We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The liver is a multi-functional organ that regulates major physiological processes and that possesses a remarkable regeneration capacity. After loss of functional liver mass the liver grows back to its original, individual size through hepatocyte proliferation and apoptosis. How does a single hepatocyte ‘know’ when the organ has grown to its final size? This work considers the initial growth phase of liver regeneration after partial hepatectomy in which the mass is restored. There are strong and valid arguments that the trigger of proliferation after partial hepatectomy is mediated through the portal blood flow. It remains unclear, if either or both the concentration of metabolites in the blood or the shear stress are crucial to hepatocyte proliferation and liver size control. A cell-based mathematical model is developed that helps discriminate the effects of these two potential triggers. Analysis of the mathematical model shows that a metabolic load and a hemodynamical hypothesis imply different feedback mechanisms at the cellular scale. The predictions of the developed mathematical model are compared to experimental data in rats. The assumption that hepatocytes are able to buffer the metabolic load leads to a robustness against short-term fluctuations of the trigger which can not be achieved with a purely hemodynamical trigger.

Os insulinomas são os tumores neuroendócrinos pancreáticos funcionantes mais frequentes, entretanto, os aspectos moleculares envolvidos em sua tumorigênese precisam ser melhor esclarecidos. As características morfológicas e histoquímicas dos insulinomas não conseguem predizer completamente seu comportamento biológico, apenas o fenótipo invasivo local e a presença de metástase são as formas confiáveis do diagnóstico maligno. A presente investigação teve por objetivos analisar a expressão gênica por reação em cadeia da polimerase (PCR) em tempo real e a expressão protéica por imuno-histoquímica dos componentes da via de sinalização do fator de crescimento hepatocítico (HGF) e seu receptor (c-MET) em 27 amostras de insulinomas, sendo: 16 tumores benignos grau 1 (G1), seis tumores benignos grau 2 (G2), dois insulinomas malignos grau 3 (G3) e três metástases hepáticas. Além disso, realizou-se a pesquisa de mutações somáticas no gene MET. Observou-se (1) o aumento da expressão dos genes HGF, MET e ST14 (codificante para a matriptase) e a baixa expressão do gene HAI-1 (codificante para a protease inibidora tipo-kunitz do tipo 1) nos insulinomas malignos e metástases quando comparados aos insulinomas benignos G1; (2) uma correlação positiva entre a expressão do mRNA do gene MET e o gene ST14 e índice proliferativo Ki-67, bem como uma correlação inversa entre a expressão do mRNA do gene HAI-1 e os genes MET, HGF, ST14 e o índice mitótico; (3) uma correlação positiva entre a expressão do gene ST14 e a expressão do mRNA do gene HGF; (4) maior expressão protéica de c-MET nos insulinomas malignos G3 em relação aos insulinomas G1/G2 e (5) ausência de mutações nos éxons 2, 10, 14, 16, 17 e 19 do gene MET. Concluiu-se que os genes HGF, MET, ST14 e HAI-1 estão diferencialmente expressos entre insulinomas malignos e benignos, o que pode ter implicações diagnósticas e terapêuticas / In an attempt to better understand the molecular processes involved in the tumourigenesis of islet beta-cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two potent inhibitors of HGF activator and of matriptase) in 27 sporadic insulinomas; 16 grade 1 (G1), six grade 2 (G2), two grade 3 (G3) and three hepatic metastases. Quantitative reverse-transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes and immunohistochemical analysis was used to evaluate the expression of MET and SPINT1. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19. Overexpression of MET was observed in grouped G3 insulinomas and metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. Positive correlations were observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene. The final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoural progression and malignancy in insulinomas

Doctor of Philosophy(PhD) / Owing to its strategic position in the liver sinusoid, pathologic and morphologic alterations of the Liver Sinusoidal Endothelial Cell (LSEC) have far-reaching repercussions for the whole liver and systemic metabolism. LSECs are perforated with fenestrations, which are pores that facilitate the transfer of lipoproteins and macromolecules between blood and hepatocytes. Loss of LSEC porosity is termed defenestration, which can result from loss of fenestrations and/ or decreases in fenestration diameter. Gram negative bacterial endotoxin (Lipopolysaccharide, LPS) has marked effects on LSEC morphology, including induction LSEC defenestration. Sepsis is associated with hyperlipidemia, and proposed mechanisms include inhibition of tissue lipoprotein lipase and increased triglyceride production by the liver. The LSEC has an increasingly recognized role in hyperlipidemia. Conditions associated with reduced numbers of fenestrations such as ageing and bacterial infections are associated with impaired lipoprotein and chylomicron remnant uptake by the liver and consequent hyperlipidemia. Given the role of the LSEC in liver allograft rejection and hyperlipidemia, changes in the LSEC induced by LPS may have significant clinical implications. In this thesis, the following major hypotheses are explored: 1. The Pseudomonas aeruginosa toxin pyocyanin induces defenestration of the LSEC both in vitro and in vivo 2. The effects of pyocyanin on the LSEC are mediated by oxidative stress 3. Defenestration induced by old age and poloxamer 407 causes intrahepatocytic hypoxia and upregulation of hypoxia-related responses 4. Defenestration of the LSEC seen in old age can be exacerbated by diabetes mellitus and prevented or ameliorated by caloric restriction commencing early in life

The liver is the major detoxifying organ, clearing the blood from drugs and other xenobiotics. The extent of hepatic clearance (CL) determines drug exposure and hence, the efficacy and toxicity associated with exposure. Drug-drug interactions (DDIs) that alter the hepatic CL may cause more or less severe outcomes, such as adverse drug reactions. Accurate predictions of drug CL and DDI risk from in vitro data are therefore crucial in drug development. Liver CL depends on several factors including the activities of transporters involved in the hepatic uptake and efflux. The work in this thesis aimed at developing new in vitro and in silico methods to predict hepatic transporter-mediated CL and DDIs in vivo. Particular emphasis was placed on interactions involving the hepatic uptake transporters OATP1B1, OATP1B3, and OATP2B1. These transporters regulate the plasma concentration-time profiles of many drugs including statins. Inhibition of OATP-mediated transport by 225 structurally diverse drugs was investigated in vitro. Several novel inhibitors were identified. The data was used to develop in silico models that could predict OATP inhibitors from molecular structure. Models were developed for static and dynamic predictions of in vivo transporter-mediated drug CL and DDIs. These models rely on a combination of in vitro studies of transport function and mass spectrometry-based quantification of protein expression in the in vitro models and liver tissue. By providing estimations of transporter contributions to the overall hepatic uptake/efflux, the method is expected to improve predictions of transporter-mediated DDIs. Furthermore, proteins of importance for hepatic CL were quantified in liver tissue and isolated hepatocytes. The isolation of hepatocytes from liver tissue was found to be associated with oxidative stress and degradation of transporters and other proteins expressed in the plasma membrane. This has implications for the use of primary hepatocytes as an in vitro model of the liver. Nevertheless, by taking the altered transporter abundance into account using the method developed herein, transport function in hepatocyte experiments can be scaled to the in vivo situation. The concept of protein expression-dependent in vitro-in vivo extrapolations was illustrated using atorvastatin and pitavastatin as model drugs.

Doctor of Philosophy(PhD) / Owing to its strategic position in the liver sinusoid, pathologic and morphologic alterations of the Liver Sinusoidal Endothelial Cell (LSEC) have far-reaching repercussions for the whole liver and systemic metabolism. LSECs are perforated with fenestrations, which are pores that facilitate the transfer of lipoproteins and macromolecules between blood and hepatocytes. Loss of LSEC porosity is termed defenestration, which can result from loss of fenestrations and/ or decreases in fenestration diameter. Gram negative bacterial endotoxin (Lipopolysaccharide, LPS) has marked effects on LSEC morphology, including induction LSEC defenestration. Sepsis is associated with hyperlipidemia, and proposed mechanisms include inhibition of tissue lipoprotein lipase and increased triglyceride production by the liver. The LSEC has an increasingly recognized role in hyperlipidemia. Conditions associated with reduced numbers of fenestrations such as ageing and bacterial infections are associated with impaired lipoprotein and chylomicron remnant uptake by the liver and consequent hyperlipidemia. Given the role of the LSEC in liver allograft rejection and hyperlipidemia, changes in the LSEC induced by LPS may have significant clinical implications. In this thesis, the following major hypotheses are explored: 1. The Pseudomonas aeruginosa toxin pyocyanin induces defenestration of the LSEC both in vitro and in vivo 2. The effects of pyocyanin on the LSEC are mediated by oxidative stress 3. Defenestration induced by old age and poloxamer 407 causes intrahepatocytic hypoxia and upregulation of hypoxia-related responses 4. Defenestration of the LSEC seen in old age can be exacerbated by diabetes mellitus and prevented or ameliorated by caloric restriction commencing early in life

Os insulinomas são os tumores neuroendócrinos pancreáticos funcionantes mais frequentes, entretanto, os aspectos moleculares envolvidos em sua tumorigênese precisam ser melhor esclarecidos. As características morfológicas e histoquímicas dos insulinomas não conseguem predizer completamente seu comportamento biológico, apenas o fenótipo invasivo local e a presença de metástase são as formas confiáveis do diagnóstico maligno. A presente investigação teve por objetivos analisar a expressão gênica por reação em cadeia da polimerase (PCR) em tempo real e a expressão protéica por imuno-histoquímica dos componentes da via de sinalização do fator de crescimento hepatocítico (HGF) e seu receptor (c-MET) em 27 amostras de insulinomas, sendo: 16 tumores benignos grau 1 (G1), seis tumores benignos grau 2 (G2), dois insulinomas malignos grau 3 (G3) e três metástases hepáticas. Além disso, realizou-se a pesquisa de mutações somáticas no gene MET. Observou-se (1) o aumento da expressão dos genes HGF, MET e ST14 (codificante para a matriptase) e a baixa expressão do gene HAI-1 (codificante para a protease inibidora tipo-kunitz do tipo 1) nos insulinomas malignos e metástases quando comparados aos insulinomas benignos G1; (2) uma correlação positiva entre a expressão do mRNA do gene MET e o gene ST14 e índice proliferativo Ki-67, bem como uma correlação inversa entre a expressão do mRNA do gene HAI-1 e os genes MET, HGF, ST14 e o índice mitótico; (3) uma correlação positiva entre a expressão do gene ST14 e a expressão do mRNA do gene HGF; (4) maior expressão protéica de c-MET nos insulinomas malignos G3 em relação aos insulinomas G1/G2 e (5) ausência de mutações nos éxons 2, 10, 14, 16, 17 e 19 do gene MET. Concluiu-se que os genes HGF, MET, ST14 e HAI-1 estão diferencialmente expressos entre insulinomas malignos e benignos, o que pode ter implicações diagnósticas e terapêuticas / In an attempt to better understand the molecular processes involved in the tumourigenesis of islet beta-cells, the present study evaluated the expression of genes belonging to the hepatocyte growth factor and its receptor (HGF/MET) system, namely, MET, HGF; HGFAC and ST14 (encode HGF activator and matriptase, respectively, two serine proteases that catalyze conversion of pro-HGF to active HGF); and SPINT1 and SPINT2 (encode serine peptidase inhibitors Kunitz type 1 and type 2, respectively, two potent inhibitors of HGF activator and of matriptase) in 27 sporadic insulinomas; 16 grade 1 (G1), six grade 2 (G2), two grade 3 (G3) and three hepatic metastases. Quantitative reverse-transcriptase polymerase chain reaction was employed to assess RNA expression of the target genes and immunohistochemical analysis was used to evaluate the expression of MET and SPINT1. Somatic mutations of MET gene were searched by direct sequencing of exons 2, 10, 14, 16, 17 and 19. Overexpression of MET was observed in grouped G3 insulinomas and metastases concomitantly with upregulation of the genes encoding HGF and matriptase and downregulation of SPINT1. Positive correlations were observed between MET RNA expression and Ki-67 proliferation index while a negative correlation was detected between SPINT1 expression and the mitotic index. No somatic mutations were found in MET gene. The final effect of the increased expression of HGF, its activator (matriptase) and its specific receptor (MET) together with a decreased expression of one potent inhibitor of matriptase (SPINT1) is probably a contribution to tumoural progression and malignancy in insulinomas

Abstract Cytochrome P450 (CYP) enzymes are the major metabolizers of xenobiotics, e.g. drugs, and environmental toxins. Thus, changes in CYP expression have an important impact on drug metabolism and susceptibility to chemical toxicity. In the present study, the transcriptional mechanisms of both constitutive and inducible regulation of the Cyp2a5 gene in mouse liver were investigated. Mouse primary hepatocyte cultures were used as the main model system together with cell and molecular biology methods. The key activation regions of the Cyp2a5 5' promoter were determined using reporter gene assays. Two major transcription activation sites of the Cyp2a5 5' promoter, called the proximal and the distal, were found. Transcription factors hepatocyte nuclear factor-4 (HNF-4) and nuclear factor I were shown to bind to the proximal promoter. Aryl hydrocarbon receptor nuclear translocator (ARNT) and upstream stimulatory factor bound to a common palindromic E-box element in the distal promoter region. All three response elements were shown to be essential for constitutive expression of CYP2A5 in murine hepatocytes. ARNT appeared to control Cyp2a5 transcription without a heterodimerization partner suggesting active involvement of the ARNT homodimer in mammalian gene regulation. Aryl hydrocarbon receptor (AHR) ligands were shown to induce Cyp2a5 transcriptionally by an AHR-dependent mechanism, and established Cyp2a5 as a novel AHR-regulated gene. The AHR response element and the E-box, identified in these studies, were located near to each other and close to a separately defined nuclear factor (erythroid-derived 2)-like 2 binding site in the distal region of the Cyp2a5 promoter, suggesting cooperation between these elements. Peroxisome proliferator-activated receptor gamma coactivator-1α was shown to up-regulate Cyp2a5 transcription through coactivation of HNF-4α. This indicates that xenobiotic metabolism can be regulated by modification of co-activation. The present results show that CYP2A5 is regulated by several different cross-regulatory pathways. The regulatory mechanisms involved in the transcription of the Cyp2a5 gene may also control other CYP genes, especially the human ortholog CYP2A6, and may explain some of the individual variations in the metabolism of xenobiotics.

