Дисертації з теми "Cancer-On-Chip"

De nombreux gènes suppresseurs de tumeurs sont inactivés par hyperméthylation dans les cancers. Cette inactivation serait en partie initiée par la protéine, MBD2 (Methyl-CpG-Binding Domain protein 2). Cette protéine recrute au niveau de séquences méthylées des complexes enzymatiques capables de modifier la structure chromatinienne et crée ainsi des régions fonctionnellement inactives. Dès lors, ce répresseur apparaît être une cible potentielle pour combattre le cancer. Dans cette perspective, rechercher les cibles de MBD2 et comprendre sa capacité à contrôler l'expression génique semblent cruciales. Au cours de deux études gènes candidats, nous avons pu démontrer (i) une réelle spécificité de cible du répresseur méthylationdépendant MBD2 pour les loci hTERT et pS2/TFF1 ; et (ii) un nouveau rôle de la protéine MBD2 en tant que modulateur de l'expression génique. De plus, les actions antagonistes entre le répresseur MBD2 et le trans-activateur naturel du gène pS2, le récepteur aux oestrogènes α, ont été explorées. Puis, l'analyse globale des profils de distribution de MBD2, de la méthylation de l'ADN, ainsi que de l'ARN polymérase II, sur puce promoteur a montré que MBD2 possède toutes les caractéristiques d'un répresseur trancriptionnel méthylation-dépendant. En effet, 74% des promoteurs fixés par MBD2 sont méthylés et cette liaison est associée dans 65% des cas à une répression transcriptionnelle.

Pour croître et proliférer, les cellules cancéreuses de la prostate activent des voies de signalisation dépendantes des androgènes. L'intervention thérapeutique en première ligne du cancer de la prostate (CaP) s'appuie donc d’abord sur le blocage de l'axe androgènes-récepteur aux androgènes (RA) mais rapidement, les patients développent des tumeurs résistantes (CRPC, Castration Resistant Prostate Cancer).Les récepteurs à activité tyrosine kinase de la famille ErbB semblent jouer un rôle dans cette résistance, en particulier le récepteur ErbB3. En effet, l'inactivation des voies en aval d'ErbB1 et ErbB2, en association avec les anti-androgéniques n'empêche pas la progression vers l'hormono-indépendance, et une accumulation nucléaire d'ErbB3 est observée dans les CRPC en même temps que la voie PI3K-Akt est réactivée.Dans ce contexte, nous avons validé l'expression d'une isoforme nucléaire ErbB380kDa chez les patients et dans des lignées hormono-sensible (LNCaP) et hormono-résistante (PC3). Par ChIP-on-chip, nous avons isolé 353 promoteurs cibles communs aux deux lignées, 245 spécifiques à la lignée LNCaP et 925 à la lignée PC3, et montré qu'ErbB380kDa est un co-régulateur transcriptionnel des gènes étudiés, parmi lesquels GATA2. L'analyse in silico de ces promoteurs révèle des sites de liaison pour les facteurs de transcription GATA2 et MZF1 au niveau des régions liant ErbB380kDa. Un complexe nucléaire GATA2-MZF1-ErbB380kDa est retrouvé dans les cellules LNCaP et PC3.Des travaux récents montrent que GATA2 s'associe au RA pour réguler l'expression de gènes et qu'il pourrait être participer à la dissémination métastatique dans le CaP.Nos résultats suggèrent qu'ErbB380kDa pourrait jouer un rôle régulateur, en amont de GATA2, dans les processus de résistance et l'apparition de métastases. Cette isoforme nucléaire insensible aux traitements actuels apparaît donc comme une cible privilégiée pour le ciblage thérapeutique
Prostate cancer (PCa) is dependent on androgens and functional androgen-receptor (AR) for growth and proliferation. Androgen-directed therapy is used at the first stages of the disease but cancer cells frequently become resistant (CRPC) by inappropriate reactivation of AR activity. As ErbB receptors are expressed in PCa cells, therapies aiming at inactivate the pathways downstream have been tested in advanced prostate cancers alongside hormone-based therapy. Still, a significant proportion of CRPC treated by ErbB1/2 inhibitors resist to treatment. ErbB3 could be responsible for this failure through both its unexpected nuclear localization and the reactivation of the PI3K-Akt pathway in those advanced tumors.We have described a nuclear ErbB380kDa isoform, expressed in hormone-sensitive (LNCaP) and hormone-resistant (PC3) PCa cell lines that accumulates in the nucleus of tumor cells during cancer progression. ChIP-on-chip experiments led us to characterize 353 target promoters binding ErbB380kDa in both cell lines; 245 promoters specific to LNCaP and 925 specific to PC3 cells, among which the promoter of GATA2. We show that ErbB380kDa functions as a transcriptional co-regulator for the studied genes, potentially through its interaction with transcription factors. In silico analysis revealed binding sites for GATA2 and MZF1 transcription factors on the target promoters, and a complex GATA2-MZF1-ErbB380kDa has been found in LNCaP and PC3 cells. Recent publications have reported a role for GATA2 in the regulation of RA responsive-genes and in metastatic spreading. We propose that ErbB380kDa could act, upstream of GATA2, to induce resistance mechanisms and facilitate cancer progression. Thus, ErbB380kDa emerges as a putative target for the development of new therapies in prostate cancer

This research utilizes Electrical Impedance Spectroscopy, a technique classically used for electrochemical analysis and material characterization, as the basis for a non-destructive, label-free assay platform for three dimensional (3D) cellular spheroids. In this work, a linear array of microelectrodes is optimized to rapidly respond to changes located within a 3D multicellular model. In addition, this technique is coupled with an on chip micro-pH sensor for monitoring the environment around the cells. Finally, the responses of both impedance and pH are correlated with physical changes within the cellular model. The impedance analysis system realized through this work provides a foundation for the development of high-throughput drug screening systems that utilize multiple parallel sensing modalities including pH and impedance sensing in order to quickly assess the efficacy of specific drug candidates. The slow development of new drugs is mainly attributed to poor predictability of current chemosensitivity and resistivity assays, as well as genetic differences between the animal models used for tests and humans. In addition, monolayer cultures used in early experimentation are fundamentally different from the complex structure of organs in vivo. This requires the study of smaller 3D models (spheroids) that more efficiently replicate the conditions within the body. The main objective of this research was to develop a microfluidic system on a chip that is capable of deducing viability and morphology of 3D tumor spheroids by monitoring both the impedance of the cellular model and the pH of their local environment. This would provide a fast and reliable method for screening pharmaceutical compounds in a high-throughput system.

A micro-electrophysiological analysis system (-EPAS) using various microfabrication techniques for single cell study was developed. Conventional microfabrication techniques combined with plastic and polymer microfabrication techniques have been used to realize the system. The system is capable of performing patch clamp recording and whole cell electrical impedance spectroscopy (EIS) on a single cell. Methodologies for single cell manipulation were developed. The ion channel activities of primary cultured bovine chromaffin cells were measured in both the patch clamping mode and the whole cell EIS mode. Membrane capacitance of the chromaffin cell was calculated from these measurements. Increases in the capacitances were observed when certain ion channels were blocked using toxins. The dielectric properties of human breast cancer cell lines from different pathological stages were measured and compared to a normal human breast cell line in the whole cell EIS mode. The measured properties were correlated to the pathological stages of the breast cancer cell lines. Decreases in the membrane capacitances were observed for the more pathologically progressed cancer cell lines.

