Les carcinoïdes pulmonaires, les carcinomes neuroendocriniens à grandes cellules (LCNEC) et les mésothéliomes malins sont des tumeurs thoraciques rares, dont l'incidence a augmenté au cours des dernières années. Le diagnostic de ces tumeurs est soumis à la variabilité inter-observateur et les opportunités thérapeutiques sont limitées. De grandes études génomiques visant à les caractériser au niveau moléculaire pourraient aider à mieux comprendre les mécanismes sous-jacents à leur développement et faciliter le diagnostic et le traitement de ces maladies. Mon projet de thèse visait à combler les lacunes dans la compréhension des carcinoïdes pulmonaires, des LCNEC et du mésothéliome péritonéal malin. À la suite des travaux entrepris au cours de ma thèse, nous avons constaté que (1) de la même manière que le mésothéliome pleural, les mésothéliomes péritonéaux sont également caractérisés par des mutations conduisant à la perte d'expression de BAP1, facteur de bon pronostic, (2) les patients atteints d’un LCNEC conservant une expression de RB1 présentent de meilleurs résultats lorsqu’ils sont traité avec une chimiothérapie du cancer du poumon non à petites cellules par rapport à une chimiothérapie du cancer du poumon à petites cellules, (3) les carcinoïdes pulmonaires peuvent être classés en trois groupes moléculaires pertinents sur le plan clinique, et (4) , l'identification de supra carcinoïdes confirme l'existence d'un lien moléculaire entre les néoplasmes neuroendocriniens pulmonaires de faible et de haut grade / Pulmonary carcinoids, large-cell neuroendocrine carcinomas (LCNEC), and malignant mesotheliomas are rare thoracic tumours, which incidence has been increasing over the past years. The diagnosis of these tumours is subjected to inter-observer variability and the therapeutic opportunities are limited. Large genomic studies to characterize them at a molecular level might help to better understand the mechanisms underlying their development, and to help the diagnosis and treatment of these diseases. My thesis project aimed to fill the gap in the understanding of pulmonary carcinoids, LCNEC, and malignant peritoneal mesothelioma. As result of the work undertaken during my thesis, we found that (1) similarly to pleural mesothelioma, peritoneal mesotheliomas are also characterised by mutations leading to the loss of expression of BAP1, which is a factor of good prognostic, (2) LCNEC patients with a remaining expression of RB1 have a better outcome when treated with non-small cell lung cancer chemotherapy in comparison to small-cell lung cancer chemotherapy, (3) pulmonary carcinoids can be classified in three clinically-relevant molecular groups, and (4), the identification of supra carcinoids supports a molecular link between the low and high-grade lung neuroendocrine neoplasms

Os produtos derivados de asbesto são amplamente utilizados pelo setor industrial, sendo descritas diversas doenças relacionadas à sua exposição, entre elas, o tumor primário da pleura, ou mesotelioma. O mecanismo fisiopatológico da lesão pelas fibras de asbesto no espaço pleural ainda não está totalmente estabelecido. Entre os fatores possivelmente implicados estão os efeitos provocados por uma resposta inflamatória com migração celular e liberação de mediadores moleculares levando à necrose, apoptose e alterações na proliferação e fibrogênese. No entanto, existem dificuldades no estudo da resposta in vivo ao asbesto, principalmente em virtude da população multicelular da cavidade pleural. Neste sentido, tem sido preconizado na literatura o estudo envolvendo animais geneticamente modificados ou selecionados, a fim de melhor compreender o papel das diversas populações envolvidas neste processo. Neste trabalho, tivemos como objetivo estudar comparativamente a resposta inflamatória aguda no líquido pleural e em células mesoteliais em cultura expostas a diferentes fibras de asbesto. Para tanto, animais controle e geneticamente selecionados para alta (AIR max) e baixa (AIR min) resposta inflamatória, e células mesoteliais em cultura foram expostas às fibras