Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Patrícia Čečková Supervisor: PharmDr. Ivan Vokřál, Ph.D. Title of diploma thesis: Study of drug interactions with OATP family transporters using intestinal tissue slices An essential role in the action of orally administered drugs is their absorption through the intestinal barrier. It expresses a variety of transporters, including the OATP2B1 and OATP1A2 influx transporters, belonging to the SLC family. They are located on the apical membrane of enterocytes and allow the flow of endogenous and exogenous substances from the lumen of the intestines to the enterocyte. They affect not only the pharmacokinetics of drugs, but also their safety and efficacy. They represent sites of drug interactions with other drugs/food components that may altered drug efficacy or toxicity. Since FDA (The Food and Drug Administration) and EMA (European Medicines Agency) do not have intestinal OATP transporters included in their guidelines for preclinical studies, there is no single model of interaction study. The limitations of cell models and genetically modified organisms lead to the development of new methods such as the ex vivo method of precision cut intestinal slices (PCIS), which represents a tissue model...

A promising approach for the study of Glioblastoma are organotypic murine brain tissue slices as a substrate for the glioma cells to invade into. Current 3D assays based on this principle involved the use of tumor spheroids or cell suspensions in co-culture with the brain tissue slices. While this allowed for the study of glioma cell invasion, tumor spheroids lack the microarchitecture of patient-derived tumor tissue or glioma xenografts. This study has expanded this type of assay by investigating the viability of glioma xenograft slices in co-culture with organotypic murine brain tissue slices, proposing facile quantification methods of tumor growth and invasion and using this system for studying the effects of small molecule inhibitors. The organotypic murine brain tissue slices were prepared by slicing mouse brains in the coronal axis, using a vibratome, to a thickness of 300 µm. The slices were then transferred onto tissue culture membrane inserts for growth in air-liquid interface culture. A single mouse brain allowed for the production of multiple organotypic murine brain tissue slices, thus drastically reducing the number of animals needed for the study. GBM tumor xenografts from mice were sliced similarly on a vibratome, and circular portions with a diameter of 2 mm were placed on top of the murine brain tissue slices. After 7 days in culture, tissue slice co-cultures were analyzed by immunohistochemical staining of vertical sections, containing both the tumor and the murine brain tissue, for Type III intermediary filament proteins Vimentin and GFAP and the neural crest cell marker S-100. Independent of the cell line used for xenograft preparation, tumor tissues stained strongly positive for Vimentin, while the normal mouse brain tissue stained negative (in the studied region), so Vimentin was used as the primary tumor marker. For the quantification of the data acquired from micrographs, the tumor height, depth as well as the area of the invading cells and the tumor upper area (situated above the air margin of the brain tissue slice), the tumor lower area and the recipient tissue area were measured. From these parameters, a number of indices for each sample were derived, such as the tumor invasion index (TI-index), the tumor space occupying growth index (SOG-index), the tumor invasion depth index (TID-index), the space occupying growth depth index (SOGD-index) and the total tumor depth index (TTD-index). To validate the proposed quantification method, the results were compared with tumor spheroid tandem co-cultures. It was shown to be possible for GBM tumor xenografts to maintain their viability and invasive properties in co-culture with organotypic murine brain tissue slices. This was demonstrated immunohistochemically with xenografts from GBM cell lines G55T2, U-87 MG, LN-229 and T98G, displaying progressive tumor cell invasion from day 3 to 7 in co-culture. Tumor cell viability and proliferation in the ex vivo setting were also confirmed by Ki-67 staining. The usage of GBM tumor xenografts was also advantageous compared to spheroid-based assays. The direct comparison between the tumor spheroid and tumor xenograft co-cultures showed stark differences between assays even when using the same cell line. U-87 MG cells showed little or no invasiveness in the spheroid model