.MARIDONNEAU-PARINI, C.VEROLLET

IPBS, INSERM, Paris, Harvard Medical Sch, Multiscale Electron, ELIA, INSERM, Centre de Physiopathologie, ENS Lyon, IRCM, Montreal

Revue Scientifique : Proceedings of the National Academy of Sciences, Vol.115, N°11, pp.E2556-E2565, Mars 2018 , N° 18104

Lien : https://hal.laas.fr/hal-01764831

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Abstract

Bone deficits are frequent in HIV-1–infected patients. We report here that osteoclasts, the cells specialized in bone resorption, are infected by HIV-1 in vivo in humanized mice and ex vivo in human joint biopsies. In vitro, infection of human osteoclasts occurs at different stages of osteoclastogenesis via cell-free viruses and, more efficiently, by transfer from infected T cells. HIV-1 infection markedly enhances adhesion and osteolytic activity of human osteoclasts by modifying the structure and function of the sealing zone, the osteoclast-specific bone degradation machinery. Indeed, the sealing zone is broader due to F-actin enrichment of its basal units (i.e., the podosomes). The viral protein Nef is involved in all HIV-1–induced effects partly through the activation of Src, a regulator of podosomes and of their assembly as a sealing zone. Supporting these results, Nef-transgenic mice exhibit an increased osteoclast density and bone defects, and osteoclasts derived from these animals display high osteolytic activity. Altogether, our study evidences osteoclasts as host cells for HIV-1 and their pathological contribution to bone disorders induced by this virus, in part via Nef.

143360
18022
27/02/2018

Two-photon lithography and microscopy of 3D hydrogel scaffolds for neuronal cell growth

A.ACCARDO, MC.BLATCHE, R.COURSON, I.LOUBINOUX, C.VIEU, L.MALAQUIN

ELIA, I2C, TEAM, INSERM

Revue Scientifique : Biomedical Physics & Engineering Express, Vol.4, N°2, 027009p., Février 2018 , N° 18022

Lien : https://hal.laas.fr/hal-01698486

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Abstract

3D fabrication techniques are rapidly expanding in the field of scaffold development for cell culture and tissue engineering. Herein we report the realization of free-standing PEGDA hydrogel architectures by using two-photon lithography. The morphological and immunofluorescence characterization of neuro2A cells revealed a tridimensional colonization featuring multiple neuritic extensions per cell as well as the expression of β-tubulin neuronal marker and actin microfilaments. The results open new perspectives in the continuous quest for structured biomaterials able to provide a favorable environment to cells and at the same time not interfering with imaging protocols necessary for a clear scenario of the cell seeding.

142468
18379
31/01/2018

Microenvironnements pour l’analyse biologique et l’ingéniérie des tissus

L.MALAQUIN

ELIA

Habilitation à diriger des recherches : 31 Janvier 2018, 149p., Président:, Rapporteurs: M.C.JULLIEN, S.LEGAC, C.NIZAK, Examinateurs: O.FRANCAIS, L.CASTEILLA, Directeur: G.LANDA, Membre invité: C.VIEU , N° 18379

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Résumé

Au sein des tissus vivants, les cellules forment une communauté organisée et en interaction dans d’une matrice extra cellulaire. De ce microenvironnement cellulaire, naissent les mécanismes responsables de l’homéostasie ou de la dégénérescence des tissus qui sont étroitement liés à la topologie ainsi qu’à la distribution spatiale des propriétés physico-chimiques et biologiques. Ainsi, la fabrication de modèles mimétiques permettant de reproduire, en trois dimensions, les paramètres physiques et chimiques essentiels du microenvironnement, est devenu un enjeu majeur en biologie et en médecine. Dans cet objectif, les technologies microfluidiques et de bioimpression apparaissent comme des outils particulièrement performants pour créer des modèles de microenvironnements propices à la manipulation de liquides biologiques, à leur traitement à des fins d’analyse ou d’étude de la prolifération cellulaire. Cette soutenance sera dédiée, dans un premier temps, à une présentation des projets de recherches menés au sein de l’institut Curie puis au sein du LAAS CNRS autour du développement de de concepts microfluidiques pour la réalisation de dispositifs miniaturisés d’analyse (pré-concentration, capture et analyse de biomarqueurs et de cellules) ou de diagnostic cellulaire (capture de cellules tumorales circulantes, analyse génétique). Dans un deuxième temps seront présentées les évolutions de ces activités de recherche vers le développement de modèles standardisés de microenvironnements cellulaires sur la base de technologies d’impression 3D et de bioimpression.