de asbesto crocidolita ou crisotila. Após 4, 24 ou 48 horas foram avaliadas a produção das citocinas IL-1b, IL-6 e MIP-2. Adicionalmente, no modelo in vivo foi avaliado o perfil celular do líquido pleural e a expressão do Ra PDGF em RESUMO fragmentos de pleura, e no modelo in vitro a resposta celular de apoptose e necrose. Como resultados, as fibras de asbesto crocidolita e crisotila produziram, em animais AIR max, uma elevação significativa no líquido pleural de leucócitos, neutrófilos e da IL-1b em comparação aos controles e aos animais AIR min. Entretanto, não houve diferença no número de macrófagos, IL-6 e MIP-2. As células mesoteliais em cultura expostas tanto às fibras crocidolita quanto crisotila apresentaram elevados índices de apoptose e necrose e da produção das citocinas IL-1b, IL-6 e MIP-2 quando comparadas aos controles. Houve forte correlação das citocinas MIP-2 e IL-1b em cultura com os níveis no líquido pleural para ambas as fibras estudadas. Foi demonstrado, para ambas as fibras, uma forte expressão do Ra PDGF na superfície pleural tanto nos animais com alta quanto com baixa resposta inflamatória quando comparado aos controles. Como conclusão, caracterizamos o perfil da resposta inflamatória aguda a diferentes fibras de asbesto em modelos experimentais in vivo e in vitro, contribuindo para a melhor compreensão do mecanismo de agressão celular secundário a este material de uso comercial tão freqüente. / Asbestos-derived products are used thoroughly by industry. Several diseases related to asbestos exposition have been described, among them the primary tumor of the pleura mesothelioma. The mechanisms by which asbestos fibers produce injury to the pleural space are not clear. Among the factors possibly implicated are the effects secondary to an inflammatory response characterized by cellular migration and the release of molecular mediators leading to necrosis, apoptosis, cellular proliferation and fibrogenesis. However, it is difficulty to characterize the cellular response in vivo, mainly by virtue of the multi-cellular population present into the pleural cavity. Therefore, studies involving animals genetically modified or genetically selected have been proposed in the literature, in order to better understand the role of the several cellular populations involved in this complex process. In this study, our objective was to determine the inflammatory response of the pleural fluid and compare to the response of cultured mesothelial cells exposed to different asbestos fibers. Controls and mice genetically selected for high (AIR max) or low (AIR min) inflammatory response as well as mice cultured mesothelial cells were treated to crocidolite or chrysotile asbestos fibers. After 4, 24 or 48 hours the production of the cytokines IL-1b, IL-6 and MIP-2 were analyzed. In addition, the in vivo cellular profile of the pleural fluid and the Ra PDGF expression in the pleura fragments was documented. In parallel, the in vitro mesothelial cellular response of apoptosis and necrosis was quantified. Both asbestos fibers produced in AIR max mice a significant elevation in the pleural fluid total leukocytes, neutrophils and IL-1b levels in comparison to the controls and AIR min animals. However, no difference was found in the macrophage number, IL-6 and MIP-2 levels. Cultured mesothelial cells had a high apoptosis, necrosis, IL-1b, IL-6 and MIP-2 levels in comparison to the controls when exposed to either crocidolite or chrysotile. MIP-2 and IL-1b levels in cultured mesothelial cells strongly correlated with the levels of the pleural fluid for both fibers. In addition, a pronounced expression of Ra PDGF in the pleural surface was demonstrated in both high and low inflammatory selected mice when compared to the controls. In conclusion, we characterized the acute inflammatory response to the asbestos fibers crocidolite and chrysotile in an in vivo and in vitro experimental model, aiming to contribute to better understand the mechanism of cellular aggression secondary to this particulate material of such frequent commercial use.

Introdução Trata-se de um estudo descritivo das sentenças de segunda instância do Poder Judiciário que envolva o problema do trabalhador exposto ao risco do amianto como uma questão de saúde pública. Objetivo Avaliar as decisões judiciais a respeito da exposição dos trabalhadores ao risco do amianto como causa de doença do trabalho a partir da análise de acórdãos judiciais de 1999 até 2009. Métodos Os objetivos foram alcançados mediante pesquisa em acórdãos judiciais publicados no TRT (Tribunal Regional do Trabalho) da 2ª Região, TRT (Tribunal Regional do Trabalho) da 15ª Região e TST (Tribunal Superior do Trabalho), que são tribunais pertencentes à Justiça do Trabalho, bem como no TJSP (Tribunal de Justiça de São Paulo), STJ (Superior Tribunal de Justiça) e STF (Supremo Tribunal Federal), pertencentes à Justiça Comum, a partir do momento histórico em que houve a promulgação da Constituição Federal de 1988, considerando-se o ano da publicação ou do registro dos acórdãos de 1999 a 2009. Os acórdãos foram selecionados por meio da internet nos sites dos Tribunais em referência, que disponibilizam a busca on line. Resultados Os resultados indicam que de um total de 119 decisões judiciais analisadas, considerando-se todos os critérios de inclusão e exclusão, apenas em 42 casos houve a comprovação do nexo de causalidade entre a exposição do trabalhador ao risco do amianto e a doença adquirida no trabalho, o que evidencia que o trabalhador desincumbiuse de seu ônus probatório em pequena parte dos acórdãos. Conclusões Pela avaliação dos acórdãos judiciais de 1999 até 2009 a respeito da exposição dos trabalhadores ao risco do amianto como causa de acidente do trabalho, doença profissional ou morte, somente em 35,3 por cento das decisões foram declarados procedentes os pedidos dos trabalhadores / Introduction - This is a descriptive study of Judiciary second appeal decisions involving the problem of the risk of workers when exposed to asbestos as a public health issue. Objective - To evaluate judicial decisions regarding the exposure of workers to the risk of asbestos as a cause of occupational disease from the analysis of Judicial Decisions from 1999 to 2009. Methods - The objectives have been achieved by researching judicial decisions published by the TRT (Regional Court of Labor) of the 2nd Region, by the TRT (Regional Court of Labor) of the 15th Region and by the TST (Superior Labor Court), which are courts of the Labor Justice Department, as well as the ones by the TJSP (Court of Justice of São Paulo), by the STJ (Higher Justice Court) and by the STF (Supreme Federal Court), of the Common Justice, from the historical moment in which the 1988 Constitution was promulgated, considering the year of publication or record of the Judiciary Decisions from 1999 to 2009. The judicial decisions were selected with the use of the internet in the sites of the Courts previously mentioned, which made possible to make this search on line. Results - The results indicate that from a total of 119 judicial decisions, which were analyzed, and also taking in consideration all the inclusion and exclusion criteria, only in 42 cases there was proof of a causal link between the worker\'s risky exposure to asbestos and the disease acquired at work, which shows that the employee is discharged of the evidential burden on a small portion of the decisions. Conclusions - For the evaluation of the judicial decisions from 1999 to 2009 regarding the exposure of workers to the risk of asbestos as a cause of work accidents, and occupational disease or death, only 35.3 per cent of decisions were in favor of the workers claim

Os produtos derivados de asbesto são amplamente utilizados pelo setor industrial, sendo descritas diversas doenças relacionadas à sua exposição, entre elas, o tumor primário da pleura, ou mesotelioma. O mecanismo fisiopatológico da lesão pelas fibras de asbesto no espaço pleural ainda não está totalmente estabelecido. Entre os fatores possivelmente implicados estão os efeitos provocados por uma resposta inflamatória com migração celular e liberação de mediadores moleculares levando à necrose, apoptose e alterações na proliferação e fibrogênese. No entanto, existem dificuldades no estudo da resposta in vivo ao asbesto, principalmente em virtude da população multicelular da cavidade pleural. Neste sentido, tem sido preconizado na literatura o estudo envolvendo animais geneticamente modificados ou selecionados, a fim de melhor compreender o papel das diversas populações envolvidas neste processo. Neste trabalho, tivemos como objetivo estudar comparativamente a resposta inflamatória aguda no líquido pleural e em células mesoteliais em cultura expostas a diferentes fibras de asbesto. Para tanto, animais controle e geneticamente selecionados para alta (AIR max) e baixa (AIR min) resposta inflamatória, e células mesoteliais em cultura foram expostas às fibras de asbesto crocidolita ou crisotila. Após 4, 24 ou 48 horas foram avaliadas a produção das citocinas IL-1b, IL-6 e MIP-2. Adicionalmente, no modelo in vivo foi avaliado o perfil celular do líquido pleural e a expressão do Ra PDGF em RESUMO fragmentos de pleura, e no modelo in vitro a resposta celular de apoptose e necrose. Como resultados, as fibras de asbesto crocidolita e crisotila produziram, em animais AIR max, uma elevação significativa no líquido pleural de leucócitos, neutrófilos e da IL-1b em comparação aos controles e aos animais AIR min. Entretanto, não houve diferença no número de macrófagos, IL-6 e MIP-2. As células mesoteliais em cultura expostas tanto às fibras crocidolita quanto crisotila apresentaram elevados índices de apoptose e necrose e da produção das citocinas IL-1b, IL-6 e MIP-2 quando comparadas aos controles. Houve forte correlação das citocinas MIP-2 e IL-1b em cultura com os níveis no líquido pleural para ambas as fibras estudadas. Foi demonstrado, para ambas as fibras, uma forte expressão do Ra PDGF na superfície pleural tanto nos animais com alta quanto com baixa resposta inflamatória quando comparado aos controles. Como conclusão, caracterizamos o perfil da resposta inflamatória aguda a diferentes fibras de asbesto em modelos experimentais in vivo e in vitro, contribuindo para a melhor compreensão do mecanismo de agressão celular secundário a este material de uso comercial tão freqüente. / Asbestos-derived products are used thoroughly by industry. Several diseases related to asbestos exposition have been described, among them the primary tumor of the pleura mesothelioma. The mechanisms by which asbestos fibers produce injury to the pleural space are not clear. Among the factors possibly implicated are the effects secondary to an inflammatory response characterized by cellular migration and the release of molecular mediators leading to necrosis, apoptosis, cellular proliferation and fibrogenesis. However, it is difficulty to characterize the cellular response in vivo, mainly by virtue of the multi-cellular population present into the pleural cavity. Therefore, studies involving animals genetically modified or genetically selected have been proposed in the literature, in order to better understand the role of the several cellular populations involved in this complex process. In this study, our objective was to determine the inflammatory response of the pleural fluid and compare to the response of cultured mesothelial cells exposed to different asbestos fibers. Controls and mice genetically selected for high (AIR max) or low (AIR min) inflammatory response as well as mice cultured mesothelial cells were treated to crocidolite or chrysotile asbestos fibers. After 4, 24 or 48 hours the production of the cytokines IL-1b, IL-6 and MIP-2 were analyzed. In addition, the in vivo cellular profile of the pleural fluid and the Ra PDGF expression in the pleura fragments was documented. In parallel, the in vitro mesothelial cellular response of apoptosis and necrosis was quantified. Both asbestos fibers produced in AIR max mice a significant elevation in the pleural fluid total leukocytes, neutrophils and IL-1b levels in comparison to the controls and AIR min animals. However, no difference was found in the macrophage number, IL-6 and MIP-2 levels. Cultured mesothelial cells had a high apoptosis, necrosis, IL-1b, IL-6 and MIP-2 levels in comparison to the controls when exposed to either crocidolite or chrysotile. MIP-2 and IL-1b levels in cultured mesothelial cells strongly correlated with the levels of the pleural fluid for both fibers. In addition, a pronounced expression of Ra PDGF in the pleural surface was demonstrated in both high and low inflammatory selected mice when compared to the controls. In conclusion, we characterized the acute inflammatory response to the asbestos fibers crocidolite and chrysotile in an in vivo and in vitro experimental model, aiming to contribute to better understand the mechanism of cellular aggression secondary to this particulate material of such frequent commercial use.

Le mésothéliome pleural malin (MPM) est un cancer au pronostic sombre, en lien avec des traitements décevants. Quand la chirurgie fait partie d’un traitement multimodal, il est essentiel de l’associer avec un traitement adjuvant local pour tuer les cellules tumorales résiduelles. Récemment, la thérapie photodynamique (PDT) intrapleurale intra-opératoire, après exérèse tumorale chirurgicale, est apparue comme un traitement prometteur. Son succès repose sur l’illumination la plus complète et homogène de la cavité pleurale, contrôlée par une dosimétrie de lumière. Celle utilisée aujourd’hui en pratique clinique aux Etats-Unis repose sur la dosimétrie ponctuelle de sept capteurs intrathoraciques, mais ne permet pas d’avoir un reflet de la lumière délivrée sur l’ensemble des surfaces pleurales. Mon projet de recherche se décline sur un axe clinique et un axe expérimental : 1) mettre en place un essai de clinique de phase II au CHRU de Lille, associant la PDT intrapleurale au traitement multimodal du MPM, 2) développer une méthode de dosimétrie de lumière peropératoire innovante par la caractérisation du dispositif lumineux et l’utilisation de l’imagerie.L’essai clinique a démarré en Février 2016, en utilisant la dosimétrie de référence avec les sept capteurs en suivant le protocole américain. Une baguette lumineuse innovante a été conçue au laboratoire, ainsi que le système de dosimétrie. Quatre patients ont bénéficié d’une pleurectomie/décortication suivie d’une PDT intrapleurale (avec le photosensibilisateur Photofrin®) et d’une chimiothérapie adjuvante, sans toxicité majeure. Concernant le projet expérimental, un profil d’illumination de la baguette lumineuse a été défini en combinant les mesures de puissance ponctuelles (Watt) et celles de pixel à partir d’une photographie digitale. Un coefficient d’atténuation effectif a été calculé : µeff = 0,705 cm-1. A l’aide d’un système de repérage spatial électromagnétique (TrackStar®), la position de la baguette lumineuse est connue à l’intérieur de la cavité pleurale en temps réel, et ses coordonnées spatiales sont projetées sur le TDM thoracique en 3D. Après avoir intégré le profil d’illumination de la baguette au système de repérage, un logiciel dédié permet la représentation spatiale sur imagerie de la dose cumulée de lumière délivrée. Ce travail a été réalisé et validé sur un fantôme de cavité thoracique intra opératoire, puis notre méthode de dosimétrie a été comparée à celle de référence.Les perspectives sont de pouvoir tester la faisabilité cette nouvelle méthode de dosimétrie chez l’homme et de développer des dispositifs lumineux innovants pour une meilleure standardisation de la PDT pour le MPM. / Malignant pleural mesothelioma (MPM) is a cancer with a poor prognosis due to deceiving treatments. When surgery is part of a multimodal treatment for MPM, it is crucial to combine it with a local adjuvant treatment to kill residual tumour cells. Recently, intrapleural photodynamic therapy (PDT) after surgical resection has appeared to be a promising treatment for MPM. Its success relies essentially on the most complete and homogeneous illumination of the pleural cavity, monitored by light dosimetry. The dosimetry method used today in the United States, with intrathoracic probes collecting the light at 7 strategic locations, does not give information about the light delivered on the whole pleural surface. This research project has a clinical and experimental axis: 1) to set up a phase II clinical trial in Lille University Hospital, combining intrapleural PDT to surgery within a multimodal treatment, 2) to develop an innovative peroperative light dosimetry method by the characterization of a light device and use of imaging.Clinical trial has started in February 2016, with the seven probes dosimetry method of reference, following the American protocol. An innovative light wand was conceived within the laboratory, as well as the dosimetry sytem. Four patients have undergone pleurectomy/decortications, intrapleural PDT (with photosensitizer Photofrin®) and adjuvant chemotherapy, without major toxicity. Regarding the experimental project, an illumination profile of the light wand was defined using two complementary methods: power measurements (Watt) and pixel intensity from digital photography. An effective attenuation coefficient was calculated: µeff = 0.705 cm-1. With an electromagnetic spatial tracking system (TrackStar®), we localized in real time the position of the light wand within the pleural cavity and projected its spatial coordinates to the 3D CT scan images. After having inserted the illumination profile of the light wand to the tracking system, a dedicated software allowed the spatial representation on CT images of the cumulated light dose. This work has been completed on an intraoperative thoracic cavity phantom. Our dosimetry method was validated on this phantom and compared to the one of reference.The perspectives are to test the feasibility of this new dosimetry method in humans, and to develop alternative light devices for a better standardization of the intrapleural PDT technique for MPM.

Le mésothéliome pleural malin (MPM) est une tumeur rare et agressive qui se développe au niveau de la plèvre.L'Adrénomedulline (AM) est un peptide de 52 acides aminés. Il intervient dans plusieurs processus physiologiques et physiopathologiques. Il est surexprimé dans plusieurs tumeurs où il joue un rôle important dans la croissance tumorale.Dans un premier temps nous avons montré que l'AM et ses récepteurs sont exprimés dans des biopsies de patients atteints de MPM suggérant son implication dans la croissance tumorale du MPM. In vitro l'incubation de lignées de MPM: H2452 et MSTO_211H avec des anticorps αAM ou αAMR inhibent la prolifération, l'invasion et la migration cellulaires. In vivo, le traitement avec un anticorps αAM ou l'antagoniste AM22-52, inhibe la croissance tumorale des xénogreffes de MSTO_211H par rapport au groupe contrôle. L'analyse histologique montre une augmentation significative de l'apoptose et une diminution importante de la vascularisation chez les tumeurs traitées par rapport aux tumeurs contrôles. Ces résultats démontrent le rôle important joué par l'AM dans la croissance tumorale du MPM et fait du système de l'AM une cible thérapeutique potentielle.Dans un deuxième temps, Nous avons émis l'hypothèse de la transactivation de l'EGFR par l'AM. Notre hypothèse s'est concrétisée dans la mesure où L'inhibition de l'EGFR par un inhibiteur spécifique l'AG1478 abolie l'activation de ERK par l'AM,La phosphorylation de l'EGFR par l'AM,La neutralisation de l'EGFR avec son propre anticorps inhibe la phosphorylation par l'AM suggérant une activation ligand dépendante. Ces résultats nous permettent de mieux comprendre le mécanisme d'action de l'AM. / Malignant pleural mesothelioma (MPM) grows aggressively in the thoracic cavity without curative possibilities, underlining the need for new therapeutic targets. Adrenomedullin (AM), a multifunctional peptide, is highly expressed in several tumors and plays an important role in angiogenesis and tumor. In the first part of our work, QRT-PCR showed an increase of AM mRNA levels in MPM when compared to normal pleura tissue. Immunohistochemically, AM and its receptors were localized in the carcinomatous epithelial compartment of MPM. The MPM cell lines H2452 and MSTO_211H expressed AM with a significant increase under hypoxia. The proliferation, migration and invasion of MPM cells are decreased by anti-AM and anti-AM receptors antibodies (αAM and αAMR) supporting that MPM cells can be regulated by AM. In vivo, αAM and AM22-52 antagonist therapies of MSTO_211H xenografts blocked angiogenesis and stimulated apoptosis, resulting in tumor regression. Histologic examination of treated tumors showed evidences of disruption of tumor vasculature. These findings highlight the implication of the AM pathway in the MPM growth and in neovascularization by supplying/amplifying signals essential for pathologic neoangiogenesis and lymphangiogenesis.In the second part of this work, we reported that the EGFR becomes rapidly tyrosine-phosphorylated upon stimulation of lung cancer cells lines with AM, suggesting that there is an intracellular mechanism for transactivation. Specific inhibition of EGFR function by the AG1478 or EGFR blocking antibody suppressed MAPK activation. These results suggest strongly a ligand-dependent mechanism of EGFR transactivation by AM.

Cancer is a leading cause of death in the western world and is increasing in frequency world-wide. Although diagnosis, treatment and therapeutic approaches to cancer have improved, many types of cancer are still lethal due to the lack of radical treatment. One of the fatal neoplastic disease types with poor prognosis is represented by malignant mesothelioma (MM). MM is characterised by very high mortality rate and limited therapeutic options. The etiology of the disease is mainly associated with exposure to asbestos fibres. The incidence of MM is increasing in many countries. The search for novel molecular targets, anti-cancer strategies and drugs, which would considerably improve the treatment is of great importance. Certain new drugs, especially those with specific molecular targets, show high selectivity in their action to cancer cells, and have considerably increased the cure rate in some types of cancer. Mitochondria have recently emerged as a very promising target for anti-cancer agents. A group of compounds with anti-cancer activity that induce apoptosis by way of mitochondrial destabilisation, termed 'mitocans', have been a recent focus of research. Several mitocans have been shown to selectively induce apoptosis in cancer cells and suppress the growth of many types of carcinomas in...

Even though anti‐cancer chemotherapy has been continuously improved during the last decades. problems with adverse effects and drug resistance still constitutes a considerable obstacle and sets a demand for new effective treatment options. Tissue homeostasis in multi‐cellular organisms is maintained through intrinsic cell death, apoptosis, which removes unwanted or damaged cells. Disrupted apoptosis is an important factor in tumorgenesis and drug resistance, therefore induction or restoration of apoptotic pathways is also important for the treatment of cancer. Several naturally occurring bacterial toxins have the ability to induce apoptosis and could thus be candidates to complement or improve the therapeutic effect of other anticancer drugs. The bacterial toxins, adenylate cyclase (AC) toxin from Bordetella pertussis, α‐toxin from Staphylococcus aureus and verotoxin‐1 (VT‐1) from Escherichia coli were investigated for their ability to induce apoptosis in different tumor cell lines. Toxin induction of cell death was investigated by cell viability assays, end‐stage apoptosis induction by DNA‐fregmentation (TUNEL) assay. Toxin receptor expression and signal transduction pathways to apoptosis were investigated by flow cytometry, caspase enzyme activity assays and western blot. Immunohistochemistry was used for identification of toxin receptor expression in tumor tissue samples. AC‐toxin was cytotoxic and induced apoptosis in cultured malignant plural mesothelioma (MPM) and small‐cell lung cancer (SCLC) cells. Low‐toxic concentrations of AC‐toxin enhanced cisplatin cytotoxicity and apoptosis in both cell lines. MPM‐cells with acquired cisplatin resistance were more sensitive to α‐toxin than the less resistant parental MPM cell line. A low‐toxic concentration of α‐toxin re‐sensitized resistant MPM cells to cisplatin cytotoxicity by apoptosis induced through the mitochondrial pathway without detectable activation of common up‐stream apoptosis signalling proteins. VT‐1 was highly cytotoxic and induced apoptosis in globotriosylceramide (Gb3) ‐expressing glioma, breast cancer and non‐small‐cell lung cancer (NSCLC) cells but was not cytotoxic to non‐Gb3‐expressing cells. PPMP, an inhibitor of glucosylceramide synthesis which makes exposed cells unable to synthesize Gb3 rendered Gb3‐expressing cells resistant to VT‐1. MPM cells with acquired‐cisplatin resistance expressed Gb3 in contrast to the absent of expression in the less resistant parental cell line. Gb3, could however be up‐regulated by cisplatin in Gb3‐negative MPM‐cells. Presence of a low‐toxic concentration of VT‐1 potentiated cisplatin‐induced cytotoxicity and apoptosis in the cisplatin‐resistance MPM cell line. VT‐1 was a potent inducer of apoptosis, probably via stress‐induced Mitogen‐activated protein kinase (MAPK)‐signaling involving c‐Jun N‐terminal kinase (JNK) and p38, leading to disruption of the mitochondrial membrane integrety, activation of caspase‐9 and ‐3, and ultimately DNA fragmentation and cell death. Gb3 expression was demonstrated in clinical specimens of glioblastoma and breast cancer making these tumor types interesting for further VT‐1 studies. We conclude that bacterial toxins may be used to induce apoptosis in several types of cancer cells. Low concentrations of verotoxin‐1 and α‐toxin may potentially be used to overcome acquired cisplatin‐resistance in cancer patients.

[Truncated abstract] Previous studies in the field of tumour immunology had identified regulatory T (Treg) cells as important suppressors of the anti-tumour immune response as the presence of Treg cells in the peripheral blood of cancer patients was correlated with worse disease outcomes. Other studies had identified Treg cells to be active at sites of immune regulation such as the gut of colitis patients. It was therefore hypothesised that Treg cells would be present and active within tumours to suppress the cellular antitumour immune response. ... This treatment targeting multiple pathways of Treg cell mediated immuno-suppression and resulted in tumour regression in 50% of treated animals. Finally, the anti-tumour immune response is complex and a potentially synergistic multi-modality treatment designed to inactivate intra-tumoural Treg cells but to also induce apoptosis in tumour cells themselves was investigated. Alpha-tocopheryl succinate (α-TOS), an analogue of vitamin E, had previously been shown to induce apoptosis in human MM xenografts implanted into immuno-deficient (nude) mice. Administration of α-TOS was therefore examined as a potentially synergistic treatment to be coupled with Treg cell inactivation. At the previously published doses used to treat immuno-deficient mice, α-TOS was found to be toxic to the immuno-competent mice used in this study. A marked depleting effect on total T cells was seen in the treated animals. The results of this thesis demonstrated the high potential for an adjunct immunotherapy of MM. They did however also highlight the importance of future studies into anticancer therapies to be conducted using clinically relevant tumour models and clinically relevant treatment regimes. The need to consider synergistic multi-modal therapies was also emphasised.

Malignt mesoteliom är en ovanlig och aggressiv cancerform. Den är vanligast i pleura och kallas då pleuralt mesoteliom. Vanligaste orsaken till utveckling av pleuralt mesoteliom är exponering för asbest. Insjuknande patienter har dålig prognos och begränsade behandlingsmöjligheter. Röntgen kan i ett första stadie ge diagnos av sjukdomen. Oftast behövs också en biopsi tas för fastställande av diagnos. Även provtagning av pleuravätskan kan ge värdefull diagnostisk information. Immunhistokemi är en viktig tilläggsanalys för diagnos med biopsier och cellblock. Immunhistokemi innebär färgning med antikroppar. För pleuralt mesoteliom kan exempelvis antikropparna PD-L1 och calretinin användas för diagnostisering. I denna studie färgades 10 parade pleurabiopsier och cellblock från pleuravätska från patienter med malignt mesoteliom. Antikropparna PD-L1 och calretinin användes. Syftet var att utvärdera uttrycket av PD-L1 och calretinin. Alla infärgade glas undersöktes i ljusmikroskop. För PD-L1 ansågs enbart membranfärgning som positivt infärgad och för calretinin ansågs cytoplasmatisk och/eller kärnfärgning som positiv. Procentuellt antal positiva tumörceller undersöktes vid två cutoff värden, ≥1 % och ≥10 %. Procentuellt antal <1 % vid cutoff ≥1 % och procentuellt antal <10 % vid cutoff ≥10 % ansågs negativt. Detta gällde för båda antikropparna. Antal positiva och negativa biopsier samt cellblock och konkordansen redovisades. Overall percentage agreement (OPA), Cohen’s kappa (κ) och konfidensintervall (CI) beräknades också. Resultatet visade på lägre antal positiva färgningar för cellblocken jämfört med biopsierna för båda antikropparna. För konkordansen visades att den vara lika för PD-L1 och calretinin vid cutoff ≥1 % men högre för PD-L1 än för calretinin vid cutoff ≥10 %. / Malignant mesothelioma is a rare and aggressive cancer. It is most common in the pleura, called pleural mesothelioma. The most common reason for developing pleural mesothelioma is exposure to asbestosis. Patients that get sick have a poor prognosis and limited treatment options. X-ray can in a first stadium give diagnose of the disease. A biopsy is often necessary to confirm the diagnosis. Even sampling of pleural fluid can give valuable diagnostic information. Immunohistochemistry is a common additional analysis for diagnosis with biopsy and cell block. Immunohistochemistry implies staining with antibodies. For example, for pleural mesothelioma the antibodies PD-L1 and calretinin can be used for diagnosis. In this study 10 paired pleural biopsies and cell blocks from pleural fluid from patients with malign mesothelioma were immunostained. The study aimed to evaluate expression of PD-L1 and calretinin. All the stained glasses were evaluated by a light microscope. For PD-L1, only membranous staining was considered positive and for calretinin cytoplasmic and/or nuclear staining was considered positive. Percentage number of positive tumor cells were evaluated at two cutoff values, ≥1 % and ≥10 %. Percentage number <1 % at cutoff ≥1 % and percentage number <10 % at cutoff ≥10 % were considered negative. The same criteria were applied for both antibodies. The number of positive and negative biopsies as well as cell blocks and the concordance were reported. Overall percentage agreement (OPA), Cohen’s kappa (κ) and confidence interval (CI) was also calculated. Result showed lower number of positive staining for the cell blocks than for the biopsies for both antibodies. The concordance was shown to be equal for PD-L1 and calretinin at cutoff ≥1 % but higher for PD-L1 than for calretinin at cutoff ≥10 %.