but the glioma cells were diffusely spread into the murine brain tissue in the xenograft model. G55T2 xenograft co-cultures on the other hand displayed a significant increase of the SOG-index compared to their spheroid counterparts. Results were also compared to previous findings in an orthotopic tumor xenograft model, concluding that the proposed ex vivo model showed significant advantages compared to the orthotopic model by being more facile and cost-effective to implement and displaying comparatively more profound tumor invasiveness when studying the same cell lines. The strong increase of the invasive and space assuming properties of glioma cells in xenograft tissue slice tandem cultures also supports the hypothesis that they are superior to spheroid-based assays in studying tumor invasiveness. The developed GBM xenograft tissue slice tandem-cultures were also used for the ex vivo analysis of drug effects. The treatment with the Pim1 small molecule inhibitor SGI-1776 revealed after 7 days a decrease in the TI-index and SOG-index compared to the untreated group. A similar experiment was performed on spheroid co-cultures using a combination of SGI-1776 and the STAT3 inhibitor Stattic, also resulting in reduced TI- and SOG-indices. From this work, it can be concluded that the developed 3D ex vivo method is a facile and cost-effective platform to study the growth and invasiveness of GBM xenograft tumors in an in vivo-like environment. Owing to the large number of samples that can be generated from a single mouse, it has the potential to drastically reduce the number of animal experiments, addressing the 3R principle. It also showed more profound tumor cell invasiveness compared to spheroid-based ex vivo or orthotopic in vivo xenograft models and provides the quantification tools needed for preclinical drug testing. The model also has the potential to be expanded towards the usage of patient-derived tumor tissue as well as the preclinical testing of non-drug-based therapy options.:1 Introduction 1 1.1 Definition of Glioblastoma 1 1.2 Epidemiology, presentation 2 1.3 Etiology 5 1.4 Molecular pathways relevant in Glioblastoma tumorigenesis 5 1.5 Treatment options 9 1.6 Research models 11 1.7 Tissue slice models 15 1.8 Aims and research objectives 18 2 Publication 19 3 Summary 34 4 References 39 5 Supplementary materials 60 5.1 Additional materials 65 5.2 Comparison of immunohistochemical data to available literature 66 6 Darstellung des eigenen Beitrags 67 7 Erklärung über die eigenständige Abfassung der Arbeit 69 8 Publications 70 9 Curriculum vitae 71 10 Acknowledgements 72

Development of a three dimensional (3D) HYBROT model with targeted in vivo like intact cellular circuitry in thick brain slices for multi-site stimulation and recording will provide a useful in vitro model to study neuronal dynamics at network level. In order to make this in vitro model feasible, we need to develop several associated technologies. These technologies include development of a thick organotypic brain slice culturing method, a three dimensional (3D) micro-fluidic multielectrode Neural Interface system (µNIS) and the associated electronic interfaces for stimulation and recording of/from tissue, development of targeted stimulation patterns for closed-loop interaction with a robotic body, and a deep-tissue non-invasive imaging system. To make progress towards this goal, I undertook two projects: (i) to develop a method to culture thick organotypic brain slices, and (ii) construct a multiphoton imaging system that allows long-term and deep-tissue imaging of two dimensional and three dimensional cultures. Organotypic brain slices preserve cytoarchitecture of the brain. Therefore, they make more a realistic reduced model for various network level investigations. However, current culturing methods are not successful for culturing thick brain slices due to limited supply of nutrients and oxygen to inner layers of the culture. We developed a forced-convection based perfusion method to culture viable 700µm thick brain slices. Multiphoton microscopy is ideal for imaging living 2D or 3D cultures at submicron resolution. We successfully fabricated a custom-designed high efficiency multiphoton microscope that has the desired flexibility to perform experiments using multiple technologies simultaneously. This microscope was used successfully for 3D and time-lapse imaging. Together these projects have contributed towards the progress of development of a 3D HYBROT. ----- 3D Hybrot: A hybrid system of a brain slice culture embodied with a robotic body.

