Introdução: O carcinoma espinocelular (CEC) representa 7% de todos os novos casos de câncer no mundo, sendo o carcinoma espinocelular o tipo mais frequente. Tanto o comportamento biológico quanto o crescimento dos tumores devem ser melhores entendidos, uma vez que a sobrevida dos pacientes apresentou discreta melhora nas últimas décadas. Os modelos PDX foram desenvolvidos para estudar a biologia tumoral e principalmente os mecanismos de crescimento e proliferação através da manutenção da arquitetura e microambiente tumoral do tumor original. Os retinóides possuem a capacidade de restaurar o crescimento e a diferenciação de células normais através da ação dos receptores retinóides nucleares (RARs e RXRs) que são os principais mediadores destas ações que ao sofrerem alterações na sua expressão podem levar ao desenvolvimento e manutenção de tumores. No estudo da carcinogênese o modelo PDX é uma importante ferramenta pois mantém a arquitetura e microambiente do tumor original melhorando a compreensão de algumas vias, entre estas o processo de EMT/MET, na diferenciação das células tronco tumorais e quais receptores nucleares podem estar influenciando nestas vias. Objetivos: Analisar os padrões de comportamento biológico - tempo de formação e expansão do tumor e a manutenção dos padrões histológicos e de arquitetura do tumor original - em F0 e F1 no modelo PDX (xenoenxerto derivado de paciente) das amostras de centro de tumor e epitélio adjacente em camundongos Balb C/nude e avaliar a expressão gênica dos receptores retinóides, ALDH1 e marcadores do processo de EMT/MET por RT-PCR em PDX de carcinoma espinocelular oral em comparação com a amostra dos pacientes doadores nas passagens F(0) e F(1). Método: 24 camundongos Balb C/Nude, divididos em 2 grupos TG(I) – tumor graft paciente (I) e TG(II) – tumor graft paciente (II), subdivididos em 4 grupos de 3 animais: (A) – receberam PDX do centro do tumor; (B) – receberam PDX de epitélio adjacente ao tumor (margem de segurança cirúrgica); (C) receberam PDX de um animal do grupo (A); (D) receberam PDX de um animal do grupo (B). E Após estas fases, as amostras coletadas serão avaliadas por RT-PCR para comparação das expressões gênicas entre a amostra original (CT e EA) com os PDX´s nas passagens F(0) e F(1). Resultados: formação de tumores em todos os grupos – tanto do PDX de fragmento de centro do tumor quanto do PDX do epitélio adjacente. E A expressão gênica dos parâmetros observados não diferem no tumor original e passagem F(0) significativamente diferentes em F(1) (p<0,05). Conclusões: A técnida do PDX para o CEC é possível de ser realizada em menor tempo com a implantação de apenas um fragmento do tumor. Os tumores resultantes do PDX apresentaram tamanho suficiente para novas passagens, bem como para seu 6 uso em estudos de comportamento biológico das células neoplásicas. Quanto ao epitélio adjacente ao tumor (margem de segurança cirúrgica) constatou-se a presença de células tumorais com potencial de promover o crescimento de tumores devendo portanto ser melhor observada nas ressecções. O PDX de primeira passagem F(0) é o que mais se assemelha com o tumor original sendo o melhor para testes terapêuticos e estudos da carcinogênese do CEC oral. Keywords: CECP, modelo PDX, xenoenxerto, margem de segurança cirúrgica, , receptores retinóides, microdissecção a laser. / Introduction: Squamous cell carcinoma (SCC) represents 7% of all new cases of cancer in the world, with squamous cell carcinoma being the most frequent type. Both the biological behavior and the growth of the patients should be better understood, since the patients' survival show unobtrusive improvement in the last decades. PDX models were developed to study a tumor biology and especially the mechanisms of growth and proliferation through maintenance of the architecture and tumor microenvironment of the original tumor. Retinoids have a capacity to restore normal cell growth and differentiation through the action of nuclear retinoid receptors (RARs and RXRs) that are the main mediators and maintenance actions of tumors. In the study of carcinogenesis, the PDX model is an important tool because it maintains an architecture and microenvironment of the original tumor, improving an understanding of some pathways, among them in the EMT / MET process, the difference in tumor stem cells and which nuclear receptors may be influencing these routes. Objectives: To analyze changes in methodology and patterns of biological behavior - time of tumor formation and expansion and maintenance of histological and architectural patterns of the original tumor - in F0 and F1 without PDX model (patient derived xenograft) tumor and adjacent epithelium in Balb C / nude mice and to evaluate the gene expression of retinoid receptors, ALDH1 and EMT / MET process markers by RT-PCR in PDX of