Le cancer du poumon non à petites cellules (CPNPC) est l'une des rares maladies tumorales, avec mélanome et carcinome vésical, pour lesquelles les médicaments immuno-oncologiques ont conduit à une révolution thérapeutique. Seuls 20 à 30% des patients atteints de CPNPC bénéficient de la monothérapie avec inhibiteurs des points de contrôle immunitaires (ICP) avec des réponses durables, tandis que les combinaisons ont conduit à réponse longue dans jusqu'à 40% des patients. Notre étude vise à mieux caractériser la modulation du microenvironnement tumoral lors d'un traitement ICP, plus ou moins une chimiothérapie concomitante, afin de guider des stratégies d'immunothérapie plus convaincantes. Inspiré par la technologie d'organes sur puce, nous avons reconstitué ex vivo un microenvironnement de tumeur pulmonaire immunocompétent simplifié en réalisant des co-cultures 3D dans des dispositifs microfluidiques. Cette approche nous a permis de réaliser une imagerie en direct et une quantification des effets de l'ICP sur l'écosystème tumoral. L’architecture de la puce se compose de trois micro-chambres parallèles, séparées par des micro-piliers qui permettent le confinement d'un hydrogel biomimétique dans le canal central par capillarité. En co-cultivant des cellules CPNPC et des lymphocytes T cytotoxiques autologues (récoltés à partir des TIL du même patient et amplifiés ultérieurement in vitro), nous pourrions récapituler, visualiser et quantifier une activité cytotoxique efficace et spécifique des cellules T contre les cellules cancéreuses autologues. Pour cela, nous avons développé un nouvel algorithme qui pourrait localiser les cellules cancéreuses et, grâce à un rapporteur fluorescent de l'activité caspase, mesurer leur mort d'une manière spécifique au temps et à l'espace. Dans ces co-cultures 3D, l'activité cytotoxique des cellules T a été renforcée par le traitement avec l'inhibiteur PD-1 et l'inhibiteur PD-L1, reconstituant ainsi sur puce une réponse ICI. De plus, cette méthode nous a permis d'extraire un paramètre, le potentiel d'induction de la mort, qui estime mathématiquement la «contagiosité de la mort» en calculant la proximité dans l'espace et le temps des signaux de mort. Fait intéressant, cette analyse nous a révélé que la mort des cellules cancéreuses causée par la chimiothérapie ou par les cellules T cytotoxiques est contagieuse, alors que dans les conditions témoins, la mort des cellules cancéreuses est stochastique. Cette observation peut avoir des implications biologiques et cliniques, par exemple en ce qui concerne « l'effet spectateur », observé après un traitement de radiothérapie. De plus, afin d'avoir un aperçu moléculaire de l'impact de la co-culture sur les cellules T, en présence ou en l'absence d'ICI, nous avons analysé par cytométrie de flux l'expression de plusieurs marqueurs de cellules T. Après 3 jours de co-culture sur puce, les lymphocytes T ont montré une expression accrue des marqueurs d'activation, tels que CD69 et CD25, ainsi qu'une augmentation de l’expression des marqueurs d'épuisement, notamment PD-1, TIGIT, TIM-3, LAG- 3, CD137 et OX-40. Le couplage de l'analyse d'image et de l'étude de la plasticité des lymphocytes T, nous a permis d'associer pour la première fois l'activité cytotoxique finement quantifiée des lymphocytes T avec leur statut d'activation / épuisement et de décrire un phénotype réactif aux immunothérapies. Dans cette thèse, nous avons démontré que la tumeur-sur-puce peut être exploité pour évaluer l'efficacité des inhibiteurs de points de contrôle immunitaires, potentiellement pour déterminer l'effet de médicaments combinés et enfin pour étudier les mécanismes de résistance primaire des cellules cancéreuses
Non-small cell lung cancer (NSCLC) is one of the few tumor diseases, with melanoma and vesical carcinoma, for which immuno-oncology drugs led to a therapeutic revolution. Only 20 to 30% of the NSCLC patients benefit from immune checkpoint inhibitors (ICI) monotherapy with durable responses, while combinations led up to 40% of long responder patients. Our study aims to better characterize the modulation of the tumor microenvironment upon ICI treatment, plus or minus concurrent chemotherapy, in order to guide more compelling immunotherapy strategies. Inspired by the organ-on-a-chip technology, we implemented the reconstitution ex vivo of a simplified immunocompetent lung tumor microenvironment by performing 3D co-cultures in microfluidic devices. This approach allowed us to perform live-imaging and quantification of the effects of ICI on the tumor ecosystem.The design of the chip consists of three parallel micro-chambers, separated by micro-pillars that allow the confinement of a biomimetic hydrogel in the central channel by capillarity. By co-culturing autologous NSCLC cells and cytotoxic T lymphocytes (harvested from the TILs of the same patient and furtherly amplified in vitro) we could recapitulate, visualize and quantify an efficient and specific cytotoxic activity of the T cells against the autologous cancer cells. For this purpose, we developed a novel algorithm that could localize the cancer cells and, thanks to a fluorescent reporter of the caspase activity, measure their death in a time- and space-specific manner. In these 3D co-cultures the cytotoxic activity of T cells was enhanced by the treatment with PD-1 inhibitor and PD-L1 inhibitor, therefore reconstituting on-chip an ICI response. Furthermore, this method allowed us to extract a parameter, the potential of death induction, which mathematically estimates the “contagiousness of death” by computing the proximity in space and time of death signals. Interestingly, this analysis revealed us that the death of cancer cells caused by either chemotherapy or cytotoxic T cells is contagious, whereas in control conditions the cancer cells death is stochastic. This observation may have biological and clinical implications, for instance regarding the bystander effect, observed after radiotherapy treatment. Furthermore, in order to have a molecular insight on the impact of the co-culture on T cells, in presence or absence of ICI, we analyzed by flow cytometry the expression of several T cell markers. After 3 days of co-culture on chip, the T cells showed an increased expression of activation markers, such as CD69 and CD25, as well as an increased expression of exhaustion markers, notably PD-1, TIGIT, TIM-3, LAG-3, CD137 and OX-40. The coupling of image analysis and the study of T cell plasticity, allowed us to associate for the first time the finely quantified cytotoxic activity of the T cells and their activation/exhaustion status and describe a responsive phenotype to immunotherapies. In this thesis, we demonstrated that the tumor-on-chip is suitable to evaluate the efficacy of immune checkpoint inhibitors, to potentially assess the effect of combined drugs and to study the mechanisms of cancer cell primary resistance

Applications for lab-on-a-chip devices have been expanding rapidly in the last decade due to their lower required volume of sample, faster experiments, smaller tools, more control, and ease of parallelization compared to their macroscale counterparts. Moreover, lab-on-a-chip devices provide important capabilities, including isolating rare cells from body fluids, such as isolating circulating tumor cells from blood or peritoneal fluid, which are not feasible or at least extremely difficult with macroscale devices. Particles experience different forces (and/or torques) when they are suspended in a fluid in a microdevice. A dominant force is the drag force on the particle as it flows through the fluid.  External forces such as dielectrophoresis, the motion of a particle due to its polarization in the presence of a non-uniform electric field, may also be applied. For instance, well-specified mixing or separation of particles can be achieved by using the combination of drag and dielectrophoretic forces. Two major mechanisms for manipulating particles in a microdevice include control of forces applied to the particles, such as those due to electric and velocity fields, and the geometry of the device that affects the nature of these fields. The coupling between the geometry of the microdevices and applied fields makes the prediction of associated forces inside the microdevice challenging and increasingly difficult when the applied field is time-dependent. Understanding the interaction of external forces and particles and fluid is critical for engineering novel microsystems. Determining this interaction is even more complicated when dealing with bioparticles, especially cells, due to their complex intrinsic biological properties which influence their electrical and mechanical properties. Particles with non-spherical geometries further increase the complexity, making drag and other shape-dependent forces, such as dielectrophoretic force, less predictable and more complicated. In order to introduce more complexity to the system and maintain precise control over particle movement and fluid flow, it is essential to have a comprehensive understanding about the mechanics of particles-fluid interaction and the dynamics of the particle movement. Although microfluidics has been investigated extensively, unanswered questions about the effect of forces on the particle remain. Answering these questions will facilitate designing novel and more practical microdevices for medical, biological, and chemical applications.
Microfluidics devices were engineered for differentiation of subpopulations of cells based on their bioelectrical properties. These microdevices were utilized for separating prostate, leukemia, and three different stages of breast cancer cells from hematologic cells with concentrations as low as 1:106 with efficiency of >95%. The microfluidic platform was also utilized to isolate prostate cancer stem cells (CSCs) from normal cancer cells based on their electrical signature. Isolating these cells is the first step towards the development of cancer specific therapies. The signal parameters required to selectively isolate ovarian cancer cells at different cancer stages were also compared with peritoneal cells as the first step in developing an early diagnostic clinical system centered on cell biophysical properties. Moreover, the effect of non-toxic concentrations of two metabolites, with known anti-tumor and pro-tumor properties, on the intrinsic electrical properties of early and late stages of ovarian cancer cells was investigated. This work is the first to show that treatment with non-toxic doses of these metabolites correlate with changes in cells electrical properties.
Ph. D.