145273
18008
29/01/2018

On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation

B.VENZAC, M.DIAKITE, D.HERTHNEK, I.CISSE, U.BOCKELMANN, S.DESCROIX, L.MALAQUIN, J.L.VIOVY

UPMC, Univ of Stockholm, ESPCI, ELIA

Revue Scientifique : The Analyst, Vol.143, N°1, pp.190-199, Janvier 2018 , N° 18008

Lien : http://hal.upmc.fr/hal-01679664

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Abstract

Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.

142183
18416
01/01/2018

Nanoscale structural mapping as a measure of maturation in the murine frontal cortex

G.SMOLYAKOV, E.DAGUE, C.ROUX, M.H.SEGUELAS, C.GALES, J.M.SENARD, D.ARVANITIS

ELIA, IMRCP, I2MC, INSERM

Revue Scientifique : Brain Structure and Function, Vol.223, N°1, pp.255-265, Janvier 2018 , N° 18416

Lien : https://hal.laas.fr/hal-01907682

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Abstract

Atomic force microscopy (AFM) is emerging as an innovative tool to phenotype the brain. This study demonstrates the utility of AFM to determine nanome-chanical and nanostructural features of the murine dorso-lateral frontal cortex from weaning to adulthood. We found an increase in tissue stiffness of the primary somatosensory cortex with age, along with an increased cortical mechanical heterogeneity. To characterize the features potentially responsible for this heterogeneity, we applied AFM scan mode to directly image the topography of thin sections of the primary somatosensory cortical layers II/III, IV and V/VI. Topographical mapping of the cortical layers at successive ages showed progressive smoothing of the surface. Topographical images were also compared with his-tochemically derived morphological information, which demonstrated the deposition of perineuronal nets, important extracellular components and markers of maturity. Our work demonstrates that high-resolution AFM images can be used to determine the nanostructural properties of cor-tical maturation, well beyond embryonic and postnatal development. Furthermore, it may offer a new method for brain phenotyping and screening to uncover topographical changes in early stages of neurodegenerative diseases.

145547
17437
01/01/2018

Cell biology of microbes and pharmacology of antimicrobial drugs explored by Atomic Force Microscopy

C.FORMOSA, R.DUVAL, E.DAGUE

SRSMC, ELIA

Revue Scientifique : Seminars in Cell & Developmental Biology, Vol.73, pp.165-176, Janvier 2018 , N° 17437

Lien : https://hal.laas.fr/hal-01907671

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Abstract

Antimicrobial molecules have been used for more than 50 years now and are the basis of modern medicine. No surgery can nowdays be imagined to be performed without antibiotics; dreadful diseases like tuberculosis, leprosis, siphilys, and more broadly all microbial induced diseases, can be cured only through the use of antimicrobial treatments. However, the situation is becoming more and more complex because of the ability of microbes to adapt, develop, acquire, and share mechanisms of resistance to antimicrobial agents. We choose to introduce this review by briefly drawing the panorama of antimicrobial discovery and development, but also of the emergence of microbial resistance. Then we describe how Atomic Force Microscopy (AFM) can be used to provide a better understanding of the mechanisms of action of these drugs at the nanoscale level on microbial interfaces. In this section, we will address these questions: (1) how does drug treatment affect the morphology of single microbes?; (2) do antimicrobial molecules modify the nanomechanical properties of microbes, or do the nanomechanical properties of microbes play a role in antimicrobial activity and efficiency?; and (3) how are the adhesive abilitites of microbes affected by antimicrobial drugs treatment? Finally, in a second part of this review we focus on recent studies aimed at changing the paradigm of the single molecule/cell technology that AFM typically represents. Recent work dealing with the creation of a microbe array which can be explored by AFM will be presented, as these developments constitute the first steps toward transforming AFM into a higher throughput technology. We also discuss papers using AFM as NanoMechnanicalSensors (NEMS), and demonstrate the interest of such approaches in clinical microbiology to detect quickly and with high accuracy microbial resistance.

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