oral squamous cell carcinoma compared to a sample of donor patients in F ( 0) and F (1). Method: 24 Balb C / Nude mice, divided into 2 groups TG (I) - patient tumor graft (I) and TG (II) - patient tumor graft (II) subdivided into 4 groups of 3 animals: (A) - received PDX from the center of the tumor; (B) - received epithelial PDX adjacent to the tumor (surgical margin of safety); (C) received PDX from one animal of group (A); (D) received PDX from one animal of group (B). E After these phases, as samples collected for RT-PCR evaluation for comparison of gene expressions between an original sample (CT and EA) with F passages of PDX F (0) and F (1). Results: tumor formation in all groups - both the PDX of the tumor center fragment and the PDX of the adjacent epithelium. E The gene expression of the observed parameters did not differ without original tumor and F (0) differential passage in F (1) (p <0.05). Conclusions: The PDX technique for CPB is possible to be performed in a shorter time with a tumor fragment implantation. Tumors resulting from PDX presented the solution for new passages, as well as for their use in studies of the biological behavior of neoplastic cells. As for the epithelium adjacent to the tumor (surgical margin of safety), a presence of tumor cells with the potential to promote the growth of tumors has been observed and should therefore be better observed in the resections. The first pass PDX F (0) is the one that most closely resembles the 8 original tumor being the best for therapeutic tests and studies of oral SCC carcinogenesis.

Small cell lung cancer (SCLC) is an extremely aggressive disease characterized by early metastasis and acquired resistance to therapy. SCLC is distinguished by its neuroendocrine (NE) component; the role of which is not fully understood in metastasis and response to therapy. Patients respond exceptionally well to first round chemotherapy; however, relapse with therapy-resistant tumours is virtually inevitable. Hypoxic regions within tumours can contribute towards metastasis and therapy resistance, highlighting hypoxia-targeted therapy as a novel approach for improving treatment for SCLC patients. Tumours are highly phenotypically heterogeneous, raising debate over the roles played by each cell type. Analysis of NE and epithelial markers in SCLC cell lines highlighted this inter-tumour heterogeneity. Further heterogeneity is displayed in SCLC xenograft tumours that show areas of dual epithelial and NE marker expression as well as regions negative for both markers. Irradiating xenograft tumours enhanced heterogeneity of the NE marker, pro-opiomelanocortin (POMC), which is ectopically secreted by a subset of SCLC tumours. Examining changes in marker expression post-therapy could provide vital information regarding transitions that can serve to guide therapy. SCLC is a highly metastatic disease. The role of the NE phenotype in human SCLC is not fully understood, but is considered essential for metastasis in murine models. Sub-cutaneous, intravenous and intra-splenic injection were carried out and resulted in no metastasis, spontaneous tumour generation and peripheral liver tumour growth, respectively. POMC expression was present and extremely heterogeneous within the liver, suggesting that NE properties are maintained in metastases; however, further work is necessary to develop a more consistent metastatic model that can be used to assess responses to therapy in a more clinically relevant setting. SCLC tumours proliferate rapidly and outgrow their nutrient and oxygen supplies, resulting in hypoxic conditions. Here, carbonic anhydrase IX (CA IX) becomes up-regulated in order to maintain pH levels suitable for survival. The specific CA IX inhibitor, S4, induces hypoxia-specific cell death in vitro and impairs tumour growth in vivo. This response is further accentuated by combining S4 with single or repeated cisplatin doses. Combination treatment reduced gene expression of S-phase kinase-associated protein (Skp2), associated with cisplatin resistance. CA IX inhibition combined with cisplatin chemotherapy therefore presents a novel treatment for SCLC tumours that could reduce therapy resistance. In summary, heterogeneity is extremely important when choosing treatment options for SCLC and must be considered when basing treatment on single biopsies. NE and epithelial markers are present within sub-cutaneous and liver tumours; however, a reliable multi-organ metastatic model is necessary to fully appreciate the role of these markers in the spread of SCLC. Hypoxic regions within sub-cutaneous xenograft tumours upregulate CA IX. Inhibition of this enzyme resulted in impaired tumour growth, particularly when used together with cisplatin. Combining CA IX inhibition with cisplatin presents a much-needed novel therapy for SCLC.