Le foie joue un rôle essentiel dans les régulations métaboliques complexes qui participent largement à l'homéostasie de l'organisme. De plus, cet organe orchestre les changements qualitatifs et quantitatifs intervenant dans la production de protéines de défense immédiatement nécessaires à la réponse à un syndrome inflammatoire aigü systémique et au retour progressif à l'homéostasie qui s'ensuit. Le facteur de transcription CCAAT enhancer-binding protein beta (C/EBPβ) enrichi dans le foie est très impliqué dans tous ces processus. Toutefois, il existe de nombreuses incohérences quant au rôle précisément joué par ce facteur de transcription. En effet, la traduction de son ARNm conduit à la formation de plusieurs isoformes de la protéine qui peuvent être activatrices (LAP) ou inhibitrices (LIP) et peu d'études, à notre connaissance, ont tenu véritablement compte de l'isoforme présente. Pour mieux comprendre le rôle de chacune de ces isoformes, nous avons mis en place, dans la lignée d'hépatome humain Hep3B, un modèle d'expression inductible de la forme activatrice (LAP) ou dominante-négative (LIP) de C/EBPβ. Le rôle antagoniste de ces 2 isoformes dans la transcription des gènes cibles de C/EBPβ a été utilisé comme stratégie pour mieux cerner les gènes régulés par ce facteur de transcription. L'identité et la transcription (directe ou indirecte) de ces ensembles de gènes-cibles de C/EBPβ ont ensuite été mises en évidence grâce à l'utilisation de deux méthodologies à grande échelle : l'analyse du transcriptome par puce à ADN et l'immunoprécipitation de chromatine sur puce (ChIP on chip). L'analyse du transcriptome nous a permis d'identifier 676 gènes inversement régulés par LAP et LIP dans la lignée d'hépatome Hep3B. L'analyse des fonctions biologiques régulées par ces gènes a mis en évidence une induction par la forme activatrice et une répression par la forme dominante-négative de C/EBPβ des voies du métabolisme hépatique (lipides, détoxication), de la transcription, de la traduction, de l'apoptose et des voies impliquées dans la régulation du processus de prolifération cellulaire. De plus, l'étude par ChIP on chip nous a permis d'identifier 38 nouvelles cibles directes de C/EBPβ. Compte tenu des résultats issus de l'analyse du transcriptome, plusieurs études fonctionnelles ont été réalisées. Ces études nous ont permis de démontrer pour la première fois que, non seulement LAP était capable de réprimer la prolifération en l'absence de RB et P53 mais que cette isoforme favorisait également l'apoptose des cellules induite par la staurosporine alors que LIP, à l'inverse, avait un effet protecteur. Par ailleurs, les cellules Hep3B permettant la surexpression contrôlée de LAP ou LIP ont été stimulées par du milieu conditionné riche en cytokines proinflammatoires afin de mimer la réponse hépatique à l'inflammation aigüe systémique. Dans ce contexte expérimental, et, toujours par analyse du transcriptome, nous avons mis en évidence un ensemble de 77 gènes régulés par LAP et LIP. Ces gènes semblent donc être de bons candidats susceptibles de participer activement à la réponse de l'organisme lors d'une inflammation aiguë. En conclusion, notre approche tout à fait originale, caractérisée par l'identification des gènes inversement régulés par les isoformes LAP et LIP de C/EBPβ, a permis de mieux comprendre comment ces deux isoformes régulent plusieurs processus physiologiques et pathologiques du foie
The liver plays an essential part in complex metabolic regulations which widely contribute to the body homeostasis. Moreover, this organ conducts the qualitative and quantitative changes in the production of specific proteins immediately induced during the acute phase response and allowing a progressive come back to homeostasis. The liver-enriched transcription factor CCAAT enhancer-binding protein beta (C/EBPβ) is widely involved in these processes, but its precise the role isnot still defined. Conflicting studies have described contradictory functions for this transcription factor which could be explained by the complex mechanisms regulating the C/EBPβ activity. Indeed, C/EBPβ encodes an intronless gene that generates a single mRNA that is alternatively translated into two major isoforms : an active LAP (liver-enriched activator protein) and a dominant negative LIP (liver-enriched inhibitory protein). Today, few studies have really taken into account the present isoform. In order to better understand the precise role of each isoform, we first engineered the Hep3B human hepatoma cell line with a Tet-off inducible LAP or LIP isoform. The antagonistic role of the both isoforms in C/EBPβ target-genes transcription has been used as a strategy to better define the C/EBPβ-regulated genes. Then, the identity and the transcription (direct or indirect) of all these target-genes were determined by two functional genomic approaches : the transcriptome analysis by cDNA arrays and the chromatine immunoprecipitation on chip (ChIP on chip). Using a cDNA microarray which provides a complete coverage of the liver transcriptome, we identified 676 genes inversely regulated by LAP and LIP in the Hep3B hepatoma line. The analysis of the biological functions regulated by these genes brought into the flore an induction by LAP and a repression by LIP of several pathways including hepatic metabolism (fat, detoxification), transcription, translation, apoptosis and regulation of the cell proliferation. Moreover, the ChIP on chip study allowed the identification of 38 C/EBPβ new direct targets. According to the data resulting from the transcriptome analysis, several functional studies have been carried out. They allowed us to prove, for the first time, that LAP was, not only able to suppress the cell proliferation in the absence of RB and P53, but that this isoform also increased the staurosporine-induced apoptosis in Hep3B cells while LIP had a protector effect. Furthermore, the Hep3B cells expressing LAP or LIP have been stimulated by a conditioned medium rich in proinflammatory cytokines in order to mimic the hepatic response to the acute phrase of inflammation. In this experimental context, and still by transcriptome analysis, we brought into the fore a group of 77 genes regulated by LAP and LIP which interestingly seem to be involved during the acute phrase response. To conclude, our original approach characterized by the identification of genes inversely regulated by LAP and LIP allowed us to better understand how these two isoforms of C/EBPβ manage several physiological and pathological liver processes

90% de la mortalité par cancer provient de tumeurs disséminées, ou métastases. Ces métastases se forment à partir de cellules tumorales qui s'échappent d'une tumeur primaire, circulent dans le sang, puis quittent les vaisseaux sanguins pour enfin aller nicher dans des organes distants et former des tumeurs secondaires. Les processus par lesquels ces cellules circulantes envahissent les organes distants, remodèlent leur environnement pour créer une «niche micrométastatique», prolifèrent pour produire des métastases macroscopiques, sont mal connus, principalement en raison d'un manque de modèles expérimentaux. En effet ces événements sont rares, se produisent à une échelle microscopique et à des localisations à priori inconnues. La perte d'adhérence cellulaire des cellules tumorales se détachant des tissus tumoraux primaires est associée à un phénomène de transformation connu sous le nom de transition épithéliale-mésenchymateuse (EMT) conduisant à la perte des caractéristiques épithéliales. Dans ce travail, nous avons souhaité aborder la question du processus métastatiques par l'étude de l'influence de l'étape de circulation dans le flux sanguin sur différentes caractéristiques de cellules tumorales. Pour cela, des modèles microfluidiques contenant des constrictions mécaniques afin d'imiter la microcirculation sanguine ont été conçus et fabriqués. Nous avons soumis des cellules provenant de tumeurs primaires du sein dans des situations de confinement périodiques à l'intérieur de ces canaux microfluidiques en utilisant un système de contrôle de flux. Nous avons étudiés l'impact des déformations induites par les constrictions des canaux microfluidiques sur l'expression génétique des marqueurs EMT, la morphologie ainsi que la dynamique des changements morphologiques. Nous montrons que ces paramètres cellulaires sont touchés par la déformation mécanique imposée sous flux, suggérant que l'étape de circulation des cellules tumorales dans le sang a un rôle important dans la capacité de celles-ci à produire des métastases
90% of cancer mortality arises from metastases, due to cells that escape from a primary tumor, circulate in the blood as circulating tumor cells (CTCs), leave blood vessels and nest in distant organs. The processes by which CTCs invade distant organs, remodel their environment to create a “micrometastatic niche”, the eventual triggering of a proliferation leading to a macroscopic metastases, are poorly known, mostly because of a lack of experimental models. These events are rare; occur in the body at unknown places and on a microscopic scale. The loss of cell adhesion of tumor cells detaching from the primary tumor tissues will undergo a transformation phenomenon known as epithelial-to mesenchymal transition (EMT) leading to the loss of epithelial characteristics with different expression patterns of EMT markers (E-cadherin, N-cadherin, Vimentin, Snail1/2, Twist1/2, ZEB1/2). The changes in mechanical and physical properties of interacting cells during morphological and malignant transformation are investigated and their quantifications measured. Here, microfluidic models containing mechanical constrictions in order to mimic the blood microcirculation have been designed and fabricated. Metastatic breast cancer cells are subjected and confined to the microfluidic channels using a flow control system. These cells are circulated under optimal culture conditions, and monitored in the channels for the observance of biophysical occurrences from continuous mechanical cellular deformations. The biophysical effects of circulation and confinement on tumor cell morphogenesis will be investigated