Astrocytes need to preserve constant volume in the face of osmolarity perturbations to function properly. To regain their original volume after hyposmotically induced swelling, they extrude intracellular electrolytes and organic osmolytes, such as inorganic ions, excitative amino acids or polyols, accompanied by osmotically driven water. This process is termed regulatory volume decrease and is ensured by various ion channels and transporters. Recently, much attention has been focused on the ubiquitous volume-regulated anion channels activated by cell swelling. VRACs are moderately outwardly rectifying with intermediary conductance, permeable to inorganic anions and organic osmolytes and sensitive to broad-spectrum anion channels blockers. Using patch-clamp technique we aimed to characterize VRACs in cultured cortical astrocytes isolated from neonatal Wistar rats and to elucidate the effect of intracellular Na+ on VRAC activity. In addition, we also intended to characterize these channels in situ in brain slices of 10 - 12 days old rats, focusing mainly on hippocampal astrocytes. To induce astrocytic swelling, we exposed astrocytes to hypotonic solution (250 mOsm). In agreement with previous findings, we showed that cultured cortical astrocytes activate VRAC currents upon exposure to hypotonic stress, which...

Background: Glucose homeostasis is tightly regulated by hormones secreted within the pancreatic islets of Langerhans. The most important are insulin and glucagon produced by beta and alpha cells respectively. Changes in beta cell mass and/or their functional deficit can lead to hyperglycemia, a major hallmark of both type 1 (T1D) and type 2 (T2D). Moreover, a dysregulation in glucagon secretion is thought to also play a major role in patients with diabetes, suggesting a failure in the counterregulatory mechanisms of glucose homeostasis in disease pathogenesis. Dysfunction at the alpha cell level in T1D manifests are blunt glucagon response to low glucose levels, which can cause severe hypoglycemic events in patients with T1D. Furthermore, exaggerated glucagon responses to glucose or amino acid intake significantly contributes to dysglycemia in both T1D and T2D patients. Most of our knowledge about glucagon and alpha cell physiology in the human setting was generated using in vivo systemic assessments or in vitro investigations of isolated human islets or dispersed single cells. Despite the increasing knowledge regarding alpha cells and glucagon biology, the underlying mechanisms of alpha cell dysfunction are still uncertain. Studies on alpha cell physiology were hindered by limited human tissue accessibility, technical methodologies and translational value of findings from rodents to humans. To fill the gap between the currently available in vivo and in vitro approaches and a more precise understanding of mechanisms of diabetes pathogenesis detailed investigation of islet cells within their native environment is needed. Aim The overall objective of this thesis was characterize alpha cell function in diabetes pathogenesis. To this end, the human pancreas slice preparation would to be adapted and advanced for the study of alpha cell physiology. These adjustments would be then used to investigate changes in alpha cell mass and function in T1D and T2D. Methods: Pancreas tissue slices were prepared from donor organs with and without T1D and from tissue donors after pancreatectomy at different stages of T2D. Immunofluorescent staining with subsequent 3D morphometry was used to quantify alpha cell volumes from 120μm thick tissue slices. Furthermore, human tissue slices were subjected to dynamic slice perifusion for the assessment of glucagon and insulin secretion kinetics in response to specific stimuli. Finally, functional and morphometrical analysis was performed on the same tissue slices to enable direct correlation of glucagon secretion and alpha cell volume in a subset of cases in the