Hsp90 (heat shock protein 90) est une protéine chaperonne ubiquitaire et conservée impliquée dans le repliement et la réparation de protéines dites « clientes ». Parmi ces protéines, de nombreuses sont impliquées dans des phénomènes oncogéniques, faisant de hsp90 une cible d’intérêt dans le traitement du cancer. Hsp90 est constituée de trois domaines, un domaine N-terminal site de l’hydrolyse de l’ATP, nécessaire à sa fonction ; un domaine intermédiaire où vient se fixer la protéine cliente et un domaine C-terminal impliqué dans la dimérisation, étape indispensable pour le repliement de la protéine cliente. De nombreux inhibiteurs ont été synthétisés en ciblant ces différents domaines. Les inhibiteurs N-terminaux sont efficaces, à l’instar de la Geldanamycine en termes d’activité anti-tumorale mais des effets secondaires ainsi que des résistances au traitement ont limité leur utilisation en pratique clinique. En effet, l’inhibition N-terminale induit une réponse au stress caractérisée par une augmentation de hsp90 et de ses co-chaperonnes, souvent associée à une résistance au traitement et un pronostic défavorable. La novobiocine, un antibiotique coumarinique, est capable d’inhiber le domaine C-terminal de hsp90, sans induire de réponse au stress. Ainsi de nombreux dérivés de cette molécule ont été synthétisés, parmi lesquels on trouve le 6BrCaQ. Cette molécule induit l’apoptose et le blocage dans le cycle cellulaire sur plusieurs lignées cellulaires (dont MCF-7 et MDA-MB-231) et provoque la dégradation de plusieurs protéines clientes impliquées dans le développement tumoral mais sa faible solubilité limite son administration in vivo.Dans cette thèse, une forme liposomale du 6BrCaQ a été développée et étudiée sur des lignées cellulaires de cancer de prostate, de sein et de leucémie aigüe myéloïde in vitro et in vivo sur un modèle orthotopique de cancer du sein (MDA-MB-231 luc-GFP). Le 6BrCaQ liposomal est capable d’ induire de l’apoptose, de bloquer le cycle cellulaire sur différentes lignées cellulaires (PC-3, MDA-MB-231 et MOLM-13) mais également de ralentir la migration cellulaire sur PC-3 (test de comblement de blessure). De plus, le 6BrCaQ liposomal entre en synergie avec la doxorubicine (cellules PC-3) et la daunorubicine (cellules MOLM-13). Au niveau moléculaire, les liposomes de 6BrCaQ modifient l’expression protéique de Hsp90 sans modifier celle d’Hsp70 sur PC-3 alors que les gènes codant pour les Hsp70 semblent être légèrement induits dans MDA-MB-231. Les résultats in vivo ont montré un ralentissement de la croissance tumorale sur un modèle de cancer du sein orthotopique (MDA-MB-231-luc2-GFP) dès 13 jours de traitement pour une dose de 1 mg/kg injectée une fois par semaine. Des analyses histologiques ont révélé une augmentation de la proportion des zones nécrotiques dans le groupe traité par rapport au contrôle et une diminution significative de la prolifération cellulaire (marquage au KI67) intra-tumorale.Par ailleurs, Hsp90 possède également des isoformes et des analogues localisés dans des organites intracellulaires, parmi lesquelles, TRAP-1, localisée au niveau de la mitochondrie, impliquée dans la rgulation du métabolisme mitochondrial et qui pourrait jouer un rôle dans la progression tumorale et les métastases. Le déqualinium (DQ) est capable de cibler la mitochondrie. Dans une seconde partie du travail, des liposomes encapsulant le DQ ont été formulés dans le but de vectoriser le 6BrCaQ vers la mitochondrie. Toutefois, face à la difficulté d’encapsuler le DQ dans des liposomes, une étude de physico-chimie sur l’interaction DQ/liposomes a été mise en place pour comprendre comment le DQ agit sur les bicouches phospholipidiques. Cette étude a révélé que, malgré une capacité de ciblage mitochondrial des liposomes, le DQ était difficile à encapsuler dans les milieux salins et n’était pas inerte sur les bicouches lipidiques ce qui limite son utilisation pour la formulation de liposomes ciblant la mitochondrie. / Hsp90 (Heat shock protein 90) is an ubiquitous and well-conserved chaperone protein involved in the folding and the repair of « client » proteins. Among these