Le cancer de l’ovaire constitue un véritable enjeu de santé public. Les nouveaux traitements se heurtent par ailleurs à des taux d’échec très élevés. Ceci s’explique notamment pour le manque de fiabilité des modèles précliniques classiques tels que la culture cellulaire en 2D. De nouveaux outils basés sur la culture cellulaire en 3D ont alors fait leur apparition tels que les sphéroïdes et les organoïdes. Or ces modèles ont leurs propres limites (coûts, difficultés d’application). La bio-impression 3D est une nouvelle approche permettant de créer des modèles tumoraux de manière contrôlée et reproductible. Néanmoins, elle a encore très peu été appliquée au cancer ovarien. En plus de la troisième dimension, il est important de prendre en compte les conditions dynamiques associées à l’environnement tumoral. Ceci est possible depuis quelques années grâce à la technologie des cancers sur puce basée sur la microfluidique. Cependant, cette technologie ne permet pas, à l’heure actuelle, de simuler le trajet vasculaire du médicament en amont de son interaction avec le tissu tumoral. Dans ce projet de thèse, nous avons souhaité créer un modèle tridimensionnel et dynamique du cancer ovarien en combinant les approches de bio-impression 3D et de microfluidique. Dans un premier temps, la bio-impression 3D a été utilisée pour créer la structure tumorale à proprement parlé. Pour y parvenir, nous avons formulé un hydrogel de gélatine et d’alginate de sodium dans lequel nous avons intégré des cellules cancéreuses ovariennes (SKOV-3) et des fibroblastes cancéreux (MeWo). Le tissu tumoral bio-imprimé a ensuite été caractérisé par différentes techniques pour démontrer sa viabilité et sa pertinence biologique. Sa réponse au cisplatine a également été évaluée. Dans un second temps, nous avons intégré le modèle tumoral bio-imprimé au sein d’un support microfluidique. Le rôle de ce support était de permettre la mise en culture du tissu bio-imprimé sous flux physiologique. Il devait également permettre de simuler le trajet vasculaire du médicament avant son interaction avec le tissu tumoral. Par la suite, nous avons fait appel à la simulation en mécanique des fluides pour concevoir une version améliorée du premier système. L’objectif étant de pouvoir tester, en même temps, plusieurs concentrations différentes de médicament sur un même dispositif microfluidique. Ce projet de thèse a démontré la capacité de la bio-impression 3D à créer des tissus tumoraux ovariens viables et fonctionnels. Il a par ailleurs ouvert des perspectives de recherche très intéressantes par rapport aux possibilités de combiner la bio-impression 3D et de la microfluidique en vue d’améliorer la modélisation préclinique des cancers ovariens
Ovarian cancer is a major public health issue. Moreover, new treatments still face very high failure rates. This is mainly due to the unreliability of conventional preclinical models such as 2D cell culture. Thus, new tools based on 3D cell culture have emerged such as spheroids and organoids. However, these models have their own limitations (cost, difficulty of application). 3D bioprinting is a new approach to create tunable and reproducible tumor models. However, very few bioprinted tumor models have been reported so far. Besides the “third dimension”, it is important to consider the dynamic conditions of the tumor environment. This has been possible for some years now thanks to microfluidics-based cancer-on-a-chip technology. However, this technology currently does not simulate the drug vascular transport before its interaction with the tumor cells. In this PhD project, we set out to create a dynamic, three-dimensional model of ovarian cancer by combining 3D bioprinting and microfluidics. First, 3D bioprinting was used to create the tumor structure itself. For that, we formulated a bio-ink comprising SKOV-3 ovarian cancer cells and MeWo cancer fibroblasts embedded in a gelatin – alginate hydrogel. The bioprinted tumor structures were then characterized by various techniques to demonstrate their viability and biological relevance. Their response to anticancer drug cisplatin was also assessed. In the second step, we integrated the bioprinted tumor model into a microfluidic support for culture under physiological flow. This support was also intended to simulate the drug's vascular transport prior to interaction with the tumor tissue. We then used computational fluid dynamics to design an improved version of the first system. The aim of this improved version was to simultaneously assess multiple drug concentrations. This PhD project demonstrated the ability of 3D bioprinting to create viable and functional ovarian tumor models. It has also brought interesting research prospects with regard to the possibilities of combining 3D bioprinting and microfluidics to improve preclinical modeling of ovarian tumors