Le cancer de la prostate (PCa) est le deuxième cancer le plus courant chez les hommes au niveau mondial. Le suppresseur de la signalisation des cytokines 1 (SOCS1) est considéré comme un suppresseur de tumeur en raison de la fréquente répression épigénétique de ce gène dans de nombreux cancers. Il a été reporté que SOCS1 inhibait l’activation de STAT3 induite par l’IL-6, ainsi que les cyclines et les kinases dépendantes des cyclines dans les cellules malignes de la prostate. D’autre part, il a été montré que SOCS1 n’était pas essentiel lors du contrôle de la signalisation de l’IL-6 dans les hépatocytes dépourvus de cette protéine, cependant elle est essentielle pour atténuer la signalisation du facteur de croissance des hépatocytes (HGF) via son récepteur MET. MET est un récepteur de tyrosine kinases qui est surexprimé dans le PCa agressif et métastatique. Notre hypothèse de recherche propose que la répression de SOCS1 par méthylation du promoteur et la dérégulation de l’expression de MET et de sa signalisation, sont des mécanismes pathogéniques liés au développement et à la progression du PCa. Nous avons généré des lignées de cellules PC3 et DU145 stables exprimant SOCS1. Les cellules ont été stimulées avec HGF et l’activation des voies de signalisation a été évaluée par immunobuvardage. Des essais in vitro de migration, de prolifération et d’invasion ont été effectués en présence de HGF. Des gènes de transition épithélio-mésenchymateuse ont été évalués par PCR quantitatif en présence ou non du facteur de croissance. Les cellules du PCa transfectées ou pas avec SOCS1 ont été inoculées dans des souris NOD SCID gamma de façon sous-cutanée ou orthoptique afin d’évaluer respectivement la croissance tumorale et la formation de métastases. Les tumeurs reséquées ont été analysées histologiquement et biochimiquement. Nos résultats montrent que SOCS1 atténue l'activation de MET induite par HGF et la phosphorylation d’ERK dans les cellules PC3, ainsi que la phosphorylation d’ERK et d’AKT dans les cellules DU145. SOCS1 inhibe également la prolifération cellulaire induite par HGF, ainsi que la migration et l’invasion in vitro. De plus, SOCS1 réduit l’expression des gènes de transition épithélio-mésenchymateuse impliqués dans la dégradation des composants de la matrice extracellulaire dans les cellules DU145 mais pas dans les cellules PC3. La surexpression de SOCS1 a stimulé l’augmentation de déposition de collagène, in vivo. Les tumeurs formées par les cellules exprimant SOCS1 étaient de taille significativement plus petites avec une réduction de la prolifération comparé aux tumeurs provenant des cellules contrôles. En outre, SOCS1 a inhibé la formation de métastases à distance dans un modèle orthotopique. En conclusion, nous suggérons que SOCS1 est un suppresseur de tumeur indispensable de la prostate, et qu’au moins une partie de sa fonction a lieu via la régulation négative de la signalisation du récepteur MET. / Abstract : Prostate cancer (PCa) is the second most common cancer among men worldwide. Suppressor of cytokine signaling 1 (SOCS1) is considered a tumor suppressor due to frequent epigenetic repression of the SOCS1 gene in several human malignancies. Inactivation of SOCS1 also occurs in PCa by gene methylation and micro-RNA-mediated repression. SOCS1 has been reported to inhibit IL-6-induced STAT3 activation and down-regulates cyclins and cyclin-dependent kinases in PCa cells. It has been shown that SOCS1 is not required to control IL-6 signaling in SOCS1-deficient hepatocytes, but is essential to attenuate hepatocyte growth factor (HGF) signaling via its receptor MET. This protein is a receptor tyrosine kinase (RTK), overexpressed in aggressive and metastatic PCa. Thus we hypothesized that the repression of SOCS1 via promoter methylation and deregulated MET expression and signaling are inter-related pathogenic mechanisms in PCa development and progression. We generated stable SOCS1-expressing PCa cell lines (PC3 and DU145) using lentiviral transduction followed by clonal selection via limiting dilution. Cells were stimulated with HGF and downstream signaling events were assessed by Western blot. Proliferation, migration and invasion assays were also conducted in the presence of HGF in vitro. Epithelial mesenchymal transition genes were evaluated by qPCR in the presence or absence of the growth factor. The PCa cells transfected with SOCS1 and non-transfected controls were inoculated into NOD SCID gamma mice as xenografts or as orthotopic tumors to assess tumor growth and metastasis formation, respectively. Resected tumors were further analyzed histologically and biochemically. Our results showed that SOCS1 attenuates HGF-induced MET activation and ERK phosphorylation in PC3 and DU145 PCa cell lines. SOCS1 inhibited HGF induced cell proliferation, migration and invasion in vitro. Additionally, SOCS1 decreased epithelial mesenchymal transition genes involved in the degradation of extracellular matrix components in DU145 cells but not in PC3. In vivo, SOCS1 overexpression leads to an increase of collagen deposition. Tumors formed by SOCS1 expressing cells were significantly smaller in size with reduced cell proliferation compared to tumors arising from control cells. Furthermore, SOCS1 inhibited distant metastasis formation in the orthotopic model. Overall our results suggest that SOCS1 has a tumor suppressor role in PCa evolution and part of this function is mediated by the negative regulation of MET receptor signalling and down-regulation of genes supporting migration and invasion processes such as matrix metalloproteinases.

Le virus de l’hépatite C (HCV) infecte les hépatocytes. Il est remarquable par le fait q’ilperturbe fortement le metabolisme lipidique de l’hôte, conduisant à des dysfonctions majeures telles qu’une stéatose ou une résistance à l’insuline. In vivo, les virions sériques présentent une densité faible et variable reflétant leur association aux lipoprotéines de faible et de tres faible densité (LDL et VLDL). Ainsi, l'existence de lipo-viro-particules (LVP), contenant à la fois les constituants viraux et les apolipoprotéines B (apoB) et E (apoE) a été suggerée. Ces LVP joueraient un rôle important dans la persistance virale. Cependant, cette association entre HCV et apoB n'a pas été retrouvée in vitro avec les modeles cellulaires disponibles.Mes travaux de thèse se sont donc concentrés sur la mise en place d'un nouveau modèle deculture cellulaire capable de produire a la fois des VLDL et des particules virales HCV, permettantainsi d’étudier l’interaction entre les deux voies de synthèse. Dans un premier temps, lacaracterisation de la production de lipoproteines par differentes lignees d'hepatocytes a permis demontrer l'existence d'un défaut de secretion de VLDL en cellules Huh7.5, classiquement utiliséespour étudier HCV in vitro, alors que les cellules HepG2 et HepaRG sont capables de produire des VLDL physiologiques. Dans un second temps, des cellules HepG2 repliquant HCV de manière persistante ont été utilisées pour caractériser les particules virales produites. Etonnamment, a l'instar des cellules Huh7.5 et malgré leur capacité a produire des VLDL, les cellules HepG2 ne secreteraient pas de LVP mais des particules virales positives pour apoE et négatives pour apoB. / Hepatitis C virus (HCV) mainly infects hepatocytes. It is unique in its ability to impair host lipidmetabolism, leading to major liver dysfunctions as, for instance, hepatic steatosis or insulinresistance. In vivo, serum virions have a low and variable density, reflecting their association withlow- and very-low-density lipoproteins (LDL and VLDL). Hence, the existence of lipo-viro-particles(LVP), containing both viral components as well as apolipoprotein B (apoB) and E (apoE), has beensuggested. These LVPs could play an important role in viral persistence. However, this associationbetween HCV and apoB has not been observed in vitro, using the currently available cell culturemodels.Therefore, during my PhD, I have worked at setting up a new cell culture model, which wascapable of producing both VLDL and HCV particles, and therefore would enable the study of theinterplay between both synthesis pathways. First of all, we characterized lipoprotein production indifferent hepatocyte cell lines and confirmed that Huh7.5 cells, usually used to study HCV in vitro,were deficient for VLDL secretion, whereas two other cell lines, HepG2 and HepaRG, were able toproduce quasi-physiological VLDLs. Therefore, in a second time, we used HepG2 cells to replicate aHCV strain containing a selection gene and to characterize viral particle production. Surprisingly,VLDL-producing HepG2 cells were also unable to secrete LVPs, but rather secreted apoE-positive andapoB-negative viral particles, which were similar to ones Huh7.5 cells produced. This suggests thatthe ability to produce LVPs does not correlate with the ability to produce VLDL.