proteins, several are involved in oncogenic phenomena making hsp90 an interesting target for cancer therapy. Hsp90 consists of three domains ; a N-terminal domain as the ATP hydrolysis site ; a middle domain where the client proteins binds and a C-terminal domain involved in the dimerization, a necessary step to refold the client protein. Several inhibitors were synthesized to target these different domains. N-terminal inhibitors such as Geldanamycin ; were shown to be very efficient but side effects and resistance to the treatment limited their clinical use. Indeed, N-terminal inhibition induces a stress response characterized by an increase of hsp90 and its co-chaperones which is often associated with resistance to the treatment and poor prognosis. Novobiocin, a coumarin antibiotic, is capable of inhibiting the C-terminal domain of hsp90, without inducing a stress response. Several derivatives of this molecule have been synthesized, including 6BrCaQ. The latter was effective in terms of apoptosis induction and cell cycle blockade on several cell lines (MCF-7, MDA-MB-231) and induced pro-tumoral client protein degradation but its low solubility limits its in vivo administration. In this thesis, a liposomal formulation of 6BrCaQ has been developed and studied on in prostate cancer cell lines and acute myelogenous leukemia in vitro and in vivo in an orthotopic model of breast cancer (MDA-MB-231 luc-GFP). Liposomal 6BrCaQ showed ability to induce apoptosis, to block the cell cycle on several cell lines (PC-3 and MDA-MB-231) and slow down migration of PC-3 cells (wound healing assay). Liposomal 6BrCaQ is able to synergize with doxorubicine or daunorubicine in PC-3 cells and in MOLM-13 cells, respectively. Moreover, protein and RNA expression profiles show that in PC-3 cells liposomal 6BrCaQ downregulates Hsp90 protein and in MDA-MB-231 cells slightly upregulates Hsp70 gene expression. Results obtained during in vivo experiments on the breast orthotopic model revealed a slow downslowdown of tumor growth after 13 days for a dose of 1 mg/kg injected weekly. Histological analysis revealed necrosis in treated groups and aassociated with a decreased cell proliferation (ki67 staining).Hsp90 also has isoforms and analogues localized in intracellular organelles, including, TRAP-1, localized in the mitochondrion and probably implicated in malignant progression through its role in the regulation of the mitochondrial metabolism. Dequalinium (DQ) demonstrated ability to target the mitochondrion. In a second part of the work, liposomes encapsulating DQ have been formulated in order to target 6BrCaQ to mitochondria. However, faced with the difficulty of encapsulating the DQ in liposomes, a physical chemistry study on the interaction DQ / liposomes was established to understand how DQ acts on phospholipid bilayers. Though a mitochondrial targeting capacity, this study revealed DQ was difficult to encapsulate in liposomes in saline medium and not completely inert on lipid bilayers limiting its use to target liposomes to mitochondria.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22882号 / 医博第4676号 / 新制||医||1047(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 濵﨑 洋子, 教授 羽賀 博典 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

INTRODUCTION: SARS-CoV-2 is a novel virus from the coronavirus family that emerged in the Hubei province of China in December 2019 and rapidly spread throughout the world. On March 11, 2020, the World Health Organization declared a global pandemic. Infection with SARS-CoV-2 causes coronavirus disease 19 (COVID19) which can be fatal. There is an obvious and pressing need for research surrounding SARS-CoV-2 that will aid in eradication of this pandemic. OBJECTIVE: The goal of this study was to absolve the dire need for small animal models of human disease that demonstrate hallmark pathological features of infection. Due to ethical and financial obstacles, the use of animals that closely resemble human immunity, such as non-human primates, is often not a viable option. For this reason, there is a push to develop small animal models that can mimic human disease