Le cancer du pancréas est l’un des cancers les plus mortels avec un pronostic extrêmement sombre. En 2020, le taux de survie à 5 ans reste très faible (de 3 à 9 % seulement) et la médiane de survie est de moins de 6 mois. Malgré des progrès dans la prise en charge des patients, les thérapies actuelles n’ont pas l’efficacité espérée. Ceci est dû à la forte chimiorésistance observée dans ce cancer. Le facteur clé de cette résistance est le microenvironnement tumoral complexe composé majoritairement de stroma et d’une matrice extracellulaire dense limitant l’accès des thérapies à la tumeur. Les limitations des modèles d’études actuels, notamment en termes de pertinence physiologique, sont un frein important dans la compréhension de cette chimiorésistance. En réponse à cette problématique, les chercheurs se tournent vers de nouvelles approches en développant de nouveaux modèles d’études alternatifs à ceux déjà disponibles (in vitro et in vivo).Les objectifs de ce travail ont été : (i) de développer un dispositif de culture in vitro 3D microfluidique permettant de reproduire le microenvironnement tumoral sur les plans biologique et mécanique, ainsi que de modéliser les écoulements et transports de masse présents dans une tumeur pancréatique, et (ii) d’aborder les changements morphologiques de la co-culture par l’étude de marqueurs épithélio-mésenchymateux et d’étudier l’impact de la chimiothérapie FOLFIRINOX dans ce modèle.Dans un premier temps nous avons montré numériquement et expérimentalement la faisabilité d’un tel modèle in vitro. La matrice extracellulaire choisie est une association de collagène I et d’acide hyaluronique créant une structure rigide proche des conditions in vivo. Elle permet le maintien de la culture à long terme en ne se dégradant pas ou peu sous l’effet de la perfusion ainsi que l’activation des cellules stellaires pancréatiques. La vitesse de perfusion choisie permet quant à elle d’appliquer un flux interstitiel équivalent à celui observé dans le microenvironnement in vivo, induisant une pression hydrostatique et une contrainte de cisaillement sur les cellules.Nous avons ensuite mis en évidence l’apport biologique de cette modélisation en montrant une chimiorésistance accrue au protocole FOLFIRINOX des cellules tumorales à la fois en mono- et en co-culture dans le dispositif microfluidique. Nous montrons également la mise en place d’un processus ayant des caractéristiques de la transition épithélio-mésenchymateuse et d’une possible promotion d’un phénotype dédifférencié des cellules tumorales par les cellules stellaires pancréatiques activées.En conclusion, nous présentons dans cette thèse un modèle d’étude microfluidique original permettant de modéliser une tumeur (co-culture de cellules épithéliales et mésenchymateuses) et d’étudier la cinétique d’une chimiothérapie multidrogues complexe. Ce dispositif devrait à l’avenir nous permettre d’étudier plus en profondeur les mécanismes de chimiorésistance et les interactions tumeur-stroma dans le cancer du pancréas
Pancreatic cancer is one of the deadliest cancers with an extremely poor prognosis. In 2020, the 5-year survival rate remains very low (only 3 to 9%) and the median is less than 6 months. Despite significant progress in the patient care, current therapies do not have the expected effectiveness. This is due to the strong chemoresistance observed in this cancer. The key factor of this resistance is the complex tumor microenvironment mainly composed of stroma and a dense extracellular matrix limiting the access of therapies to the tumor. The current models’ limitations, particularly in terms of physiological relevance, are a major obstacle in understanding this chemoresistance. In response to this issue, researchers are turning to different approaches by developing brand new models that are alternatives to those already available (in vitro and in vivo).The objectives of this work were: (i) to develop an in vitro 3D microfluidic culture device allowing to reproduce the tumor microenvironment both biologically and mechanically, as well as to model the flows and mass transport present in a pancreatic tumor, and (ii) to approach the morphological changes of the co-culture by studying epithelio-mesenchymal markers and to study the impact of FOLFIRINOX chemotherapy in this model.First, we have shown numerically and experimentally the feasibility of such an in vitro model. The chosen extracellular matrix is a combination of collagen I and hyaluronic acid creating a rigid structure close to in vivo conditions. It allows long-term culture maintenance under the effect of the perfusion as well as the activation of pancreatic stellate cells. The chosen perfusion rate allows to apply an interstitial flow in the model equivalent to the one observed in the in vivo microenvironment, inducing hydrostatic pressure and shear stress on the cancerous cells.Then, we demonstrated the biological contribution of this model by showing an increased chemoresistance to the FOLFIRINOX protocol of tumor cells both in mono- and in co-culture in the microfluidic device. We also show the establishment of a process presenting characteristics of epithelial-mesenchymal transition and a possible promotion of a dedifferentiated phenotype of tumor cells by activated pancreatic stellate cells.In conclusion, we present in this thesis an original microfluidic model allowing to mimic a tumor (co-culture of epithelial and mesenchymal cells) and to study the kinetics of a complex multidrug chemotherapy. In the future, the device should allow us to further study the mechanisms of drug resistance and tumor-stroma interactions in pancreatic cancer

Les méthodes habituelles de diagnostic du cancer sont invasives et coûteuses et permettent rarement de dépister la maladie à un stade précoce. L'apparition de biomarqueurs de cancer a été mise en évidence à un stade précoce de la maladie, bien avant le développement des symptômes. Ces marqueurs sont encore peu utilisés, notamment du fait de leur présence en faible quantité chez des individus sains, mais aussi de leur manque de spécificité pris individuellement. De nos jours, les analyses cliniques pour le diagnostic ou le suivi de certains paramètres sont effectués par division des prélèvements et mesures séparées de la concentration des différents analytes cliniquement pertinents. Dans ce contexte, le développement de dispositifs permettant la détection simultanée de ces marqueurs dans les fluides biologiques représente un véritable défi. Par ailleurs, la miniaturisation des dispositifs de mesure et leur intégration sur puce ouvrent la voie vers des unités d'analyse automatisables, transportables au chevet des patients et répondant aux besoins des milieux hospitaliers en termes d'analyses rapides, sur de faibles volumes d'échantillons, à bas coût et haut débit, sensibles et spécifiques. L’objectif de cette thèse est de développer des méthodes permettant l'immobilisation localisée de ligands sélectifs basés sur la reconnaissance moléculaire (aptamères) sur différents matériaux de microsystèmes pour la détection simultanée de plusieurs biomarqueurs. Les aptamères sont une nouvelle classe de molécules d'ADN/ARN synthétiques, reconnus pour leur haute affinité et leur excellente spécificité pour une cible sélectionnée. Ils présentent de plus l'intérêt d'être manipulables à température ambiante, et facilement regénérable après dénaturation. Malgré ces avantages, très peu de publications portent sur les technologies à base d'aptamères pour l'extraction sélective et l'analyse de traces de biomarqueurs en dispositif microfluidique. La création de telles zones de confinement permet d'assurer la surconcentration des analytes cibles pour une meilleure performance de la détection
Common methods of cancer diagnosis are invasive, expensive and rarely allow early cancer diagnosis. The discovery of cancer biomarkers present in the early stage of the disease, well before the development of symptoms make them interesting candidate for early cancer diagnosis. These markers are still not widely used, particularly because of their low concentration in healthy patient samples, but also because of their lack of specificity taken individually. Nowadays, clinical analyses for the diagnosis are performed by dividing samples and separate measurements of the concentration of the various clinically relevant analytes. In this context, the development of devices for the simultaneous detection of these markers in biological fluids is a real challenge. In addition, the miniaturization of measurement devices and their integration on a chip opens the way to automated analysis units that can be transported to the bedside and meet the needs of hospitals for fast, low-volume, low-cost, high throughput, sensitive and specific analyses. The objective of this thesis is to develop methods for the localized immobilization of selective ligands based on molecular recognition (aptamers) on different microsystem materials for the simultaneous detection of several biomarkers. Aptamers are a new classe of synthetic DNA/RNA molecules, known for their high affinity and excellent specificity for a selected target or family of targets. They also have the advantage of being easy to handle at room temperature and easily regenerated after denaturation. Despite these advantages, very few publications have been published on aptamer-based technologies for the selective extraction and analysis of biomarker traces in microfluidic devices. The creation of such localized zones ensures the overconcentration of target analytes to improve detection performance

Changes in the human gastrointestinal microbiome are associated with several diseases. To infer causality, experiments in representative models are essential, but widely used animal models exhibit limitations. Here we present a modular, microfluidics-based model (HuMiX, human-microbial crosstalk), which allows co-culture of human and microbial cells under conditions representative of the gastrointestinal human-microbe interface. We demonstrate the ability of HuMiX to recapitulate in vivo transcriptional, metabolic and immunological responses in human intestinal epithelial cells following their co-culture with the commensal Lactobacillus rhamnosus GG (LGG) grown under anaerobic conditions. In addition, we show that the co-culture of human epithelial cells with the obligate anaerobe Bacteroides caccae and LGG results in a transcriptional response, which is distinct from that of a co-culture solely comprising LGG. HuMiX facilitates investigations of host-microbe molecular interactions and provides insights into a range of fundamental research questions linking the gastrointestinal microbiome to human health and disease.

Tese de Doutoramento em Engenharia de Tecidos, Medicina Regenerativa e Células Estaminais
By 2030, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths simply due to the growth and aging of the population. Among all types of cancer, colorectal cancer is a major cause of morbidity and mortality worldwide, and accounts for over 9 % of all cancer incidence. It is the third most common cancer worldwide and affects men and women equally. In order to win the battle against cancer, further advances are in great need to unveil identification of cancer-causing agents in in vitro and in vivo animal models, as well as for the development of personalized therapies, drug screening, and to provide insightful knowledge on the mechanisms of tumor growth and metastasis. Microfluidic devices, together with tissue engineering strategies and nanotechnology have emerged as a powerful platform to tackle the previously mentioned hurdles. These themes are the focus of Section 1, in Chapters I, II and III. In this thesis we focus on the application of CMCht/PAMAM dendrimer nanoparticles.as the synthesis, uptake efficiency/internalization and cytotoxic effect of fluorescent-labeled CMCht/PAMAM dendrimer nanoparticles was investigated using different cancer cell lines in both traditional culture flasks and under physiological flow inside a microfluidic platform (Chapter V). Other than nanoparticles, the use of different biomaterials to modulate 3D microenvironment of tumors is of the utmost importance. Therefore, the use of HRP-crosslinked SF hydrogels with spatial tunable properties was proposed in a colorectal cancer extravasation 3D model on a commercial chip Vena4™ (Chapter VI). To increase the complexity of the cancer models, a microfluidic chip was designed and fabricated for the incorporation of tumor and vascular parts, allowing for gradients of gemcitabine released from CMCht/PAMAM nanoparticles to be tested (Chapter VII). Chemical modification in the CMCht/PAMAM dendrimer nanoparticles in order to target colorectal cancer was also achieved in Chapter VIII. In Chapter IX, a new proof of concept consisting of a microfluidic silk platform, flexible and implantable, was developed in house. The results and platforms developed in this thesis are discussed in Chapter X and represent a strong advance in the field of lab-on-chip research and will be a useful tool for drug discovery and study of migration/metastasis phenomena, allowing for a versatile choice of tissues, biomaterials and biological assays.
Até 2030, o número global de novos casos de cancro deverá crescer para 21,7 milhões, com cerca de 13 milhões de mortes, simplesmente devido ao crescimento e envelhecimento da população. Entre todos os tipos de cancro, o cancro colorretal é uma das principais causas de morbidade e mortalidade em todo o mundo, representando mais de 9 % entre todos os tipos de cancro. Com o objectivo de ganhar a guerra contra o cancro, novos avanços são rapidamente precisos, a fim de identificar, em modelos animais in vitro e in vivo os agentes causadores de cancro, bem como para o desenvolvimento de terapias personalizadas, teste de novos fármacos e aumentar o conhecimento actual sobre os mecanismos de crescimento tumoral e metástases. Dispositivos de microfluídica juntamente com estratégias de engenharia de tecidos e nanotecnologia surgiram como uma poderosa plataforma para lidar com os obstáculos mencionados anteriormente. Esses temas são o foco da Secção 1, nos Capítulos I, II e III. Nesta tese damos enfâse à aplicação de nanopartículas CMCht/PAMAM. A síntese, eficiência, internalização e efeito citotóxico das mesmas foram investigados usando diferentes linhas de células tumorais em frascos de cultura tradicionais e dentro de uma plataforma microfluídica (Capítulo V). Além das nanopartículas, o uso de diferentes biomateriais para modular o microambiente 3D de tumores é de extrema importância. Portanto, o uso de hidrogéis SF reticulados com HRP com propriedades mecânicas ajustáveis foi proposto num modelo 3D de extravasamento de cancro colorretal num chip comercial VenaT4™ (Capítulo VI). Para aumentar a complexidade dos modelos de cancro em chips, uma plataforma microfluídica foi projetada e fabricada para a incorporação de ambas as partes tumorais e vasculares, permitindo testar gradientes de gemcitabina libertados das nanopartículas CMCht/PAMAM (Capítulo VII). Modificações químicas nas nanopartículas para atingir especificamente células cancerígenas também foram alcançadas no Capítulo VIII. No Capítulo IX, uma nova prova de conceito consistindo numa plataforma microfluídica de seda, totalmente flexível e implantável, foi desenvolvida. Os resultados e plataformas desenvolvidos nesta tese representam um forte avanço no campo da investigação do cancro, e serão uma ferramenta útil para a descoberta de drogas e estudo de metastases, permitindo uma escolha versátil de tecidos, hidrogéis e ensaios biológicos.

Development of in-vitro 3D cellular disease models is emerging at a fast pace. 2D culture systems are limited due to minimal cell-cell interactions and poor stromal intervention. Hence, there is a need to develop 3D models which are more physiologically relevant than 2D models & improves the prediction of drug candidates. 3D models possess more realistic multicellular complexity (cell-cell, cell-matrix interactions) and mimics in-vitro microenvironment more closely. Present study delineates the development of a novel in-vitro 3D cancer model by incorporating cancer aggregates into porous scaffolds. This serves as a portable lab-on-a-chip platform for screening antitumor chemotherapeutic agents. For this purpose, gelatin–chitosan scaffolds were prepared by freeze-drying, crosslinked using glutaraldehyde and investigated for physiochemical and biological characteristics. Microtissue comprising HeLa cells was prepared by Hanging drop technique and embedded into porous scaffolds and cultivated in standard culture conditions. Microtissue viability in the 3D microenvironment of polymeric scaffold was assessed by MTT assay. Cryopreservation performance of microtissue seeded scaffold construct was examined by cooling using liquid N2. Resuscitation of the constructs 2 days of cryopreservation showed that system retained 76% of cell viability and metabolic activity. The constructs were then used as a cancer tissue mimetic to compare the efficacy of two anticancer drugs namely 5-FU and Cisplatin. Not all cells of the microtissue are exposed to the same drug concentration due to poor drug diffusion through the biomimetic scaffold which resembles native ECM. As predicted, percentage cell death is less in case of our model compared to conventional monolayer culture. These results clearly imply that the proposed gelatin-chitosan scaffold based 3D cancer model closely mimics in-vivo tumor conditions and can be used as an in-vitro screening system for anticancer drug screening.

Ovarian cancer is a high mortality disease where early stage detection may have significant survival benefits. Promising next-generation non-invasive, biomarker-based screening modalities involve longitudinal monitoring of serum biomarkers and multi-marker panel detection. Here, rapid, sensitive, precise and multiplexable diagnostic platforms can facilitate biomarker validation along with early detection and screening, and this work attempts to exploit the programmable bio-nano-chip (p-BNC) immunosensor to address these specific translational needs in ovarian cancer. First, the p-BNC was adapted for Cancer Antigen 125 (CA125) quantitation, the current FDA standard, with prominent implications in novel early detection and screening modalities. Antibody pairs binding to distinct epitopes on CA125 were identified and the p-BNC operating variables (incubation times, flow rates and reagent concentrations) were attuned to deliver optimal analytical performance (inter- and intra-assay precision of 1.2% and 1.9% and Limit-of-Detection (LOD) 1.0 U/mL), competitive with current gold standards, but with a short analysis time of 43 minutes. Further validation of the system with advanced stage patient sera (n=20) demonstrated good correlation with 'gold standard' ELISA (R² = 0.97). Next, the p-BNC was adapted for concomitant analysis of CA125 and Human Epididymis Protein 4 (HE4), a novel multiplexed biomarker panel for early detection and screening. The HE4 immunoassay was developed to perform optimally with the 'rate determining' CA125 assay. Cross-reactivity analysis demonstrated high specificity multiplexing. The dose-response curves for the multiplexed CA125 and HE4 immunoassays were congruous with their singleplex counterparts with respective LODs of 0.51 U/mL and 4.18 pM and a total analysis time of 44 minutes. A small pilot scale clinical study was conducted to discriminate between surgically confirmed patient sera (n=8) and corresponding age-matched healthy controls (n=8) utilizing the multiplexed p-BNC, interpreted with a risk of ovarian malignancy algorithm. Successful discrimination was achieved between the groups with Receiver Operating Characteristic (ROC) curve AUC (Area Under the Curve) values of 1.00, 0.984 and 1.00 respectively for CA125, HE4 and the composite marker combination. Taken together, the analytical and clinical performance, multiplexing capabilities and the short turn-around times on the p-BNC offer methodological advancements over current gold standard techniques, indicating strong promise for ovarian cancer diagnostics.
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Notch signaling plays a key role in development, tissue homeostasis, and cancer. High expression levels of Notch signaling components are associated with aggressive disease and poor patient prognosis in breast cancer. Mesenchymal‐epithelial transition factor (MET) is a receptor tyrosine kinase with an established prognostic significance correlating with poor disease outcome in breast cancer patients as a result of high metastatic rate. We performed expression array analysis to identify candidate Notch target genes; we identified and validated MET as a target of NOTCH1 signaling in breast cancer. We found that NOTCH1 knockdown significantly reduces MET promoter activity, as well as expression levels of MET transcript and protein. The mechanism of NOTCH1 regulation of MET expression will be the focus of future work. To further identify candidate target genes of NOTCH1 signaling, we generated and validated a NOTCH1 antibody for use in chromatin immunoprecipitation experiments.

In the following work, nanoparticle quantum dot (QD) fluorophores have been exploited to measure biologically relevant analytes via a miniaturized sensor ensemble to provide key diagnostic and prognostic information in a rapid, yet sensitive manner—data essential for effective treatment of many diseases including HIV/AIDS and cancer. At the heart of this “nano-bio-chip” (NBC) sensor is a modular chemical/cellular processing unit consisting of either a polycarbonate membrane filter for cell-based assays, or an agarose bead array for detection of biomarkers in serum or saliva. Two applications of the NBC sensor system are described herein, both exhibiting excellent correlation to reference methods ((R² above 0.94), with analysis times under 30 minutes and sample volumes below 50 [mu]L. First, the NBC sensor was employed for the sequestration and enumeration of T lymphocytes, cells specifically targeted by HIV, from whole blood samples. Several different conjugation methods linking QDs to recognition biomolecules were extensively characterized by biological and optical methods, with a thiol-linked secondary antibody labeling scheme yielding intense, specific signal. Using this technique, the photostability of QDs was exploited, as was the ability to simultaneously visualize different color QDs via a single light pathway, effectively reducing optical requirements by half. Further, T-cell counts were observed well below the 200/[mu]L discriminator between HIV and AIDS and across the common testing region, demonstrating the first reported example of cell counting via QDs in an enclosed, disposable device. Next, multiplexed bead-based detection of cancer protein biomarkers CEA, Her-2/Neu, and CA125 in serum and saliva was examined using a sandwich immunoassay with detecting antibodies covalently bound to QDs. This nano-based signal was amplified 30 times versus molecular fluorophores and cross talk in multiplexed experiments was less than 5%. In addition, molecular-level tuning of recognition elements (size, concentration) and agarose porosity resulted in NBC limits of detection two orders of magnitude lower than ELISA, values competitive with the most sensitive methods yet reported (0.021 ng/mL CEA). Taken together, these efforts serve to establish the valuable role of QDs in miniaturized diagnostic devices with potential for delivering biomedical information rapidly, reliably, and robustly.
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Oral cancer is the sixth most common cancer worldwide and has been marked by high morbidity and poor survival rates that have changed little over the past few decades. Beyond prevention, early detection is the most crucial determinant for successful treatment and survival of cancer. Yet current methodologies for cancer diagnosis based upon pathological examination alone are insufficient for detecting early tumor progression and molecular transformation. Development of new diagnostic tools incorporating tumor biomarkers could enhance early detection by providing molecular-level insight into the biochemical and cellular changes associated with oral carcinogenesis. The work presented in this doctoral dissertation aims to address this clinical need through the development of new automated cellular analysis methods, incorporating lab-on-a-chip sensor techniques, for examination of molecular and morphological biomarkers associated with oral carcinogenesis. Using the epidermal growth factor receptor (EGFR) as a proof-of-principle biomarker, the sensor system demonstrated capacity to support rapid biomarker analysis in less than one-tenth the time of traditional methods and effectively characterized EGFR biomarker over-expression in oral tumor-derived cell lines. Successful extension from in vitro tumor cell lines to clinically relevant exfoliative brush cytology was demonstrated, providing a non-invasive method for sampling abnormal oral epithelium. Incorporation of exfoliative cytology further helped to define the important assay and imaging parameters necessary for dual molecular and morphological analysis in adherent epithelium. Next, this new sensor assay and method was applied in a small pilot study in order to secure an initial understanding of the diagnostic utility of such biosensor systems in clinical settings. Four cellular features were identified as useful indicators of cancerous or pre-cancerous conditions including, the nuclear area and diameter, nuclear-to-cytoplasm ratio, and EGFR biomarker expression. Further examination using linear regression and ROC curve analysis identified the morphological features as the best predictors of disease while a combination of all features may be ideal for classification of OSCC and pre-malignancy with high sensitivity and specificity. Further testing in a larger sample size is necessary to validate this regression model and the LOC sensor technique, but shows strong promise as a new diagnostic tool for early detection of oral cancer.
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碩士
亞洲大學
生物資訊學系碩士班
98
Background: Lung cancers are among the neoplastic diseases with the worst prognosis. More treatment modalities are needed to improve clinical outcome. It’s being a new trend to search for bioactive traditional Chinese medicines (TCM). Ganoderma, also known as Lingzhi, is a TCM and has been widely used for improving human health for centuries. We have addressed the anticancer effects of Ganoderma on various cancers. In this study, a comprehensive genomic profiling was used to further explore the signal pathway for the anticancer effects of Tien Shen Lingzhi(TSL),an improved strain of Ganoderma tsugae. Methods: A comparative genomics study was performed to search for TSL-mediated differential expressions on H23/0.3 cells. Bioinformatics tool, e.g., GeneGo Meta CoreTM ,was used to analyze the experimental results based on Affymetrix Human Genome U133 Plus 2.0 microarray. Results: The whole-genome expression profiles of H23/0.3 with or without TSL treatment were resolved by multidimentional scaling (MDS) analysis. It indicated a TSL-mediated cell cycle perturbation. Bioinformatics analysis showed cell cycle was the most affected pathway. GeneGo Meta CoreTM derived cellular pathway map revealed dramatic hits (17/26) on the signal transduction of DNA replication. The microarray results were confirmed by Q-PCR. Conclusion: The genomic microarray data demonstrates the TSL-induced cell cycle deregulation in H23/0.3 cells. TSL-mediated cell cycle perturbation on H23/0.3 cells is consistent with the cellular pathway map resulting from GeneGo Meta CoreTM analysis.

碩士
國立清華大學
動力機械工程學系
102
Because people lifestyle changed, cancer has been the top ten causes of death for 13 years. In every 100 people, 28 people were dead because of cancer. Cancer is caused by either the congenital mutations or the overexpression of oncogenes. It has a powerful ability to transform proliferative and transfer. Because the activation of oncogenes mutations takes months, the diagnosis need a long time. Therefore, the cancer was considered a terminal illness. However, with the medical improvement, the surgery and chemotherapy have been used to cure malignant tumors. Although surgery is easy to remove the tumor directly, this method can only postpone the disease if the cancer has moved to other organs. The chemotherapy can effective cure the cancer, but the patients might usually suffer from the significant side effects. However, both of these methods are unable to effectively cure the cancer. There are many kinds of therapies proposed in recent years, such as gene therapy, inhibition of angiogenesis, inhibition of pro-cancer protein and immunotherapy. Immunotherapy has the potential among a variety of treatments. Cancer immunotherapy is a popular medical technology. On April 29, 2010, the FDA approved cancer treatment vaccine which was called Provenge. Provenge is used for the treatment of advanced prostate cancer. It represents the customization of new medical era. It can be used to make different vaccines according to different types of cancer. Among all strategies, cancer immunotherapy of dendritic cells / tumor fusion vaccine is a therapeutic option. Dendritic cells are phagocytes and have the strongest antigen expression. When dendritic cells swallow the antigen, they will activate the adaptive immune system. Therefore, the fusion of dendritic cells / tumor can make dendritic cells divide faster and mass-produce the vaccine. In tradition, either polyethylenglycol or randomness method was used for electric fusion. The efficiency and the quality of the vaccine were both unstable. Through this research we develop a cell electrofusion lab chip which combined the rapid and precise cell pairing micro-structures and the high yield electrofusion micro-electrodes to improve the cell fusion techniques and overcome the key barriers. We wish to build and develop an automated, mass-produced and efficient dendritic cell / tumor fusion vaccine.

碩士
國立陽明大學
生醫光電工程研究所
99
In the tumor microenvironment, a lot of stromal cells secret growth factors to enhance the metastasis, proliferation, and survival of cancer cells. A number of cells including fibroblasts and macrophages are involved in the metastasis of a primary tumour. We develop microfluidic cell culture chips that contain three culture chambers to co-culture lung cancer cells, macrophages, and fibroblasts to study the interactions amount them. The results showed that cancer cells were activated fibroblasts into myofibroblasts by conditional medium of cancer cell. The myofibroblast conditional medium will promote the mobility of cancer cells, but macrophages secreted TNF-? will inhibit the myofibroblastic ability of autocrine TGF-?? The ??SMA fluorescence intensity and aspect ratio both decreased, so myofibroblasts to promote cancer cell the ability of migration speed will be reduced. But myofibroblasts and macrophages conditional medium together effect on the cancer cells, that will synergies enhance the metastasis of cancer cells. We find that the effect of myofibroblast to increase the migration speed of cancer cells is suppressed by the conditioned medium of the macrophage. Therefore, macrophages interactions between stromal cells in the tumor micro-environment play a very important role in tumor growth and metastasis.