context of T1D. Results: Here we developed a semi-automatic 3D approach to quantify total endocrine cell volumes within a given volume of pancreas tissue. In addition, we established an in situ method for dynamic insulin release measurements from islets preserved in their native environment. We successfully modified this protocol to allow the measurement of glucagon release in slices from organ donors. After further optimizations, we were additionally able to also measure alpha cell function from surgical specimens after pancreatectomy. To gain insight into alpha cell pathophysiology in T1D we investigated alpha cell volume in donor organs with different disease duration and age at onset. Alpha cell volumes in slices of individuals with T1D did not show a dramatic change (neither increase nor decrease) in comparison to slices generated from non-diabetic (ND) pancreata. Furthermore, functional assessment of glucagon release using a specific stimulation protocol for alpha cells suggests preserved stimulatory capacity of these cells in slices from autoantibody positive donors. Interestingly, this is also the case in the so far studied slices from donors with different durations of diabetes. Nevertheless, normalization of secreted glucagon to the total alpha cell mass within the slice indicated reduced glucagon release in the here investigated two cases of T1D. In the context of T2D, 3D morphometrical analysis revealed that overall endocrine cell volume, including alpha cell volume, is maintained in our cohort of IGT and T2D individuals. Glucagon release can also be measured in tissue procured from patients undergoing pancreatectomy, given the presence of amino acids in the perifusion media and increased trypsin inhibitors. While we provided proof of concept using tissue from ND individuals, we are confident that the approach will give valuable insight in different states of diabetes. Conclusion: These results demonstrate that human pancreas tissue slices represent a complementary platform to study alpha cell pathophysiology in both major types of diabetes. We provide evidence that this approach can be used to study alpha cell pathophysiology in T1D and T2D. Our preliminary data indicates no defect in the stimulatory capacity in slices from Aab+ and T1D donors, however more cases need to be investigated given the heterogeneous nature of the disease. We anticipate that the here proposed protocol for measurement of glucagon release from tissue slices will help us to gain insight in the role of alpha cells in diabetes pathophysiology. / Hintergrund: Die Aufrechterhaltung der Glukosehomöostase wird durch die Hormonsekretion der Langerhans’schen Inseln im Pankreas reguliert. Die wichtigste Rolle hierbei spielen die Insulin-produzierenden Betazellen und die Glukagon-produzierenden Alphazellen. Der Verlust der Betazellmasse und/oder der Funktion kann zur Entwicklung einer Hyperglykämie führen, die ein Hauptmerkmal des Typ 1 (T1D) und Typ 2 (T2D) Diabetes ist. Darüber hinaus wird vermutet, dass auch der Mechanismus der Gegenregulierung durch die Sekretion von Glukagon eine wichtige Rolle in der Pathogenese des Diabetes spielt. Während eine fehlende Glukagonsekretion zu schweren hypoglykämischen Phasen bei Typ 1 Diabetikern führen kann, geht man zusätzlich davon aus, dass eine erhöhte Reaktivität von Alphazellen sowohl auf Glukose als auch Aminosäuren ebenfalls zum Verlust der Glukosehomöostase im T1D und T2D beitragen kann. Trotz der stetig wachsenden Erkenntnisse über die Physiologie der Alphazellen, die vor allem durch systemische Untersuchungen in vivo oder an isolierten Langerhans’schen Inseln und Einzelzellen in vitro durchgeführt wurden, sind die zugrundeliegenden Mechanismen für deren Fehlfunktion beim Menschen noch nicht eindeutig aufgeklärt. Dies beruht hauptsächlich auf nur bedingt vorhandenem humanem Gewebe, technischen Schwierigkeiten bei der Isolation der Zellen, sowie der nur bedingten Vergleichbarkeit zu Studien in Nagern. Um diese Wissenslücken zwischen in vivo und in vitro Studien schließen zu können, ist es notwendig detaillierte Untersuchungen der Zellen unter nahezu physiologischen Bedingungen und in der nativen Umgebung der Pankreas in situ durchzuführen, um Alphazell-spezifische Mechanismen in der Diabetespathogenese genauer beleuchten zu können. Ziele: Ziel dieser Dissertation war es, durch Anpassung und Weiterentwicklung der Technik zur Gewinnung von Gewebeschnitten des humanen Pankreas, die Funktion der Alphazellen sowohl unter physiologischen Bedingungen als auch in der Entwicklung des T1D und T2D genauer zu charakterisieren. Methoden: Zur Untersuchungen von Alphazellen in Gewebeschnitten in situ wurden Gewebestücke sowohl von Organspendern mit und ohne T1D, sowie von metabolisch charakterisierter Patienten in verschiedenen Stadien der T2D Pathogenese nach einer Pankreatektomie verwendet. Die aus dem Gewebe gewonnenen 120 μm dicken Schnitte wurden zum einen für immunhistochemischer Färbungen verwendet, die eine 3-dimensionale morphometrische Analyse der Alphazellmasse ermöglichen. Ferner wurden Schnitte zur Ermittlung der Kinetik von Glukagon- und Insulinsekretion nach Stimulation mittels Perifusion benutzt. Schließlich wurden sowohl die morphologischen als auch funktionellen Analysen auf denselben Gewebeschnitten durchgeführt, um die Funktion der Alphazellen mit deren Masse besser korrelieren zu können. Ergebnisse: Zusätzlich zur Mitentwicklung eines halbautomatisierten Verfahrens zur 3D Analyse von endokrinen Zellvolumina in Gewebeschnitten des Pankreas wurde die bereits vorhandene Methode zur Messung der Insulinsekretionskinetik weiterentwickelt. Außerdem erfolgte die Etablierung adäquater Protokolle zur Messung der Glukagonsekretion in humanen Gewebeschnitten, die im Kontext beider Diabetestypen verwendet wurden. Um einen besseren Einblick in die T1D Pathogenese zu erhalten, wurde das Alphazellvolumen- und die Funktion in Gewebeschnitten von Organspendern mit unterschiedlichem Diabetes Verlauf (Alter bei Diagnose, Dauer seit Diagnose) mit nicht-diabetischen, aber Autoantikörper-positiven Spendern und nicht-diabetische Kontrollen verglichen. In Bezug auf das Alphazellvolumen waren zwischen den einzelnen Gruppen keine Unterschiede zu erkennen, die auf Veränderungen in der Entwicklung und Manifestation des T1D hinweisen. Darüber hinaus ergab die funktionelle Analyse, dass die Glukagonsekretion in nicht-diabetischen, Autoantikörper-positiven Spendern erhalten bleibt. Dies konnte zusätzlich auch in den bisher untersuchten Geweben von Typ 1 Diabetikern nachgewiesen werden, obwohl die Volumen-normalisierte Sekretion auf eine geringere Glukagonausschüttung hindeutet. Im Hinblick auf die T2D Pathogenese konnte bei der 3D Morphometrie von Nichtdiabetikern, Patienten mit beeinträchtigter Glukosetoleranz und Typ 2 Diabetikern keinerlei Unterschiede in den endokrinen Zellvolumina festgestellt werden. Durch die Anpassung der Konditionen für die Perifusion von reseziertem Gewebe konnte bei Nichtdiabetikern die erfolgreiche Messung der Glukagonsekretion gezeigt werden und ermöglicht zukünftig auch die Untersuchung einer möglichen Alphazelldysfunktion in der Entwicklung eines T2D. Schlussfolgerung: Die Ergebnisse dieser Arbeit zeigen, dass die etablierte Plattform zur morphologischen und funktionellen Analyse humaner Pankreasgewebeschnitte in situ eine wichtige Rolle in der Untersuchung der Alphazellen in der Diabetes-Pathogenese spielt. Die bisher erhobenen Daten zur Untersuchung des T1D haben gezeigt, dass die Kapazität der Glukagonsekretion nicht signifikant verändert ist. Aufgrund des heterogenen Krankheitsverlaufs beider Diabetesformen ist es jedoch notwendig Gewebe von einer größeren Anzahl an Spendern/Patienten zu untersuchen, um einen besseren Einblick in die Rolle der Alphazellen in der Entstehung des Diabetes zu erhalten.

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