responses, particularly those in viral infections that have a narrow species tropism. To achieve this in the context of the novel coronavirus, SARS-CoV-2, we studied various mouse models and their capacity to become infected with and mount an immune response to SARS-CoV-2. Our goal was to identify a model that sufficiently mimics severe COVID19 seen in humans as well as provide molecular insight into pathways that prevent the development of severe disease. METHODS: NRG-L and HIS-NRGF-L mice were subcutaneously implanted with human fetal lung xenografts and infected with SARS-CoV-2. Tissues were stained with H&E for histopathological scoring. NRG-L and HIS-NRGF-L tissues were fluorescently labeled using 2 different multiplex immunohistochemistry panels. Slides were digitized by a Vectra Polaris™ fluorescent whole slide scanner and digital analysis was completed using HALO™. Statistical analysis was conducted using GraphPad Prism™ 9.0.1. RESULTS: Infected NRG-L mice present extensive histopathological manifestations when compared to uninfected controls. Cumulative histology scores at both 2 and 7DPI were increased when compared to uninoculated fLX. Neutrophil influx, intra-airspace necrosis, capillary fibrin thrombi, and presence of syncytial cells were the most significant independent observations that contributed to the increased cumulative score. Together these findings indicate that fLX inoculated with SARS-CoV-2 faithfully recapitulate several features of diffuse alveolar damage (DAD) described in severe COVID-19. HIS-NRGF-L mice displayed decreased influx of neutrophils, intra-airway necrosis, and syncytial cells when compared to NRG-L fLX. Hemorrhage was decreased at both 2 and 7 DPI for HIS-NRGF-L fLX compared to NRG-L fLX. Cumulative histology scores were decreased in HIS-NRGF-L fLX at 7 DPI when compared to NRG-L fLX. Taken together these findings support the hypothesis that human myeloid and lymphoid infiltrates suppress or prevent the disparate host response observed in NRGL-L fLX that manifested in pronounced diffuse alveolar damage. CONCLUSION: Using digital image analysis of multiplex immunohistochemistry paired with semi-quantitative histopathological scoring, this study characterized important hallmark lesions observed in severe COVID19 as seen in small animal models. NRG-L and HIS-NRGF-L mice that are subcutaneously implanted with human fetal lung xenografts are susceptible to infection with SARS-CoV-2 and can produce severe and moderate disease phenotypes respectively. Co-engraftment with human fetal lung tissue and human immune system components in HIS-NRGF-L mice suppresses the divergent host response that is observed in NRG-L mice. For this reason, NRG-L mice engrafted with fLX are an ideal small animal model of severe COVID19, whereas HIS-NRGF-L mice severe as a valuable and informative model for deciphering molecular mechanisms driving severe COVID-19 that will serve as targets for therapeutic development.

Les cancers du sein luminaux, exprimant le récepteur aux œstrogènes (RE) représentent 65-75% des cancers du sein soit environ 35.000 nouvelles patientes par an en France. Les référentiels thérapeutiques en vigueur recommandent une prescription systématique d’hormonothérapie au stade précoce, et quasiment constante au stade avancé. Néanmoins, il est admis que plus de 20% des patientes au stade précoce, et la quasi-totalité au stade avancé, vont échapper au traitement endocrinien, rendant impératif le développement de modèles précliniques permettant d’étudier les mécanismes d’hormonorésistance. Dans un contexte de modèles cellulaires anciens et très imparfaits (MCF7, T47D), et de quasi absence de modèles murins pertinents, nous avons choisi de développer des modèles murins dérivés de tumeurs fraîches, dits PDX (patient derived xenografts). Nous avons montré que ces modèles, difficiles à obtenir, récapitulaient avec une grande fidélité les caractéristiques morphologiques et biologiques des tumeurs d’origine. Les PDX se distinguent également par une grande stabilité de ces caractéristiques lors des passages successifs, les rendant utilisables au long cours. Nous avons