博士
國立交通大學
機械工程系所
101
In this dissertation, we demonstrated that the direct and accurate measurement of protein-protein interaction force can be done by atomic force microscopy (AFM) via immobilization of one protein on chip (imm-protein) and another on probe tip of AFM (afm-protein). Preliminarily, we observed the binding between T-cell receptor, CD28 (imm-CD28), and B-cell receptor, CD80 (afm-CD80), with an immuno-suppressive blocker, cynarin. Average unbinding forces were reduced from 61.4 pN to 38.9 pN with a blocking effect of ~37% as compared with ~9% by SPR. Using AFM as a detection tool, the significant quantity differences caused by drug target binding therefore can be applied as an efficient and accurate drug screening or protein-protein interaction measurement. The other subject in this dissertation is to discuss the drug effect on cell’s mechanical property. A less side-effect and non-harmful custom anti-cancer peptide (CB1a) composed by 33 amino acids was created. The results showed that the peptide drug has cytotoxicity effect on cancer cells in solution (in vitro). The elastic property differences of single cell surface while interacted with anticancer drug CB1a were estimated by AFM. The changes of elastic force of cell with/without CB1a was illustrated and reflected as Young’s modulus. After adding CB1a, the normal cell, MRC-5, almost remained its original Young’s modulus value. For cancer cell NCI-H460, the Young’s modulus reduced to only below 50%. It means that the quantity variation can be considered as a good selectivity of CB1a and provided another cytotoxicity mechanism effect on cancer cell surface. Furthermore, we also compared with the commercial anti-cancer drug, Doxorubicin (Dox), to study the differences of morphology and physical property changes under drugs treating. The results prove that CB1a prefers to damage cell membrane from outside, and Doxorubicin leads cell to death by stopping the functions inside the cells.

Nucleic acids amplification-based methods can profit from the features offered by Lab-on-a-chip technologies, in particular those that aimed for molecular diagnosis purposes. Currently, isothermal amplification approaches, more precisely LAMP, have become promising alternatives to the current gold standard technology (PCR). Regardless the amplification mechanism, accurate target quantification is still challenging. To this end, the development of digital amplification methods has helped to circumvent this limitation. This thesis focused on the development of a chip-based digital LAMP system towards the quantification of prostate cancer biomarkers. For this, LAMP was integrated with droplet-based digital amplification concept. LAMP positive amplification was achieved after 60 minutes, leading to a 2-fold increase in fluorescence when compared to the negative amplification controls, in a vortex-based droplet generation approach. However, aspects inherent to this method prevented a quantitative assessment of LAMP amplification. In order to overcome these limitations, a novel microfluidics chip-based device was developed and implemented towards dLAMP quantification of c-Myc gene. The T-junction type droplet generator chip achieved droplets of 1.5 nL with a coefficient of variation bellow 3%, in line with the standard for this technique. This system showed a sharp response to template concentration, observable by the raise in the fraction of positive droplets. Additionally, the target quantification proven to be precise (R2 =0.99) for 4 orders of magnitude of copies/µL (5 copies/µL - 5x105 copies/µL) after Poisson’s modulation. Aiming for the implementation of this chip-based dLAMP system into the detections of prostate cancer-associated biomarkers, amplification reactions of SChLAP1 and PCA3 genes were developed and further optimized for real-time fluorescence monitoring. As a result, it was possible to develop a quantitative method for cDNA amplification, that presented higher amplification efficiencies and a reduction on the overall reaction time, when compared to the gold standard RT-PCR. Furthermore, the proposed strategy is compatible with the integration into the chip-based microfluidics device, hence easily extended to the monitorization of gene expression levels.

Novel disposable optical biochips based on one-dimensional photonic crystals (1DPC) sustaining Bloch surface waves (BSW) are an attractive tool for the detection of several disease-related biomarkers. In response to growing global burden of cancer, one of the most significant public health challenges of the 21st century is the prevention and early diagnosis of this disease. By diagnosing in advance such disease permits to increase the survival probability of patients and to improve their life perspectives. Consequently, cancer biomarkers have gained considerable attention. Within this framework, the herein proposed optical biochips can quantify low concentrations (sub ng/mL) of the ERBB2 breast cancer biomarker in biological complex matrices, such as cell lysate samples. The choice of focusing on this specific breast-cancer-related biomarker lies in the global issue represented by this type of cancer. According to the data provided by the World Cancer Research Fund, its incidence rate increases year per year establishing itself as the most commonly occurring cancer in women worldwide contributing 25% of the total number of new cases diagnosed in 2018 and as the second most common cancer overall after lung cancer. However, the versatility of the system opens up new possibilities for designing different assays, depending on the specific biomarker sought. To discriminate ERBB2 levels in several different cell lysate samples, we made use of 1DPC biochips and on a reading instrument that can work in both a label-free and a fluorescence detection mode. Such combined configuration provides the advantage of complementary information and lower limit of detection (LoD) in the fluorescence mode. In the label-free mode, the BSW excitation is achieved by a prism coupling system (Kretschmann-Raether configuration), like in the surface plasmon resonance (SPR) technique, resulting in a dip in the angular reflectance spectrum. According to the interactions that take place at the surface, the angular position of such a dip shifts as a function of refractive index change at the interface. Moreover, the fluorescence operation, in which fluorescence angular spectra are acquired, is obtained by making use of fluorophores or dye labelled antibodies bound at the 1DPC surface. Furthermore, coupling between the dye labels and the BSW results in strongly directional and enhanced fluorescence emission. The advantages brought by the 1DPC, when compared to metal structures, are the smaller energy losses and the narrower resonances. Despite the great sensitivity offered by the fluorescence detection mode, the measurements are affected by a phenomenon that cannot be neglected when quantitative and accurate information is needed, as occurs in biosensing assays, i.e. photobleaching. Presently, there is no study about photobleaching in experiments with BSW sustained by 1DPC, despite its evident effects. Photobleaching denotes the irreversible loss of fluorescence emitted energy of a dye that dramatically changes its absorption and emission properties. The rate of such a fluorescence bleaching is affected by several factors such as the power of the illumination beam, the exposure time, and the photonic crystal structure itself. In addition, it is also influenced by the molecule’s transition dipole moment. In particular, fluorophores having a transition dipole moment oriented parallel to excitation polarized light will be excited preferentially, and in turn will be strongly photobleached. As a consequence, the fluorescence emission will be polarized and no more isotropic. This effect is more or less significant depending on the binding strength of the fluorophores to the surface. In this dissertation, we report for the first time on cancer detection assays, carried out with our setup, in which the trustworthiness is guaranteed by a correct approach to data analysis, which accounts in a correct way for photobleaching, which could not only affect the overall emission intensity but also its polarization distribution via the TE and TM BSW modes provided by the 1DPC. To get to such a result, the experimental data is interpreted by means of a theoretical model for the orientational distribution of dye labels over time, taking into account the density of the optical states of the 1DPC, photobleaching and rotational diffusion of surface bound emitters. The approach permits to model anisotropic fluorescence emission and to manage photobleaching effects in biosensing assays, leading to a correct data interpretation. The theoretical description permits not only to manage photobleaching but also to exploit it as a new tool for probing rotational diffusion of any protein labelled with fluorescent emitters at the surface of 1DPC endowed with different chemistries. Such a possibility is related to the polarization dependent spectroscopic role played by the 1DPC, which permits to analyse simultaneously two polarizations, TE and TM, within a relatively simple optical layout and thus accessing either the orientation or the depolarization kinetics under non-stationary conditions of the resonant fluorescence signal. Such a type of measurement is not possible with conventional fluorescence anisotropy techniques or using other types of surface electromagnetic waves, such as surface plasmon polaritons, for example, that are only TM polarized.

Thesis (Ph.D.)--University of Alberta, 2009.
Title from PDF file main screen (viewed on Apr. 1, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Electrical and Computer Engineering, University of Alberta. Includes bibliographical references.