également évalué les modèles obtenus pour leur profil de sensibilité à diverses modalités de traitement hormonal.Dans une seconde étape, nous avons développé des modèles résistants à partir des PDX précédemment obtenues. Quatre modèles ont pu être obtenus, qui nous ont permis d’avoir à disposition des modèles rendant compte de situations cliniques variées. Ces 4 modèles ont fait l’objet d’analyses biologiques extensives visant à identifier les caractéristiques moléculaires potentiellement associées à telle modalité de résistance : nos données suggèrent fortement qu’il y a autant de mécanismes de résistance que de situations, rendant illusoire une définition biologique unifiée de l’hormonorésistance. La reprogrammation fonctionnelle du RE semble être au centre de ces mécanismes.La voie PI3K/mTOR est une des plus fréquemment associée à l’hormonorésistance. De manière originale, nous avons mis en évidence que cette voie était activée aussi bien dans les modèles sensibles que dans les modèles résistants. La troisième étape a consisté à évaluer l’efficacité de l’everolimus, agent ciblant mTORC1. Nous avons pu montrer que l’everolimus était hautement actif dans toutes les situations considérées, sans argument pour une synergie entre everolimus et tamoxifène ou exemestane. En revanche, il existe une nette tendance à la synergie avec le fulvestrant, inhibiteur hautement spécifique du RE entraînant sa dégradation, et faisant suggérer des interactions avec la voie non génomique du RE.Nous testons actuellement des inhibiteurs spécifiques de la PI3KCA grâce à diverses collaborations industrielles qui permettront également de mener des analyses génomiques approfondies. De multiples projets académiques sont en cours. / Luminal breast cancer (ER+, HER2 negative) accounts for 65-75% of all breast carcinomas. Current guidelines strongly recommend endocrine treatment at both the early and advanced stages. However, more than 20% of early stage patients, and all advanced patients will eventually develop endocrine resistance.As most preclinical models (MCF7, T47D) do not recapitulate tumor biology, we have chosen to develop murine models derived from fresh tumors, hence called patient derived xenografts (PDX). We show that these models, although difficult to generate, faithfully exhibit the morphological and biological features of their parental counterpart, with high long term stability. These models have also been evaluated for their sensitivity to various endocrine treatments.In the next step, we developed from these initially endocrine sensitive models new tumors rendered resistant to endocrine therapies. We show that there is no unique biological pattern associated with endocrine resistance, although ER functional reprogramming appears to be critical. We also show that PI3K/mTOR pathway activation, may not be always related to endocrine resistance, and suggest that fulvestrant, an ER down regulator, may be highly synergistic with everolimus in specific cases.Several PI3KCA inhibitors are currently being evaluated in this setting.

Einleitung: Glioblastoma multiforme (GBM) gehören zu den häufigsten und bösartigsten Hirntumoren. Eine vollständige Heilung ist derzeit noch nicht möglich. Ziele: Es wurde ein orthotopes GBM-Xenotransplantat-Modell in der Maus etabliert, optimale Durchführungsprotokolle wurden erstellt und die Wachstumskinetik der Tumoren charakterisiert. Außerdem erfolgte eine lokale therapeutische Intervention mit Komplexen aus Polymeren und siRNAs (small interfering RNAs) bzw. AntimiRs (Anti-micro-RNAs) und der Verlauf wurde mittels Biolumineszenz-imaging (BLI) überwacht. Die tumortragenden Hirne wurden immunhistochemisch untersucht. Neben der Therapie wurden erstmals Tissue Slice Cokulturen aus Maushirnen mit orthotopen Xenotransplantaten hergestellt und durch immunhistochemische Untersuchungen bezüglich des Tumorwachstums und der Invasivität in das umliegende Normalgewebe charakterisiert. Des Weiteren wurde ein subkutanes (s.c.) Xenotransplantat-Modell etabliert, dessen Tumorwachstum mittels BLI überwacht wurde, welches eine therapeutische Applikation von PEI-komplexierten siRNAs enthielt und deren Tumoren mittels Messung der Proteinkonzentration und der Lumineszenz charakterisiert wurden. Mit dieser Arbeit wurden durch die Verwendung von siRNAs bzw. AntimiRs im Rahmen der RNA-Interferenz Strategien entwickelt, mit denen eine Grundlage für die spezifisch auf ein bestimmtes Gen gerichtete GBM-Therapie geschaffen werden konnte. Tiere, Material, Methoden: In insgesamt 190 immundefizienten Mäusen wurden s.c. und orthotope Xenotransplantate aus der Reporterzelllinie G55T2-Luc-GFP hergestellt. Eine Behandlungsgruppe mit s.c. Xenotransplantaten bestand aus 7-8 Tieren. Die Behandlungsgruppe erhielt intraperitoneale (i.p.) Injektionen des PEI-siRNA-Komplexes LP10Y-siLuc3 (LP10Y: AG Aigner, Universität Leipzig, Deutschland, siLuc3: Eurogentec, Belgien), während die Negativkontrollgruppe (NC) i.p. Injektionen mit dem Komplex LP10Y-siLuc2 erhielt. Diese Behandlung erfolgte 3 x wöchentlich über 6-12 d. Das Wachstum der Tumore wurde mit BLI in vivo verfolgt und die Lumineszenz im zeitlichen Verlauf gemessen. Die Tumore wurden lysiert und deren Lumineszenz bezogen auf die Proteinkonzentration gemessen. Für das orthotope Xenotransplantat wurde eine Schraube mit Führungskanal (Screw guide, Plastics One, USA) 1 mm rostral und 2 mm lateral des Bregmas stereotaktisch implantiert. Mit einer Mikroinfusionspumpe und einer Mikrodosierspritze wurden die Zellen über den Führungskanal mit 12 µl/ h in das rechte Striatum injiziert. 5-7 d nach der Inokulation der Zellen erfolgte das Einbringen von 3 µl der PEI-AntimiR bzw. siRNA-Komplexe PEI/antimiR-155-Komplex (PEI: AG Aigner, Universität Leipzig, Deutschland, AntimiR 155-ZEN: Integrated DNA Technologies, USA) bzw. LP10Y-siPLK1 (PLK1: Eurogentec, Belgien). Die Tiere der NC wurden mit den Komplexen PEI/antimiR-NC5-Komplex bzw. LP10Y-siLuc3 behandelt. Zum Vergleich wurde eine Gruppe gar nicht behandelt. Die Behandlung erfolgte 3 x wöchentlich über 12 d. Eine Behandlungsgruppe bestand aus 10 randomisiert verteilten Tieren. Das Wachstum wurde in vivo mit BLI verfolgt. Die Hirne wurden entnommen und mit einem Vibratom Gewebeschnitte erstellt, die mit Kresylviolett gefärbt und an denen mit einer Bildbearbeitungssoftware die Tumorflächen ausgemessen wurden. Außerdem wurden mit einem Mikrotom Paraffinschnitte hergestellt und diese immunhistochemisch beurteilt. Es wurden Tissue Slice Cokulturen aus unbehandelten Tumoren und gesundem Gewebe des Striatums bzw. Cortex erstellt und aus diesen ebenfalls Paraffinschnitte hergestellt, welche immunhistochemisch beurteilt wurden. Ergebnisse: Es konnte gezeigt werden, dass ein Tumorwachstum s.c. injizierter GBM-Zellen in vivo mit BLI verfolgt werden kann und dass die siRNA siLuc3 mit LP10Y erfolgreich in G55T2-Luc-GFP eingeschleust wurde. Bei der Untersuchung der Luciferaseaktivität bezogen auf die Proteinkonzentration der lysierten s.c. Tumoren konnte eine Reduktion der Lumineszenz in der Behandlungsgruppe nachgewiesen werden. Das orthotope Xenotransplantat-Modell wies eine Tumoranwuchsrate von bis zu 96 % und eine Mortalitätsrate von bis zu 0 % auf. Das Tumorwachstum konnte nicht in vivo mit BLI verfolgt werden, da die umliegenden Gewebe die Lumineszenz vollständig abschirmten. Bei den Behandlungen mit LP10Y-siPLK1 und dem PEI/antimiR-155-Komplex konnte histologisch eine Reduktion der Tumorgröße und immunhistochemisch Apoptose und eine Tendenz zur Reduktion der Proliferation und Migration nachgewiesen werden. In der Behandlungsgruppe mit LP10Y-siPLK1 konnte außerdem eine verminderte Expression von PLK1 im Tumorgewebe und eine gesteigerte Überlebensrate nachgewiesen werden. Mit den Tissue Slice Cokulturen konnte festgestellt werden, dass die Xenotransplantate in den verschiedenen Hirnabschnitten unterschiedliches Migrationsverhalten zeigen. Die auf dem Cortex wachsenden Tumore waren kleiner als die auf dem Striatum wachsenden Tumore, wiesen jedoch mehr Migration in das umliegende Hirngewebe und mehr raumforderndes Wachstum im Verhältnis zum nicht raumfordernden Wachstum auf. Das Verhältnis der migrierenden Zellen zur Länge des Tumors des Xenotransplantats im Cortex wies höhere Werte auf als das im Striatum. Schlussfolgerungen: LP10Y eignet sich zur Einschleusung von siRNA und könnte ein vielversprechendes Nano-Therapeutikum darstellen. Das BLI von Luciferase exprimierenden, s.c. Tumoren ist ein gutes Verfahren zur Verfolgung der Wachstumskinetik. Es scheint sich jedoch nicht zur Untersuchung der Transfektionseffizienz von PEI-Komplexen zu eignen, da die Erscheinungsform von Glioblastomen mit Einblutungen, Nekrosen und Zysten sehr verschieden sein kann. Es ist also möglich dass die Tumoren unterschiedlich viele lebende Zellen pro Volumen mit entsprechender Luciferaseaktivität aufweisen. Alternativ dazu ist das Untersuchungsmodell der Lumineszenz bezogen auf die Proteinkonzentration lysierter Tumoren ein geeignetes Verfahren für die Quantifizierung der Luciferaseaktivität und der Transfektionseffizienz von PEI-Komplexen in GBM. Das im Zuge dieser Arbeit etablierte orthotope Xenotransplantat-Modell ist ein valides Modell mit verschiedenen Eigenschaften humaner GBM. Die Ergebnisse aus den Behandlungen der orthotopen GBM mit den PEI-siRNA bzw. AntimiRKomplexen sprechen für eine gute biologische Aktivität, niedrige Zytotoxizität, eine gute Knockdowneffizienz und somit auch therapeutische Effizienz der Komplexe in vivo. Das orthotope Xenotransplantat-Modell und die Tissue Slice Cokulturen stellen geeignete, gut reproduzierbare und genetisch unmanipulierte Modelle dar, welche die Untersuchung von Wachstums- und Migrationsverhalten mit und ohne therapeutische Intervention, sowie des Behandlungserfolges erlauben. Beide Polymere könnten vielversprechende Nano-Therapeutika darstellen. Ein neues exvivo-Modell, in dem die GBM in ihrer natürlichen Umgebung gewachsen sind und sich in der Cokultur weiterhin sehr ähnlich der Originalsituation verhalten können, wurde etabliert. Für die Statistiken wurde, zum Vergleich von Gruppen, der One-Way ANOVA-Test von SigmaPlot 13 verwendet.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23601号 / 医博第4788号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 戸井 雅和, 教授 伊藤 貴浩 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Small Cell Lung Cancer (SCLC) is a highly aggressive neuroendocrine tumor with a 5% survival rate over 5 years. Though SCLC comprises 13% of all cases of lung cancer the median survival time of 14.5 months has seen little improvement over the last four decades. Standard treatment relies on DNA damaging agents such as Cisplatin/Etoposide (EP) which induce a high response rate of 60-70%. Despite this initial response, nearly all patients will relapse rendering first-line therapies ineffective. Furthermore, SCLC has been shown to develop chemotherapy cross-resistance in which resistance to first-line chemotherapies will confer resistance to additional DNA damaging agents thereby reducing treatment efficacy and duration of response. Cross-Resistance constitutes a major clinical issue whose underlying mechanisms remain a mystery. The modest improvements in SCLC patient outcomes over the decades may be partially explained by the existing systems of study. Current methodologies of SCLC study rely on cell lines, patient samples, and Genetically Engineered Mouse Models which have little functional correlation to clinical outcomes. While few sources have proposed Patient Derived Xenograft (PDX) systems as an improved alternative, significant data remains sparse. Without a robust model system which accurately recapitulates patient outcomes, molecular pathways driving resistance cannot be uncovered. Here we present the generation of 34 SCLC PDX models which maintain both genomic and functional fidelity. Furthermore, treatment of a 30-model subset with first-line chemotherapy EP and a novel chemotherapy Olaparib/Temozolomide (OT) allowed for functional and molecular comparison between groups. Our findings demonstrate incomplete independent resistance mechanisms between EP and OT treatment with a small overlap of 31 genes involved in glycolysis and xenobiotic metabolism.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21419号 / 医博第4409号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 小川 誠司, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM