Tesis sobre el tema "Tumoral cell"

INTRODUÇÃO: O carcinoma espinocelular de origem bucal (CEC) apresenta uma alta taxa de mortalidade devido à invasividade das células tumorais. A migração celular, principal evento da invasão e metástase, pode ser regulada tanto por fatores intrínsecos, como adesão e contratilidade celular, quanto extrínsecos, como composição, densidade e remodelagem da matriz extracelular (MEC). OBJETIVO: Avaliar o papel de elementos intrínsecos e extrínsecos sobre o processo invasivo do carcinoma espinocelular de origem bucal. MÉTODOS: Foi realizada imuno-histoquímica para as proteínas: Miosina II (isoformas A, B e C), metaloproteinases de matriz (1, 2, 9 e 14); imunofluorescência as proteínas: e-caderina, n-caderina, FAK, paxilina, vinculina e fibronectina em amostras de CEC oral. Foi realizado ensaio de migração nas seguintes condições: 1 – matriz 2D com o substrato de fibronectina, ou laminina ou matrigel; 2 – matriz 3D com colágeno na presença ou não de fibronectina ou laminina; 3 – matriz 3D com diferentes concentrações de colágeno (0,6; 1,2 e 1,8 mg/ml) + fibronectina na presença ou não de um inibidor de MMP. Foi realizado análise de adesão celular utilizando-se o microscópio TIRF e o microscópio confocal, tanto em matrizes 2D quanto 3D. Foram realizados esferoides celulares para avaliar a contratilidade celular, através do plaqueamento das células em gel de agarose e a utilização de drogas que inibem ou que induzem a contratilidade, bem como a partir de células transfectadas com versões fosfomiméticas para a cadeia leve de miosina. Foi realizado ainda western blotting para proteínas: e-caderina, FAK, vinculina, paxilina, N-caderina, integrinas e as isoformas de miosina II, bem como foi avaliado os níveis de ativação das proteínas da família RhoGTPase, as quais estão envolvidas no controle da migração celular. RESULTADOS: A expressão das MMPs analisadas e das isoformas de miosinas foi maior nas zonas de invasão tumoral, sendo que o CEC oral também apresenta uma maior expressão de proteínas associadas à adesão com a MEC. A migração celular foi afetada pela densidade e a composição da MEC, bem como pela atividade das MMPs. Adicionalmente, a modulação das proteínas de adesão célula-matriz altera a velocidade de migração, a direcionalidade dessa migração e também a forma de migração, mudando de uma migração coletiva para uma migração individual. O aumento na contratilidade células resulta numa dispersão celular enquanto que a diminuição da contratilidade resulta numa melhor adesão célula – célula. CONCLUSÕES: O comportamento das células tumorais pode ser modulado através de fatores extrínsecos como, por exemplo, a alteração no microambiente tumoral, seja ela por mudança no substrato ou na densidade da matriz, e também dos fatores intrínsecos como a alteração nos níveis de miosina.
INTRODUCTION: Oral squamous cell carcinoma (OSCC) presents high mortality index due to the invasive phenotype of tumor cells. Cell migration is the main event in cell invasion and metastasis and it can be regulated by intrinsic factor, such as adhesion and cell contractility, and extrinsic factors, such as density and extracellular matrix (EMC) remodeling. OBJECTIVE: Analyze the role of intrinsic and extrinsic factor during the invasive process of oral squamous cell carcinoma. METHODS: We performed immunostaining in OSCC samples for the following proteins: myosin II (isoforms A, B and C), matrix metalloproteinase (1, 2, 9 and 14) e-cadherin, n-cadherin, FAK, paxillin, vinculin and fibronectin. We also performed migration assays with OSCC cell line in the following conditions 1 – 2D matrix with fibronectin or laminin or matrigel; 2 – 3D matrix with collagen in the presence or not of fibronectin or laminin; 3 – 3D matrix with different collagen concentration (0,6; 1,2 e 1,8 mg/ml) with fibronectin in the presence or not of the MMP inhibitor. In order to analyze cell adhesion, it was performed Total Internal Reflectance Fluorescence and Confocal microscopy, in 2D and 3D matrix. To analyze cell contractility, cells were plated in agarose gel in order to produce spheroids, which were treated with drugs that inhibit or induce cell contractility or cells were previously transfected with Myosin Light Chain phosphomimetics mutants. It was also performed western blotting to: e-cadherin, n-cadherin, FAK, paxillin, vinculin and myosin II isoforms, as well as it was analyze the levels in RhoGTPase family, which are involved in cell migration control. RESULTS: The expression to MMPs and myosin II isoforms were higher at invasion zone of the tumor, and the OSCC presented higher expression of proteins associated to adhesion to ECM. Cell migration was affected by the EMC composition and density and by MMP activity. Also, the modulation of cell-matrix adhesion proteins altered migration speed, cell directionality as well as influenced the switch between collective and single cell migration. The increase in cell contractility resulted in cell dispersion while the decrease in cell contractility resulted in a better cell-cell adhesion. CONCLUSIONS: The behavior of cell tumor can be modulate by extrinsic factors, for example, the change in tumor microenvironment, by the change in the EMC substrate or density and by intrinsic factors such as the alteration in myosin levels.

Latexin es un gen de respuesta a ácido retinoico (RA) cuya función celular se conoce escasamente. Latexin se encuentra silenciada por un mecanismo de hipermetilación en numerosas líneas tumorales, sugiriendo que ésta podría estar ejerciendo una función como supresor tumoral. Con el objetivo de elucidar su potencial implicación en cáncer, empleamos líneas derivadas de neuroblastoma ya que representan modelos adecuados para estudiar tanto procesos de apoptosis como de diferenciación. En este estudio revelamos que la escasa expresión de latexin parece una característica común de las líneas de neuroblastoma y que dicha expresión puede ser modulada mediante el tratamiento con RA. La sobreexpresión estable de latexin no altera los procesos proliferación celular ni de muerte celular en la línea SH-SY5Y. Sin embargo, su sobreexpresión sí que tiene un efecto significativo promoviendo la aparición de células del tipo S (del linaje Schwanniano) cuando son tratadas con RA. Además, su expresión también facilita la emergencia del fenotipo S cuando las células se exponen al agente 5-bromo-2’-deoxyuridine (BrdU). De hecho, la inhibición del axis PI3K/Akt impide la activación de senescencia Estas evidencias sugieren que latexin podría ser un determinante en la decisión del destino celular entre apoptosis y senescencia en células de neuroblastoma, probablemente modulando dicha cascada de señalización. Estos resultados nos alentaron a tratar de desvelar el panorama de expresión génico que promovueve latexin con el objetivo de identificar piezas claves en los efectos previamente mencionados que promueve latexin. Interesantemente, latexin promueve la expresión de un gran número de genes, principalmente involucrados en procesos de adhesión celular, desarrollo y morfogénesis. Curiosamente, la mayoría de genes promovidos por latexin, ya sea en presencia o ausencia de RA, pertenecen a la matriz extracelular (ECM), sugiriendo una potencial asociación entre la ECM y los efectos favorecidos por latexin en células SH-SY5Y. Cuando extendimos nuestros resultados a otros modelos celulares, observamos que latexin también fomenta el proceso de senescencia en respuesta al estímulo adecuado en la línea de neuroblastoma SK-N-LP y en la línea derivada de Glioblastoma Multiforme (GBM) LN-18. Sorprendentemente, una bajada en la expresión de latexin en las células U87-MG resulta en diferenciación tipo astrocitario cuando las células son tratadas con el agente genotóxico doxorubicina. Cabe remarcar que las células U87-MG con una expresión disminuida de latexin tienen perturbada la activación del proceso de senescencia. De una manera general, estos descubrimientos revelan una nueva función de latexin en la regulación de la especificación celular hacia la adquisición de un fenotipo senescente en diferentes modelos celulares.
Latexin is a recently discovered and poorly known retinoic acid (RA)-responsive gene whose cellular function is scarcely known. Latexin expression appears downregulated through promoter hypermethylation in several types of cancer, suggesting it can function as a tumor suppressor. With the aim to elucidate its potential role in cancer, we employed neuroblastoma-derived cells since they represent canonical and well-established models of apoptosis and differentiation. We first reveal that the lack of latexin expression appears as a common hallmark in neuroblastoma-derived cells although it can be modulated by RA treatment. The stable overexpression of latexin does not significantly alter cell proliferation or cell death responses in SH-SY5Y cells. However, the overexpression of latexin remarkably favors the emergence of S-type cells (Schwannian lineage) upon RA treatment. Consistently, latexin overexpression also facilitates the appearance of the S-type phenotype upon exposure to 5-bromo-2’-deoxyuridine (BrdU). Moreover, latexin-overexpressing cells display enhanced Akt activation upon RA or BrdU stimuli, or even in basal growth conditions. This activation allows latexin-overexpressing cells to survive for long periods under unfavorable extracellular conditions and to undergo cellular senescence. Indeed, the inhibition of the PI3K/Akt axis impedes the activation of cellular senescence. These evidences suggest that latexin could be a critical determinant of cell fate choices between apoptosis or senescence in neuroblastoma cells likely by modulating this cascade. These results encouraged us to unveil the landscape of gene expression promoted by latexin to identify key targets involved in the aforementioned latexin-mediated effects. Interestingly, latexin upregulated a large number of genes, most of them involved in cell adhesion, cell development and morphogenesis processes. Surprisingly, the vast majority of genes upregulated by latexin either in the presence or in the absence of RA belong to the extracellular matrix (ECM), therefore suggesting the potential involvement of the ECM in latexin-promoted effects in SH-SY5Y cells. When extending our results to other cellular models, we observe that latexin also promotes cellular senescence upon the adequate stimuli in the neuroblastoma cell line SK-N-LP and in the Glioblastoma Multiforme (GBM)-derived cell line LN-18. Intriguingly, latexin knock down in U87-MG cells results in astrocytic-like differentiation upon treatment with the genotoxic drug doxorubicin. Remarkably, U87-MG cells with decreased levels of latexin expression remarkably impair the activation of cellular senescence. Altogether, these findings disclose a novel functional role of latexin in regulating cell specification towards the acquisition of a senescent phenotype in different cellular models.

Contexte : Le mélanome est un cancer agressif chez l’homme, développé aux dépens des mélanocytes. L’identification de la mutation BRAF conditionne la prescription d’une thérapie ciblée. L’objectif de ce travail a été de mettre au point dans les tissus tumoraux et dans le sang (cellules tumorales circulantes, ADN libre tumoral plasmatique) des approches technologiques de biologie moléculaire et d’immunohistochimie (IHC) pour identifier des biomarqueurs prédictifs d’une réponse ou de résistance thérapeutique. Nous montrons que l’IHC BRAF (clone VE1, Roche, Ventana) pourrait remplacer l’analyse en biologie moléculaire dans certaines indications, notamment sur un matériel tumoral de petite taille. Parallèlement, nous montrons que la présence de cellules mélanocytaires circulantes [détectées par cytomorphologie (Technique ISET)] chez des patients atteints de mélanome métastatique est un facteur prédictif indépendant de mauvais pronostic de la survie globale. Enfin, nous montrons que le système Biocartis Idylla™ (processus automatisé couplant l’extraction, le séquençage et l’analyse de l’ADN) est sensible et spécifique pour la détection plasmatique des mutations BRAF et NRAS et que cette technique pourrait être indiquée dans le suivi de la maladie résiduelle (apparition de résistance) après traitement des patients atteints de mélanomes métastatiques. Conclusion : L’identification des biomarqueurs tissulaires et sanguins (BRAF, NRAS et CTC) permettent : 1- Une optimisation des délais diagnostiques de la mutation BRAF/NRAS – 2) L’identification de facteurs de mauvais pronostic – 3) De détecter une récidive précoce et de suivre la maladie résiduelle après traitement
Background: Knowledge of the BRAFV600E status is mandatory in metastatic melanoma patients (MMP). Molecular biology is currently the gold standard method for status assessment. The aim of this work was to assess and compare several methods of molecular biology and immunohistochemistry (IHC) in tissue and blood (cell-free circulating tumor DNA, circulating tumor cell (CTC)) to identify predictive biomarkers of response or resistance to targeted treatment. Results: We showed that BRAFV600 IHC could be a substitute for molecular biology in the initial assessment of the BRAFV600E status in MPP. We also found that the presence of circulating tumor cell detetcted by a cytomorphological approach ISET (Isolation by Size of Epithelial Tumor Cell – Rarecells Diagnostics, Paris, France) in MMP is an independent predictor of shorter survival. Then, in a monocentric study conducted at the University of Nice Hospital, we evaluated a novel and fully automated CE-IVD PCR-based system (IdyllaTM, Biocartis, Mechelen, Belgium) for plasmatic BRAF and NRAS mutation detection. We showed that this technology is highly sensitive and specific and provide promising potential to assess tumor progression, identify targets for therapy, and evaluate clinical response to treatment. In conclusion, identification of tissue and blood biomarkers with these technologies allow a quick turnaround-time to BRAF/NRAS diagnosis and improve monitoring of treatment response and development of resistance in metastatic melanoma patients

Contexte : Le mélanome est un cancer agressif chez l’homme, développé aux dépens des mélanocytes. L’identification de la mutation BRAF conditionne la prescription d’une thérapie ciblée. L’objectif de ce travail a été de mettre au point dans les tissus tumoraux et dans le sang (cellules tumorales circulantes, ADN libre tumoral plasmatique) des approches technologiques de biologie moléculaire et d’immunohistochimie (IHC) pour identifier des biomarqueurs prédictifs d’une réponse ou de résistance thérapeutique. Nous montrons que l’IHC BRAF (clone VE1, Roche, Ventana) pourrait remplacer l’analyse en biologie moléculaire dans certaines indications, notamment sur un matériel tumoral de petite taille. Parallèlement, nous montrons que la présence de cellules mélanocytaires circulantes [détectées par cytomorphologie (Technique ISET)] chez des patients atteints de mélanome métastatique est un facteur prédictif indépendant de mauvais pronostic de la survie globale. Enfin, nous montrons que le système Biocartis Idylla™ (processus automatisé couplant l’extraction, le séquençage et l’analyse de l’ADN) est sensible et spécifique pour la détection plasmatique des mutations BRAF et NRAS et que cette technique pourrait être indiquée dans le suivi de la maladie résiduelle (apparition de résistance) après traitement des patients atteints de mélanomes métastatiques. Conclusion : L’identification des biomarqueurs tissulaires et sanguins (BRAF, NRAS et CTC) permettent : 1- Une optimisation des délais diagnostiques de la mutation BRAF/NRAS – 2) L’identification de facteurs de mauvais pronostic – 3) De détecter une récidive précoce et de suivre la maladie résiduelle après traitement
Background: Knowledge of the BRAFV600E status is mandatory in metastatic melanoma patients (MMP). Molecular biology is currently the gold standard method for status assessment. The aim of this work was to assess and compare several methods of molecular biology and immunohistochemistry (IHC) in tissue and blood (cell-free circulating tumor DNA, circulating tumor cell (CTC)) to identify predictive biomarkers of response or resistance to targeted treatment. Results: We showed that BRAFV600 IHC could be a substitute for molecular biology in the initial assessment of the BRAFV600E status in MPP. We also found that the presence of circulating tumor cell detetcted by a cytomorphological approach ISET (Isolation by Size of Epithelial Tumor Cell – Rarecells Diagnostics, Paris, France) in MMP is an independent predictor of shorter survival. Then, in a monocentric study conducted at the University of Nice Hospital, we evaluated a novel and fully automated CE-IVD PCR-based system (IdyllaTM, Biocartis, Mechelen, Belgium) for plasmatic BRAF and NRAS mutation detection. We showed that this technology is highly sensitive and specific and provide promising potential to assess tumor progression, identify targets for therapy, and evaluate clinical response to treatment. In conclusion, identification of tissue and blood biomarkers with these technologies allow a quick turnaround-time to BRAF/NRAS diagnosis and improve monitoring of treatment response and development of resistance in metastatic melanoma patients

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Universidade Estadual Paulista (UNESP)
O mastocitoma canino (MCT) é uma neoplasia maligna de grande importância na clínica oncológica devido ao seu comportamento biológico agressivo e alta freqüência. A COX-2 e a PGE2 têm sido associadas à promoção e progressão tumoral e seus principais mecanismos envolvem estímulos da angiogênese tumoral e a inibição da morte celular programada. O VEGF é um potente indutor da angiogênese e a caspase-3 tem um importante papel na via efetora da apoptose. Compreender o mecanismo pela qual a COX-2 pode estimular a progressão tumoral no mastocitoma, permite ampliar o conhecimento do comportamento biológico desta neoplasia e direcionar tratamentos mais eficazes. O presente trabalho fez um estudo retrospectivo em 24 casos de mastocitoma canino (MCT). As neoplasias foram classificadas de acordo com Patnaik et al. (1984) e a expressão da COX-2, PGE2, VEGF e caspase-3 foram avaliadas usando a técnica de imunoistoquímica. A expressão da COX-2 foi correlacionada à expressão do VEGF, PGE2 e caspase-3 nos diferentes graus histopatológicos. A imunomarcação da caspase-3 foi menor nos tumores indiferenciados comparados com os bem diferenciados. Comparando os dados da expressão da COX-2 com os demais marcadores foi observado a correlação positiva entre COX-2 e PGE2, COX-2 e VEGF nas graduações II e III. A correlação entre COX-2 e caspase-3 foi somente detectada no grau III.
The canine mast cell tumor (MCT) is a malignant neoplasia with great importance on the clinical practice due to its aggressive behavior and high frequency. The COX-2 and the PGE2 have been associated to the tumor initiation, promotion and progression, and its main mechanisms involve the stimuli of tumor angiogenesis and the inhibition of apoptosis. The VEGF is a powerful inductor of angiogenesis and the caspase-3 is responsible for most part of the apoptotic effects. The understanding of the mechanism by which the COX-2 stimulates the tumor progression in the mast tumor cells provides an extension through the biological behavior of this neoplasia and leads to a better and effective treatment. The present work was a retrospective study in 24 cases of MCT. The neoplasias were classified according to Patnaik et al. (1984) and the expression of COX-2, PGE2, VEGF and caspase-3 were evaluated using the immunohistochemistry technique. The expression of COX-2 was correlated to the expression of VEGF, PGE2 and caspase-3 in the different histopathologic grades. Caspase-3 immunolabeling was lower in the undifferentiated tumors compared to the more differentiated ones. Comparing the COX-2 expression data to the other markers it was observed a positive correlation between COX-2 and PGE2, COX-2 and VEGF in grade II and III. Correlation between COX-2 and caspase-3 was detected only on grade III. Keywords: COX-2, PGE2, VEGF, caspase-3, mast cell tumor.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
No presente estudo avaliou-se os efeitos da inoculaÃÃo do carcinosarcoma 256 de Walker, bem como o curso de seu desenvolvimento, sobre a reaÃÃo inflamatÃria sistÃmica. Ratos Wistar machos, pesando entre 180 a 220g, foram inoculados, por via intramuscular, com 106 cÃlulas tumorais na coxa direita. Os experimentos foram realizados apÃs o 4Â, 7Â e 10Â dias (4D, 7D e 10D) da inoculaÃÃo do carcinossarcoma 256 de Walker. O grupo controle nÃo foi inoculado as cÃlulas tumorais. Os ratos foram divididos em grupos experimentais com n = 6, nos quais foram avaliados os seguintes parÃmetros: edema de pata por carragenina (Cg; 300μg/pata direita) ou dextrana (Dxt; 500μg/pata direita), atividade da enzima mieloperoxidase (MPO), migraÃÃo de neutrÃfilos para cavidade peritoneal induzidas por carragenina (Cg; 300μg/pata direita), permeabilidade vascular cutÃnea induzidas por bradicinina (2μg/sÃtio), histamina (30μg/sÃtio), serotonina (1μg/sÃtio), substÃncia P (250ng/sÃtio), capsaicina (50μg/sÃtio) ou composto 48/80 (1μg/sÃtio) e degranulaÃÃo mastocitÃria induzida por composto 48/80. A intensidade do edema foi avaliada na pata contralateral ao tumor 1, 2, 3 e 4 hs (Cg) e 30â ,1 2, 3, ,4 hs (Dxt) por pletismometria. A migraÃÃo de neutrÃfilos foi induzida pela administraÃÃo de Cg (300μg/pata) na pata contralateral ao tumor ou na cavidade peritoneal. ApÃs 4 horas, os ratos foram sacrificados e as peles das patas foram retiradas para medir indiretamente a infiltraÃÃo de neutrÃfilos, pela tÃcnica da dosagem da atividade da MPO, e a migraÃÃo de neutrÃfilos para cavidade peritoneal foi avaliada atravÃs da contagem total e diferencial de leucÃcitos. Em relaÃÃo a permeabilidade vascular cutÃnea, imediatamente apÃs as injeÃÃes intradÃrmicas dos estÃmulos (bradicinina, histamina, serotonina, substÃncia P, capsaicina ou composto 48/80), foi administrado azul de Evans (0,1mL/100g do animal), na veia do plexo peniano. ApÃs 30 min, os ratos foram sacrificados e a pele do dorso retirada, para avaliar o extravasamento do azul de Evans por espectrometria. A degranulaÃÃo de mastÃcitos do mesentÃrio foi avaliada apÃs coloraÃÃo com azul de toluidina, sendo contados os mastÃcitos degranulados num total de 100 cÃlulas. Os animais com tumor apresentaram uma inibiÃÃo significativa do edema de pata, com efeito mÃximo observado nos 7 Â e 10 Â dias, tanto com a Cg, quanto com Dxt, quando comparados com o controle sem tumor. Em nenhum dos dias estudados, foram observadas diferenÃas na atividade da MPO na pata e nem na avaliaÃÃo da migraÃÃo de neutrÃfilos para a cavidade pÃritoneal induzidas por Cg. ApÃs 4 e 7 dias da inoculaÃÃo do tumor, o animais apresentaram uma significativa diminuiÃÃo na permeabilidade vascular cutÃnea induzida por bradicinina, serotonina, e composto 48/80. No entanto, o aumento da permeabilidade vascular induzida por histamina, substÃncia P e capsaicina nÃo foi alterada nesses dois dias. No 10 Â dia, observou-se uma diminuiÃÃo da permeabilidade vascular induzida por todos os estÃmulos quando comparado com o grupo sem tumor. A degranulaÃÃo mastÃcitÃria foi inibida em animais com tumor nos 4Â, 7Â e 10Â dias em comparaÃÃo com o grupo controle. Tais dados sugerem que o microambiente do tumor de Walker diminui o curso da resposta inflamatÃria atravÃs da inibiÃÃo da degranulaÃÃo dos mastocitos
Our objective was to evaluate the effect of the 256 Walker carcinossarcoma inoculation, as well as the time course of tumoral development, upon the acute inflammatory response in rats. Wistar rats, 180-220g, received intramuscular 106 tumor cells injections. At the end of 4, 7 or 10 days, wistar rats were separated into 4 groups, with 6 animals per group. The control group, were not inoculated with tumoral cells. Several parameters were evaluated: paw edema induced by carrageenan (Cg; 300μg/hind) or dextran (Dxt, 500μg/hind paw), myeloperoxidase activity (MPO), neutrophil migration to peritoneal cavity induced by carrageenan, cutaneous vascular permeability induced by bradykinin (2μg/site), serotonin (1μg/site), histamine (30μg/site), substance P (250ng/site), capsaicin (50μg/site) or 48/80 compound (1 μg/site) and mast cell degranulation induced by 48/80 compound. Paw edema was evaluated in the contra lateral hind paw of the tumor and measured at 0, 1, 2, 3 and 4h for Cg, and 0, 30â, 1, 2, 3 and 4h.for Dxt by plethysmometry. Neutrophil migration was induced by Cg injection in the contralateral hind paw or in the peritoneal cavity. After 4h, rats were sacrificed and the skin of the hind paw was harvested to measure neutrophil infiltration by MPO assay. Neutrophil migration induced by Cg was also evaluated in the peritoneal cavity, with the total e differential leucocytes counted. In order to measure cutaneous vascular permeability, immediately after intradermic stimulus injections (bradykinin, histamine, serotonin, substance P, capsaicin or 48/80 compound) Evans Blue dye was administrated (0,1mL/100g of per animal) by endovenous route. After 30 min rats were sacrificed and the skin was harvested to evaluate Evans Blue extravasations by spectrofotometry. Mast cells degranulation was evaluated in the in mesentery incubated with 48/80 compound and colored with toluidine blue. Our results shows that, in animals inoculated with the carcinossarcoma, there was a significant inhibition in the Cg and Dxt- induced paw edema, with maximal effect at the 7th and 10th days. There were no differences in MPO activity and neither in the peritoneal neutrophil infiltration induced by Cg in rats inoculated with the carcinossarcoma when compares to normal animals. After 4 and 7 days of the tumor inoculation, we observed a significant inhibition of the vascular permeability induced only by bradikinin, serotonin and 48/80 compound.. In the 10th day after the carcinossarcoma inoculation, there was a significant inhibition of the vascular permeability induced by all inflammatory stimulus tested, when we compared animals not inoculated. Mast cell degranulation was decreased in the 4th, 7th and 10th days after carcinossarcoma inoculation. These results suggested that the tumor microenvironment decreased the acute inflammatory response probably due to a inhibition of the mast cell degranulation.

Les polynucléaires neutrophiles ont longtemps été considérés comme des cellules n’intervenant que dans la réponse immune innée. Cependant, au cours de ces dernières années, de nombreuses publications suggèrent que ces cellules, retrouvées au sein du microenvironnement de nombreux cancers, pourraient également jouer un rôle dans la tumorigénèse et la progression tumorale. Ces études mettent en évidence leur fréquence comme marqueur pronostique dans différents cancers solides, mais peu de travaux se sont intéressés à la caractérisation fonctionnelle de ces cellules dans la progression tumorale. Dans de nombreux cancers dont les lymphomes B issus du centre germinatif, les cellules tumorales, qui sont incapables de proliférer et de survivre seules, sont dépendantes de leur microenvironnement de soutien. Dans cette étude, nous avons évalué la fonctionnalité des polynucléaires neutrophiles dans la croissance des lymphomes B. Ainsi, nous avons démontré pour la première fois que les polynucléaires neutrophiles soutiennent directement la croissance et la survie des cellules tumorales de lymphomes B. De plus, un dialogue bidirectionnel existe entre les polynucléaires neutrophiles et les cellules stromales. D’une part, les cellules stromales soutiennent la survie des polynucléaires neutrophiles, qui en retour induisent les caractéristiques d’un stroma lymphoïde. L’induction de ce phénotype permet aux cellules stromales d’acquérir de meilleures capacités de soutien envers les cellules tumorales. Cette étude confirme donc que les polynucléaires neutrophiles sont une composante importante du microenvironnement tumoral, et pourraient devenir une nouvelle cible thérapeutique pour le traitement des lymphomes B issus du centre germinatif
For long time, neutrophils have only been considered as cells involved in the innate immune response. More recently, in descriptive publications, neutrophils were found in the microenvironment of many solid cancers, hypothesizing that they could also play a role in tumorigenesis and cancer progression. These studies highlighted the prognostic value of their frequency, but few of them focused on the functional characterization of these cells in tumor growth. In many cancers, including germinal centre-derived B-cell lymphomas, tumor cells are dependent on their microenvironment to proliferate and survive. In this study, we focused on the role of neutrophils in the progression of B-cell lymphomas, and for the first time we demonstrated that neutrophils directly support the growth and survival of tumor Bcells. In addition, we highlighted the existence of bidirectional cooperation between neutrophils and stromal cells. In one hand stromal cells support the survival of neutrophils. On the other hand, neutrophils induce a lymphoid stroma phenotype which is well known to enhance their supportive effect on tumor cells. This study demonstrates that neutrophils are a significant component of the tumor microenvironment and may be considered as a potential therapeutic target for the treatment of B-cell lymphomas

A heterogeneidade entre as células tumorais e o suporte a elas proporcionado pelos componentes do microambiente tumoral (TME) são os dois principais responsáveis pela progressão do câncer e por tornar essas doenças essencialmente incuráveis. Assim, identificar as principais dependências das células malignas, sejam elas internas ou advindas do meio extracelular, é fundamental para entender seu comportamento e propor terapias mais eficientes. Nesta tese, abordamos aspectos destas duas questões separadamente. Em um primeiro trabalho, investigamos as interações de células tumorais com células-tronco mesenquimais (MSCs), um dos principais componentes do TME. MSCs participam ativamente do nicho tumoral, especialmente por serem capazes de liberar uma vasta gama de moléculas que, via sinalização parácrina, podem modular as células ao seu redor. No entanto, os principais mediadores e respectivos efeitos do secretoma dessas células nos tumores ainda precisam ser melhor elucidados. Ao investigar esses efeitos em glioblastomas (GBM), um dos tumores primários mais agressivos em adultos, mostramos que o secretoma de células-tronco mesenquimais derivadas de tecido adiposo humano (hADSCs) foi capaz de bloquear a autofagia das células malignas. Nossos dados revelaram que o secretoma de hADSCs ativou a via de sinalização de mTORC1 e reduziu a translocação nuclear de TFEB, um fator de transcrição chave que regula a autofagia e a a função lisossomal, nas células de GBM, impedindo que o fluxo autofágico fosse completado. Já em um segundo trabalho, no contexto da heterogeneidade celular em tumores, propusemos uma abordagem para análise de dados de céulas únicas focada em outliers. Minorias celulares com níveis anormalmente elevados, ou reduzidos, de expressão de determinados genes ou proteínas são em muitos casos responsáveis por resistir aos tratamentos e levar à recidiva da doença, ao mesmo tempo que, por serem outliers, são muitas vezes ignoradas ou excluídas das análises de dados. Assim, decidimos utilizar métodos estatísticos em dados de expressão de células únicas para detectar e analisar células outliers, comparando o seu comportamento com as demais células não-outliers. Denominamos essa abordagem de Single Cell OUTlier analysis (SCOUT) e a testamos em dados de células tumorais avaliadas por citometria de massas e por sequenciamento de RNA de células únicas (sc-RNA-seq). Como resultado, pudemos confirmar que, especialmente diante de determinados tratamentos, células outliers podem se comportar de maneira distinta de não-outliers, revelando informações potencialmente relevantes ao desenvolvimento de estretégias terapêuticas. Por fim, desenvolvemos uma ferramenta para automatizar a detecção e seleção de outliers em dados de célula única a fim de facilitar o estudo dessas células em diversos aspectos na pesquisa do câncer.
Intratumoral heterogeneity and the support provided by components of the tumor microenvironment (TME) to malignant cells are major contributors to cancer progression, and the two main factors that make this disease essentially incurable. Thus, identifying malignant cells dependencies, either in the intra- or extracellular environment, is fundamental to understand their behavior and propose more efficient therapies. In this thesis, we approached aspects of these two issues separately. In a first work, we investigated interactions between tumors and mesenchymal stem cells (MSCs), one of the main components in the TME. MSCs actively participate in the tumor niche, especially due to their capacity of releasing a wide range of molecules that can modulate cells in their surroundings. However, little is known about the effects of MSCs-derived molecules in tumor cells behavior. In investigating these effects on glioblastomas (GBM), one of the most aggressive primary tumors in adults, we found out that the secretome of human adipose-derived stromal cells (hADSCs) was able to block autophagy in malignant cells. Our data revealed that hADSCs secretome activated mTORC1 signaling pathway and reduced nuclear translocation of TFEB, a master transcription factor that regulates autophagy and lysosomal function, in GBM cells, preventing autophagic flux from being completed. In a second work, we addressed intratumoral heterogeneity by proposing an approach to analyze outliers in single cell data. Cellular minorities with abnormally high, or low, expression levels of certain genes or proteins are in many cases responsible for resisting treatments and lead to disease relapse, while for being outliers they are also frequently ignored or excluded from data analysis. Thus, we decided to apply statistical methods on single cell expression data to detect outliers and analyze them, comparing their behavior with the remaining non-outlier cells. We called this approach Single Cell OUTlier analysis (SCOUT) and tested it on tumor cell datasets obtained from mass cytometry and single cell RNA sequencing (scRNA-seq) experiments. Using SCOUT we were able to confirm that, especially upon specific treatments, outlier cells may behave differently from non-outliers, revealing potentially relevant information to aid in the development of novel therapeutic strategies. Finally, we developed a tool to automate detection and selection of outliers in single cell data with the aim to facilitate the study of these cells under different contexts in cancer research.

A maioria dos tumores sólidos apresentam características aneuplóides. Porém a relação entre aneuploidia e transformação maligna, ainda não está definida. Nos últimos anos diversas proteínas têm sido descritas como reguladoras de eventos durante a divisão celular, principalmente as relacionadas com a formação do fuso bipolar e segregação equacional dos cromossomos. Neste estudo propomo-nos a analisar os efeitos da interferência em dois pontos críticos da mitose, a segregação cromossômica e a citocinese, em relação à aneuploidia e à instabilidade genética tumoral. Nossos dados mostraram que o tratamento sequencial de Monastrol e Blebistatina determinou o surgimento de fusos mitóticos anormais, amplificação centrossômica, localização ectópica de Aurora A e aumento de micronúcleos. Esta interferência pode levar a um quadro de instabilidade genética e, consequentemente a progressão tumoral, abrindo novas possibilidades para o estudo dos mecanismos moleculares envolvidos na regulação do ponto de checagem mitótico e resistência a quimioterápicos.
Most solid tumors have aneuploid feature. Therefore the relationship between aneuploidy and malignant transformation is not yet understood. In the last years it has been described many proteins involved in regulation of mitosis, mainly those related to bipolar spindle and chromosome segregation. In this work we propose to study the effects of the interference on two mitotic critical points, the chromosome segregation and cytokinesis, in relation to aneuploidy and genetic tumor instability. Our data showed that sequential treatment with Monastrol and Blebbistatin led to abnormal mitotic spindle, centrosome amplification, Aurora A ectopic and micronucleus increased. This interference can lead to genetic instability and may be involved in a tumor progression, opening news possibilities to study the molecular mechanisms involved in regulation the checkpoint mitotic and resistance to chemotherapy found in genetically unstable cells.

En aquest treball de tesi hem estudiat i caracteritzat diferents aspectes relacionats amb la fisiopatologia del carcinoma basocel·lular (CBC). El CBC es tracta de la neoplàsia maligna més freqüent de l'espècie humana, tot i que aquesta dada es troba moltes vegades obviada en els estudis epidemiològics de càncer al tractar-se d'un tumor que produeix molt baixa mortalitat. El carcinoma basocel·lular és un tumor invasiu però de creixement lent i rarament deriva en metàstasi. El tractament d'elecció segueix sent avui en dia el tractament quirúrgic i en alguns casos s'utilitzen teràpies sistèmiques com vismodegib, una molècula capaç de bloquejar la principal via molecular implicada en la fisiopatogènesis del carcinoma basocel·lular, la via Sonic hedgehog (Shh). És important destacar que la freqüència de carcinoma basocel·lular és molt alta i és un problema de cost sanitari molt elevat en molts països, per exemple en els països de l'arc mediterrani o en Oceania. Tot i l'altíssima prevalença del carcinoma basocel·lular a nivell mundial, encara hi ha moltes coses que es desconeixen amb certesa, per exemple el seu origen cel·lular. La teoria més acceptada sobre el carcinoma basocel·lular situa el seu origen en les cèl.lules mare del fol·licle pilós, pel que seria d'esperar que el carcinoma basocel·lular no aparegui en àrees desproveïdes de fol·licles pilosos, com les mucoses o semimucoses. No obstant, tot i que és cert que el carcinoma basocel·lular sol trobar-se en zones on hi ha fol·licles pilosos, com constitueix la major part de la superfície cutània, en aquesta tesi hem descrit diversos casos en què el carcinoma basocel·lular neix en àrees desproveïdes de pèl com la semimucosa vulvar. A partir d'aquests primers estudis hem proposat l'existència d'un origen cel·lular alternatiu al fol·licle pilós. Amb l'objectiu d'estudiar l'origen cel·lular del carcinoma basocel·lular, en aquest treball hem dut a terme un estudi inèdit en el qual hem determinat l'expressió de diferents citoqueratines en el carcinoma basocel·lular en funció de la seva localització anatòmica, grau d'exposició solar i presència de fol·licles pilosos. Els diferents grups cel·lulars en òrgans i teixits presenten un patró específic d'expressió de citoqueratines que en molts casos es manté després de la transformació tumoral, el que permet establir en quin nínxol cel·lular s'origina el tumor. Sorprenentment en aquest estudi observem que la citoqueratina 7, present en l´epiteli simple amb diferenciació glandular, es trobava altament expressada en aquells carcinomes basocel·lulars situats en semimucosa vulvar i àrees on existia abundants glàndules sebo-apocrines com en les aixelles. A partir d'aquests resultats proposem que el carcinoma basocel·lular pugui també originar-se a partir de primordis glandulars alternativament als fol·licles pilosos. En molts carcinomes la seva capacitat invasiva es relaciona amb processos de transició d'epiteli a mesènquima (TEM). Diferents treballs mostren l'expressió de marcadors de TEM en CBC el que coincideix amb la forta capacitat invasiva d'aquest tumor, encara que no sigui capaç de disseminar-se per metàstasi. Com a part d'aquesta tesi, hem estudiat la TEM en cultius primaris de CBC. Els nostres resultats mostren que en els cultius de CBC hi ha activació de la via d'TGF-β i a més apareixen un percentatge elevat de cèl·lules amb morfologia fibroblastoide. Aquests resultats també coincideixen amb la possibilitat d'activació de la TEM en CBC, però no són concloents ja que l'activació de marcadors podria ser deguda als defectes en la via Shh. Un altre tema interessant en oncologia és com alguns tumors són capaços de presentar regressió tumoral total o parcial de manera espontània, bàsicament a causa del paper que el sistema immune té en combatre el càncer. En el carcinoma basocel·lular superficial s'observa amb certa freqüència, com existeix regressió central i una vora activa de progressió. En aquesta tesi hem estudiat mitjançant immunohistoquímica l'expressió de diferents marcadors de la immunitat innata i adaptativa a la zona de regressió tumoral i de la vora activa tumoral. Les nostres dades suggereixen una resposta adaptativa a la zona de regressió tumoral que estaria inhibida a les vores actives del tumor. El CBC superficial, es tracta d'un tumor de fàcil accés, pensem que podria utilitzar-se com un model tumoral per fer estudis de regressió que permetin donar-nos informació sobre aquest procés i extrapolar resultats a altres tumors. Finalment com a últim apartat d'aquesta tesi mostrem resultats sobre l'eficàcia i el mecanisme d'acció de ingenol mebutato per al tractament del carcinoma basocel·lular. Observem que ingenol mebutato va aconseguir fins a un 80% de curació en els carcinomes basocel·lulars superficials de la nostra sèrie, destacant la important reacció inflamatòria que van presentar gran part dels pacients, amb eritema, ampolles i erosions que sobrepassaven la zona d'aplicació del producte, però en cap cas vam obtenir efectes secundaris a nivell sistèmic. L'estudi de l'infiltrat inflamatori suggereix que hi ha una resposta innata deguda a la inflamació. Fruit d'aquests treballs hem aconseguit publicar dos articles que es troben a l'annex de la tesi, i un tercer article està sent revisat. He publicat un capítol de llibre en relació amb la temàtica de la tesi doctoral. A més altres tumors cutanis que he tingut ocasió de revisar durant la meva residència i publicar també figuren a l'annex de la tesi.
En este trabajo de tesis hemos estudiado y caracterizado distintos aspectos relacionados con la fisiopatología del carcinoma basocelular (CBC). El CBC se trata de la neoplasia maligna más frecuente de la especie humana, si bien este dato se encuentra muchas veces obviado en los estudios epidemiológicos de cáncer al tratarse de un tumor que produce muy baja mortalidad. El carcinoma basocelular es un tumor invasivo pero de crecimiento lento y raramente deriva en metástasis. El tratamiento de elección sigue siendo hoy en día el tratamiento quirúrgico y en algunos casos se utilizan terapias sistémicas como vismodegib, una molécula capaz de bloquear la principal vía molecular involucrada en la fisiopatogénesis del carcinoma basocelular, la vía Sonic hedgehog (Shh). Es importante destacar que la frecuencia de carcinoma basocelular es muy alta y es un problema de coste sanitario muy elevado en muchos países, por ejemplo en los países del arco mediterráneo o en Oceanía. A pesar de la altísima prevalencia del carcinoma basocelular a nivel mundial, todavía hay muchas cosas que se desconocen con certeza, por ejemplo su origen celular. La teoría más aceptada sobre el carcinoma basocelular sitúa su origen en las células madre del folículo piloso, por lo que sería de esperar que el carcinoma basocelular no aparezca en áreas desprovistas de folículos pilosos, como las mucosas o semimucosas. Sin embargo, aunque es cierto que el carcinoma basocelular suele encontrarse en zonas donde existen folículos pilosos, como constituye la mayor parte de la superficie cutánea, en esta tesis hemos descrito varios casos en el que el carcinoma basocelular nace en áreas desprovistas de pelo como la semimucosa vulvar. A partir de estos primeros estudios hemos propuesto la existencia de un origen celular alternativo al folículo piloso. Con el objetivo de estudiar el origen celular del carcinoma basocelular, en este trabajo hemos llevado a cabo un estudio inédito en el que hemos determinado la expresión de diferentes citoqueratinas en el carcinoma basocelular en función de su localización anatómica, grado de exposición solar y presencia de folículos pilosos. Los distintos grupos celulares en órganos y tejidos presentan un patrón específico de expresión de citoqueratinas que en muchos casos se mantiene después de la transformación tumoral, lo que permite establecer en que nicho celular se origina el tumor. Sorprendentemente en este estudio observamos que la citoqueratina 7, presente en epitelio simple con diferenciación glandular, se encontraba altamente expresada en aquellos carcinomas basocelulares situados en semimucosa vulvar y áreas donde existían abundantes glándulas sebo-apocrinas como en las axilas. A partir de estos resultados proponemos que el carcinoma basocelular pueda también originarse a partir de primordios glandulares alternativamente a los folículos pilosos. En muchos carcinomas su capacidad invasiva se relaciona con procesos de transición de epitelio a mesénquima (TEM). Distintos trabajos muestran la expresión de marcadores de TEM en CBC lo que coincide con la fuerte capacidad invasiva de este tumor, aunque no sea capaz de diseminarse por metástasis. Como parte de esta tesis, hemos estudiado la TEM en cultivos primarios de CBC. Nuestros resultados muestran que en los cultivos de CBC hay activación de la vía de TGF-β y además aparecen un porcentaje elevado de células con morfología fibroblastoide. Estos resultados también coinciden con la posibilidad de activación de la TEM en CBC, pero no son concluyentes ya que la activación de marcadores podría ser debida a los defectos en la vía Shh. Otro tema interesante en oncología es cómo algunos tumores son capaces de regresar total o parcialmente de manera espontánea, básicamente debido al papel que el sistema inmune juega en combatir el cáncer. En el carcinoma basocelular superficial se observa con cierta frecuencia, como existe regresión central y un borde activo de progresión. En esta tesis hemos estudiado mediante inmunohistoquímica la expresión de diferentes marcadores de la inmunidad innata y adaptativa en la zona de regresión tumoral y del borde activo tumoral. Nuestros datos sugieren una respuesta adaptativa en la zona de regresión tumoral que estaría inhibida en los bordes activos del tumor. El CBC superficial, al tratarse de un tumor de fácil acceso, podría utilizarse como un modelo tumoral para hacer estudios de regresión que permita darnos información sobre este proceso y extrapolar resultados a otros tumores. Finalmente como último apartado de esta tesis mostramos resultados sobre la eficacia y el mecanismo de acción de ingenol mebutato para el tratamiento del carcinoma basocelular. Observamos que ingenol mebutato consiguió hasta un 80% de curación en los carcinomas basocelulares superficiales de nuestra serie, destacando la importante reacción inflamatoria que presentaron gran parte de los pacientes, con eritema, ampollas y erosiones que sobrepasaban la zona de aplicación del producto, pero en ningún caso obtuvimos efectos secundarios a nivel sistémico. El estudio del infiltrado inflamatorio sugiere que hay una respuesta innata debida a la inflamación. Fruto de estos trabajos hemos logrado publicar dos artículos que se encuentran en el anexo de la tesis, y un tercer artículo está siendo revisado. He publicado un capítulo de libro en relación con la temática de la tesis doctoral. Además otros tumores cutáneos que he tenido ocasión de revisar durante mi residencia y publicar también figuran en el anexo de la tesis.
In this doctoral thesis we have studied and characterized different aspects related to the pathophysiology of basal cell carcinoma (BCC). BCC is the most frequent malignant neoplasm in humans, although this piece of information is often overlooked in epidemiological studies of cancer due to its low mortality rate. BCC is an invasive but slow-growing tumor that rarely results in metastasis. The standard treatment nowadays continues to be surgical excision while in some cases, systemic therapies such as vismodegib, a molecule capable of blocking the main molecular pathway involved in the pathophysiology of BCC, the Sonic hedgehog (Shh) pathway, are used. It is important to note that BCC frequency is very high and has a high cost for the health system in many countries, for example in the Mediterranean countries or in Oceania. Despite the high prevalence of BCC worldwide, there are still many not well known aspects, for instance the BCC cellular origin. The most accepted theory is that the origin of BCC is in the stem cells of hair follicle, so it would be expected that BCC should not appear in non-hairy areas, such as mucous or semi-mucous. However, although it is true that BCC tends to be found in areas where hair follicles exist, as is the case with almost all the skin surface, in this work we have described several cases of BCC located in non-hairy areas such as the vulvar semimucosa. After this initial data, we have proposed a new cellular origin alternative to that of the hair follicle. In order to study the cellular origin of BCC, in this work we have carried out a novel study in which we have determined the cytokeratin profile of basal cell carcinomas depending on the degree of sun-exposure and on the anatomical localization and the presence of hair follicles. The different cell groups in organs and tissues have a specific pattern of expression of cytokeratins that in many cases is maintained after the tumor transformation, for this reason the cytokeratin profile gives clues to establish in which cellular niche the tumor is originated. Surprisingly, in this study, we observed that cytokeratin 7, present in simple epithelium with glandular differentiation, was highly expressed in basal cell carcinomas located in anatomical areas where sebaceous apocrine glands are very rich such as the axillae and the vulvar semimucosa. Based on these results, we propose the sebaceous-apocrine unit for BCC cellular origin as an alternative to the stem cells of hair follicles. In most carcinomas the invasiveness is associated to their ability to trigger epithelial mesenchymal transition (EMT). Different studies show the presence of EMT markers in BCC according to the strong invasive capacity of this tumor, although it is not capable of spreading by metastasis. As part of this work, we have studied EMT in primary BCC cultures. Our results show that BCC cells have induction of the TGF-β pathway and in addition, there is a high percentage of cells with fibroblastoid morphology. These results also agree with the possibility of activation of EMT in BCC but are inconclusive since the activation of markers could be due to defects in the Shh pathway. Another interesting topic in oncology is to know how certain tumors can spontaneously undergo either total or partial regression basically by triggering specific immune responses. In superficial basal cell carcinomas spontaneous regression is often observed in the center area while in the active edge we can observe progression. In this work, we have studied by immunohistochemistry different markers representative of innate and adaptive immunity in the area of tumor regression and the tumor active edge. Our data suggest an adaptive response in the area of tumor regression that would be inhibited at the active edges of the tumor. Being an easily accessible tumor, superficial BCC could be used as a tumor model to make regression studies that would provide us with information about this process and extrapolate the results to other tumors. Finally, as a final section of this thesis, we show results on the efficacy and mechanism of action of mebutate ingenol for the treatment of BCC. We observed that mebutate ingenol achieved up to 80% cure in our series of superficial BCC, highlighting the important inflammatory reaction that a great part of the patients showed, with erythema, blisters and erosions that surpassed the area of application of the product. However, there were no reported side effects at a systemic level. The study of the inflammatory infiltrate suggests that there is an innate response due to inflammation. As a result of these works I have published two papers which can be found in the annex to this thesis, and a third article is under revision. I have published a book chapter to the subject of this doctoral thesis. In addition, other skin tumors that I have had the opportunity to review and publish during my residency as a dermatologist also appear in the annex to this doctoral thesis.

Os carcinomas de cabeça e pescoço representam um problema na saúde pública, sendo a oitava causa no mundo de morte por câncer. A taxa de crescimento do tumor, o seu local de expansão, bem como a metástase das células cancerígenas depende muito da vascularização do tumor, sendo que esta é a responsável pelo fornecimento constante de nutrientes e de oxigênio para o crescimento tumoral. Sendo assim a angiogênese é considerada um processo essencial dentro do processo neoplásico. A avaliação dos vasos sanguíneos tumorais neoformados em carcinomas espinocelulares de boca, usando o anticorpo CD105, mostra um crescimento significativo da densidade microvascular. Baseado nestes, o objetivo do trabalho é avaliar através da imunoistoquímica a possível correlação de aumento no número de vasos sanguíneos correlacionando com diferentes grupos de processo inflamatório divididos em: pouco, moderado e intenso infiltrado inflamatório no front tumoral. Na literatura os autores de um modo geral correlacionam infiltrados inflamatórios e angiogênese, neste trabalho tentamos correlacionar se um maior ou menor infiltrado inflamatório tem influência nessa angiogênese. As lâminas foram avaliadas microscopicamente por dois profissionais de forma que eles não sabiam da classificação dada pelo outro e só quando ambos estavam em comum acordo essas lâminas foram classificadas. Para a análise estatística foi realizado a comparação múltipla entre os 03 grupos através da Análise de variância (comparação das três médias) e também o teste de Tukey, onde se observou diferença entre os grupos I e III e nos grupos II e III, porém entre os grupos I e II não houve diferença significativa. Com isso os resultados nos mostram uma correlação positiva entre a presença de maior quantidade de vasos sanguíneos, onde se encontra maior quantidade de infiltrado inflamatório, quando comparado com áreas de menor infiltrado inflamatório.
Carcinomas of the head and neck represent a public health problem, being the eighth leading cause of worldwide cancer deaths. The growth rate of the tumor, its location expansion and metastasis of cancer cells depends greatly on tumor vascularity, and this is responsible for the constant supply of oxygen and nutrients for tumor growth. Thus angiogenesis is considered an essential process within the neoplastic process. The evaluation of newly formed tumor blood vessels in oral squamous cell carcinoma using the CD105 antibody, shows a significant increase in microvascular density. Based on these, the goal is to evaluate by immunohistochemistry the possible correlation of an increased number of blood vessels correlate with different groups of inflammatory process divided into: minor, moderate and intense inflammatory infiltrate in the tumor front. In the literature, authors generally correlated angiogenesis and inflammatory infiltrates, in this work we try to correlate to a greater or lesser inflammatory infiltrate that affects angiogenesis. The slides were evaluated microscopically by two professionals so that they did not know the rating given by others and only when both were in agreement these slides were classified. For statistical analysis, the multiple comparisons between the three groups was performed by analysis of variance (comparison of three average) and also the Tukey test, where a difference was observed between groups I and III and groups II and III, but between groups I and II significativa.Com that there was no difference in the results show a positive correlation between the presence of a larger amount of blood vessels, where it is most inflammatory infiltrate when compared to areas of lesser amounts of inflammation.

Plaquetas e células tumorais interagem em uma reação cruzada com proteínas do plasma, via integrina αIIbβ3 e αvβ3, respectivamente. A integrina αvβ3 também encontra-se presente na angiogênese tumoral. O objetivo desse trabalho foi avaliar a GST-INS, uma disintegrina recombinante do veneno de Bothrops insularis em eventos da progressão tumoral. Em condições estáticas, GST-INS foi capaz de inibir totalmente a adesão de células HUVECs e SK-MEL-28 às plaquetas em comparação ao controle e ao Aggrastat® (inibidor seletivo da integrina αIIbβ3). Além de inibir a TCIPA (agregação plaquetária induzida por células tumorais) a GST-INS também inibiu a invasão de SK-MEL-28 em substrato de matrigel. Células t.End.1 ou SK-MEL-28 pré-incubadas com GST-INS não formaram túbulos no substrato de matrigel. Análise por microscopia confocal mostrou que GST-INS liga-se a integrina αv presente nas células SK-MEL-28. Nossos resultados sugerem que essa disintegrina pode ser utilizada como potencial ferramenta no estudo e desenvolvimento de antiangiogênicos e antimetastáticos.
Platelets and tumor cells interact in a cross-react with plasma proteins via integrin αIIbβ3 and αvβ3 , respectively.The integrin αvβ3 is also strongly stimulated in tumor angiogenesis. The aim of this study was to evaluate the ability of GST-INS, a recombinant disintegrin from Bothrops insularis venom on events of tumor progression. Under static conditions, GST-INS was able to completely inhibit the adhesion of endothelial cells (HUVECs) and melanoma cells (SK-MEL-28) to platelets compared to control and Aggrastat® (selective inhibitor of integrin αIIbβ3). In addition, GST-INS inhibit TCIPA (platelet aggregation induced by tumor cells) GST-INS also inhibited SK-MEL-28 on matrigel invasion substrate. t.End.1 cells or SK-MEL-28 pre-incubated with GST-INS were not able to form tubules in matrigel substrate. Analysis by confocal microscopy showed that GST-INS binds to integrin αv present in SK-MEL-28 cells. The results suggest that disintegrin can be used as a potential tool in the study and development of antiangiogenic and antimetastatic.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Although the efforts employed by scientific community to discover new anticancer therapies suitable to the increasing cancer incidence and multidrug resistance, its necessary to develop more selective and target driven drugs. Therefore, in this work we have done a screening with LQFM030 analogues, which is a Nutlin-1 analogue, aiming to evaluate their cytotoxic potential. Furthermore, we have evaluated the cytotoxicity, the morphological alterations and the cell death induction mechanisms of the compound LQFM166 in leukemia cell line K562. In parallel, we have investigated the security profile of the compound upon 3T3 basal cell line to estimate its LD50 and the Selectivity Index. The cytotoxicity assays included the tetrazolium salt (MTT) reduction and the Neutral Red Uptake assays, to assess the cytotoxicity of LQFM166 in K562 and 3T3 cell lines, respectively. The investigation of cell death induction mechanisms was carried out using flow cytometry, whereby we have evaluated the cells biochemistry parameters, including cell cycle progression, phosphatidylserine externalization, caspases 3/7, 8 and 9 activity, cytochrome c release from mitochondria, p21, p27, Bax, Bcl-2, cyclin-B1 and NFkB expression, using specific labeling for each assay. Data were analyzed by t test and the difference between control and treated groups averages was considered statistically significant when p<0,05. Regarding leukemia cell line K562, the compound LQFM166 was cytotoxic, showing a dose-time-dependent profile. Morphological alterations were observed after treatment with the compound at cellular and nuclear levels, which corroborate with apoptotic cell death. Additionally, treatment with the IC50 for 48 hours has promoted cellular and molecular changes that characterize this process, including phosphatidylserine externalization, increase of caspases 3/7, 8 and 9 activity, expression of pro-apoptotic proteins Bax, p21and p27, as well as diminution of Bcl-2 and cyclin-B1. We have also observed increase of cytochrome-c release and NFkB expression. Concerning the security profile, the compound was considered relatively selective, once the IC50 found to basal cell line (185,3 µM) was the double of the obtained to leukemia cell line regarding the same time of exposure (56,76 µM). The outcomes allow us to conclude that LQFM166 was cytotoxic upon leukemia cells K562, promoting morphological and biochemical alterations that indicate apoptotic cell death induction.
Mesmo com os esforços da comunidade científica em descobrir ou desenvolver medicamentos adequados a crescente incidência do câncer e ainda adequados à suas formas multirresistentes, é necessário o desenvolvimento de medicamentos que sejam seletivos e alvo-dirigidos. Assim, no trabalho proposto, foi realizada uma triagem com compostos análogos ao LQFM030, análogo estrutural do Nutlin-1, visando determinar o potencial citotóxico dos mesmos. Além disso, foram investigados a citotoxicidade, as alterações morfológicas e os mecanismos de indução de morte celular do composto escolhido LQFM166 em linhagem de células leucêmicas K562. Paralelamente, foi investigado o perfil de segurança do composto sobre a linhagem basal 3T3, a fim de estimar a DL50 e o Índice de Seletividade do mesmo. Os ensaios de citotoxicidade incluíram o método de redução do sal de tetrazólio (MTT) e o método de incorporação do corante vermelho neutro, para a avaliação do potencial citotóxico sobre as linhagens K562 e 3T3, respectivamente. Para a investigação dos mecanismos de indução de morte celular foi utilizada a técnica de citometria de fluxo, por meio da qual foi realizada a avaliação de parâmetros bioquímicos incluindo progressão ciclo celular, externalização da fosfatidilserina, atividade das caspases 3/7, 8 e 9, liberação do citocromo c, expressão das proteínas p21, p27, Bax, Bcl-2, ciclina-B1 e NFkB, empregando-se técnicas de marcação específicas para cada ensaio. Os dados foram analisados pelo teste t e a diferença entre as médias dos grupos controle e tratado foram consideradas estatisticamente significativas quando p<0,05. Em relação à linhagem leucêmica K562, o composto LQFM166 foi citotóxico apresentando um perfil dose e tempo dependentes. Foram observadas alterações morfológicas a níveis celular e nuclear, após o tratamento com composto, condizentes com o processo de morte celular por apoptose. Adicionalmente, externalização da fosfatidilserina, aumento da atividade das caspases 3/7, 8 e 9, aumento da expressão das proteínas pró-apoptóticas Bax, p21 e p27, bem como diminuição da expressão das proteínas Bcl-2 e ciclina-B1, após tratamento com a CI50 por 48 horas, desencadeou alterações celulares e moleculares que reforçam a sugestão de processo de morte celular por apoptose. Observouse ainda aumento da liberação do citocromo-c e da expressão da proteína NFkB. Já em relação ao perfil de segurança, o composto mostrou-se relativamente seletivo e com menor toxicidade, uma vez que a CI50 encontrada para a linhagem basal (185,3 μM) foi cerca de duas vezes maior ao encontrado para a linhagem tumoral para o mesmo tempo de exposição (56,76 μM). Considerando os resultados obtidos, pode-se concluir que o composto LQFM166 foi citotóxico sobre a linhagem leucêmica K562, desencadeando alterações morfológicas e bioquímicas características de morte celular por apoptose.

Introducció: Els carcinomes de queratinòcits (CQ) són la neoplàsia maligna més freqüent a nivell mundial en poblacions caucàsiques. En conjunt, suposen un enorme problema de salut, tant en termes de morbiditat com de despesa sanitària. En les darreres dècades, la seva incidència ha augmentat de manera significativa. A més a mes, pel que fa al carcinoma basocel·lular (CBC), han augmentat dramàticament els casos entre la gent jove, sobretot dones. Els CQ tenen, en general, un bon pronòstic. Malgrat això, el CBC pot créixer localment destruint els teixits circumdants i causant importants seqüeles funcionals i estètiques, i el carcinoma escatós (CEC) és moderadament invasiu i es pot associar a un risc substancial de recurrències locals, metàstasis i mort (1.5-4 %). La fisiopatologia dels CQ és dependent de l’alteració de diverses vies de transducció de senyals i de control de la proliferació. Un dels efectes d’aquestes alteracions és la secreció de noves proteïnes a l’espai extracel·lular, que caracteritzaran el microambient tumoral. Les proteïnes i metabòlits senyalitzadors presents en el fluid intersticial que envolta el tumor (TIF) han estat estudiats en alguns tipus tumorals, però no hi ha res descrit pel que fa als CQ. Objectius: Aquesta tesi té dos objectius principals: 1) Estudi epidemiològic i de factors de risc del CBC a Catalunya (Capítol I), i 2) Caracteritzar el perfil proteòmic del TIF en els CQ i les implicacions en la seva fisiopatologia (Capítols II i III). Resultats i conclusions: En l’estudi epidemiològic hem observat que entre els < 60 anys el CBC ja és un tumor que té una incidència major en dones que en homes. Els factors de risc independents pel CBC en gent jove són el fototipus cutani, la història familiar de CQ, i la presència de ≥ 4 cremades a la infància. A més a més, la presència de repetides cremades a la infància és superior entre els casos de CBC en gent jove localitzats en zones cobertes que entre els localitzats en zones fotoexposades. De tots els factors de risc, entre la població general, l’ús de cabines de bronzejat és l’únic que és més freqüent entre les dones que entre els homes; per tant, la popularització del seu ús podria ser la causa de la tendència a la predominança femenina del CBC. En la segona part de la tesi, primer s’ha posat a punt el mètode d’aïllament de TIF. La centrifugació a 10 000 g permet obtenir una quantitat superior de proteïnes extracel·lulars, major sensibilitat i un maneig més ràpid i fàcil de les mostres, sense implicar un augment de la lisi cel·lular respecte als altres mètodes. Per aquests motius, la proposem com a mètode d’elecció en les mostres de tumors cutanis. L’anàlisi proteòmica mostra que el CBC, el CEC i la pell sana tenen cadascun un perfil específic de proteïnes secretades al seu TIF i suggereix l’existència d’una diferent resposta immunològica de l’organisme en front al CBC i al CEC. Hem determinat que la secreció de les proteïnes PNP, FABP5, SFN i LAD1 és un perfil propi del CEC, i que estan relacionades amb l’agressivitat tumoral i podrien ser dianes terapèutiques per al seu tractament. A més a més, el patró d’expressió de FABP5 permet distingir les zones diferenciades de les invasives en el CEC. També existeix una diferent localització cel·lular de l’SFN i la LAD1 en la pell sana vs. CEC, que podria estar relacionada amb el desenvolupament tumoral. Finalment, la secreció de cornulina és un marcador de CBC i tendeix a associar-se amb els subtipus més agressius.
Introducción: Los carcinomas de queratinocitos (CQ) son la neoplasia maligna más frecuente a nivel mundial en poblaciones caucásicas. En conjunto, suponen un enorme problema de salud, tanto en términos de morbilidad como de gasto sanitario. En las últimas décadas, su incidencia ha aumentado de manera significativa. Además, en el carcinoma basocelular (CBC), han aumentado dramáticamente los casos entre la gente joven, sobre todo mujeres. Los CQ tienen, en general, un buen pronóstico. A pesar de ello, el CBC puede crecer localmente destruyendo los tejidos circundantes y causar importantes secuelas funcionales y estéticas, y el carcinoma escamoso (CEC) es moderadamente invasivo y se puede asociar a un riesgo sustancial de recurrencias locales, metástasis y muerte (1.5-4 %). La fisiopatología de los CQ es dependiente de la alteración de varias vías de transducción de señales y de control de la proliferación. Uno de los efectos de estas alteraciones es la secreción de nuevas proteínas en el espacio extracelular, que caracterizarán el microambiente tumoral. Las proteínas y metabolitos señalizadores presentes en el fluido intersticial que rodea el tumor (TIF) han sido estudiados en algunos tipos tumorales, pero no hay nada descrito en cuanto a CQ. Objetivos: Esta tesis tiene dos objetivos principales: 1) Estudio epidemiológico y de factores de riesgo del CBC en Cataluña (Capítulo I), y 2) Caracterizar el perfil proteómico del TIF en los CQ y las implicaciones en su fisiopatología (Capítulos II y III). Resultados y conclusiones: En el estudio epidemiológico se ha observado que entre los < 60 años el CBC ya es un tumor que tiene una incidencia mayor en mujeres que en hombres. Los factores de riesgo independientes para el CBC en gente joven son el fototipo cutáneo, la historia familiar de CQ, y la presencia de ≥ 4 quemaduras en la infancia. Además, la presencia de repetidas quemaduras en la infancia es superior entre los casos de CBC en gente joven localizados en zonas cubiertas que entre los localizados en zonas fotoexpuestas. De todos los factores de riesgo, en la población general, el uso de cabinas de bronceado es el único que es más frecuente entre las mujeres que entre los hombres; por lo tanto, la popularización de su uso podría ser la causa de la tendencia a la predominancia femenina del CBC. En la segunda parte de la tesis, primero se ha puesto a punto el método de aislamiento de TIF. La centrifugación a 10 000 g permite obtener una cantidad superior de proteínas extracelulares, mayor sensibilidad y un manejo más rápido y fácil de las muestras, sin implicar un aumento de la lisis celular respecto a los otros métodos. Por estos motivos, la proponemos como método de elección en las muestras de tumores cutáneos. El análisis proteómico muestra que el CBC, el CEC y la piel sana tienen cada uno un perfil específico de proteínas secretadas en su TIF y sugiere la existencia de una diferente respuesta inmunológica del organismo frente al CBC y al CEC. Hemos determinado que la secreción de las proteínas PNP, FABP5, SFN y LAD1 es un perfil propio del CEC, y que están relacionadas con la agresividad tumoral y podrían ser dianas terapéuticas para su tratamiento. Además, el patrón de expresión de FABP5 permite distinguir las zonas diferenciadas de las invasivas en el CEC. También existe una diferente localización celular de la SFN y la LAD1 en la piel sana vs. CEC, que podría estar relacionada con el desarrollo tumoral. Finalmente, la secreción de cornulina es un marcador de CBC y muestra tendencia a asociarse con los subtipos más agresivos.
Introduction: Keratinocyte carcinomas (KC) are the most frequent malignancy in Caucasian populations worldwide. Altogether, they entail a huge health problem, both in terms of morbidity and health costs. In recent decades, their incidence has increased significantly. Moreover, basal cell carcinoma (BCC) cases have increased dramatically among young people, especially women. KC generally have a good prognosis. Despite this, BCC can grow locally, destroying surrounding tissues and causing important functional and aesthetic sequelae; and squamous cell carcinoma (SCC) is moderately invasive and can be associated with a substantial risk of local recurrences, metastasis, and death (1.5- 4 %). The physiopathology of KC depends on the alteration of several signal transduction pathways and proliferation control. One effect of these alterations is the secretion of new proteins in the extracellular space, which will characterize the tumor microenvironment. The signaling proteins and metabolites present in the interstitial fluid surrounding the tumor (TIF) have been studied in some tumor types, but nothing has been described in terms of KC. Objectives: This thesis has two main objectives: 1) Epidemiological and risk factors study of BCC in Catalonia (Chapter I), and 2) Characterization of the proteomic profile of TIF in KC and the implications in their physiopathology (Chapters II and III). Results and conclusions: In the epidemiological study, it has been revealed that, among those <60 years, BCC is already a tumor that has a higher incidence in women than in men. Independent risk factors for BCC in young people are skin phototype, family history of KC, and the presence of ≥ 4 sunburns in childhood. In addition, the presence of repeated sunburns in childhood is higher among BCC cases in young people in covered areas than among those in photo-exposed areas. Of all the risk factors, among the general population, the use of tanning beds is the only one that is more common among women than among men; therefore, the popularization of their use could be the crucial factor for the trend towards the female predominance of BCC. In the second part of the thesis, the TIF isolation method has been first developed. Centrifugation at 10 000 g allows obtaining a higher amount of extracellular proteins, greater sensitivity, and faster and easier handling of the samples, without increasing cell lysis when compared to other methods. For these reasons, we propose it as the method of choice in samples of skin tumors. The proteomic analysis shows that BCC, SCC, and healthy skin each have a specific profile of secreted proteins in their TIF, and suggests the existence of a different immune response of the organism against BCC and SCC. We have determined that the secretion of PNP, FABP5, SFN, and LAD1 proteins is a specific SCC profile, and that they are related to tumor aggressiveness and could be therapeutic targets for its treatment. Furthermore, the expression pattern of FABP5 makes it possible to distinguish differentiated from invasive areas in SCC. There is also a different cellular location of SFN and LAD1 expression in healthy skin vs. SCC, which could be related to tumor development. Finally, cornulin secretion is a BCC marker and tends to associate with the most aggressive subtypes.

Les leucémies aiguës myéloïdes (LAM) sont des maladies hématologiques hétérogènes caractérisées par une prolifération clonale des blastes myéloïdes qui s’infiltrent dans la moelle osseuse (MO), le sang et d’autres organes. Identifiée comme le type le plus courant de leucémie aiguë chez l’adulte avec 80% des cas, la LAM est synonyme de rechute et de mauvais pronostic, avec 70% des patients étant confrontés à une mortalité dans l’année suivant le diagnostic. La présence des cellules souches leucémiques (CSL) a été associée à une résistance à la chimiothérapie et à une rechute dans la LAM. Le microenvironnement tumoral a été décrit pour son rôle clé dans la régulation des CSL par l’interaction des voies de signalisation. La voie des Bone Morphogenetic Proteins (BMP) est fortement impliquée dans la régulation des cellules souches hématopoïétiques (CSH), mais elle a également été reconnue pour réguler les CSL. Ici, nous avons identifié des concentrations élevées de BMP2 et BMP4 dans la MO des patients atteints de LAM au moment du diagnostic. De plus, nous avons identifié pour la première fois une nouvelle cascade de signalisation impliquant la liaison de BMP4 au récepteur BMPR1A, qui induit l’expression de ΔNp73 et NANOG. L’activation de cette signalisation favorise un phénotype proche des cellules souches dans les cellules leucémiques. Par conséquent, nous avons émis l’hypothèse que cette voie est responsable de la capacité de résistance des cellules leucémiques à la chimiothérapie. En outre, nous avons identifié BMPR1A/ΔNp73/NANOG comme marqueurs potentiels du pronostic dans la LAM, en raison de leurs surexpressions au moment du diagnostic associé à une rechute dans les trois ans. Lorsque nous avons analysé des échantillons de LAM lors d’une rechute, nous avons constaté des taux plus élevés de l’isoforme ΔNp73 par rapport à ceux de patients au moment du diagnostic. D’autre part, nous avons identifié une forte expression du récepteur BMPR1A, ΔNp73, NANOG, SOX2 et ID1 dans les cellules leucémiques primaires résistantes à court terme. Ces résultats sont en corrélation avec ce que nous avons observé dans les cellules résistantes de LAM, où BMPR1A, ΔNp73, NANOG et ID1 semblent être impliqués dans la capacité de résistance des cellules de LAM face à la chimiothérapie. La modulation et le ciblage des éléments de la voie BMP et des gènes associés identifiés au travers de notre étude représentent donc une approche prometteuse pour le développement de stratégies thérapeutiques innovantes et plus efficaces contre les LAM
Acute myeloid leukemias (AML) are heterogeneous hematological malignancies characterized by a clonal proliferation of myeloid blasts which infiltrate the bone marrow, blood and other organs. Identified as the most common type of acute leukemia in adults with 80% of cases, AML is associated with high relapse and poor prognosis where 70% of patients face mortality within one year after diagnosis. Leukemic stem cell (LSCs) presence has been related to resistance to chemotherapeutic agents and relapse in AML. The tumor microenvironment has been described for its key role regulating LSCs through the crosstalk of signaling pathways. Bone Morphogenetic Proteins (BMP) pathway is highly involved in hematopoietic stem cell (HSC) regulation, but has also been recognized to regulate LSCs. Here, we have identified high concentrations of BMP2 and BMP4 in bone marrow (BM) AML samples at diagnosis. Furthermore, we have identified for the first time a new signaling cascade, involving the binding of BMP4 to BMPR1A receptor, which induces the expression of ΔNp73 and NANOG. Activation of this signaling promotes a stem-like phenotype in leukemic cells. Therefore, we hypothesized that this signaling is responsible for the resistant capacity of leukemic cells to chemotherapy. In addition, we have reported BMPR1A/ΔNp73/NANOG as potential AML prognosis markers, due to their overexpression at diagnosis associated to an increased rate of relapse of AML patients within three years. When we analyzed AML samples at relapse, higher levels of ΔNp73 isoform were found compared to patients at diagnosis. Moreover, we have identified high expression of the BMPR1A receptor, ΔNp73, NANOG, SOX2 and ID1 in short-term resistant primary leukemic cells. These results correlate with what we observed in AML resistant cells, where BMPR1A, ΔNp73, NANOG and ID1 seem to be implicated in driving the resistant capacity of AML cells to drug therapy. Therefore, modulation and targeting of the BMP pathway elements and related genes identified with our study, represent a promising approach towards the development of new and more effective therapeutic strategies against AML

No câncer de mama, o metabolismo tumoral, ação dos moduladores de estrógenos são fatores que podem influenciar as células dendríticas (DCs). Neste trabalho avaliou o fenótipo de DCs em amostras tumorais, a diferenciação de DCs a partir de células mononucleares do sangue periférico (PBMCs) das pacientes e comparou com voluntárias saudáveis. Os resultados mostraram que há alteração na capacidade de geração, no fenótipo, na capacidade aloestimuladora, maior produção de interleucina 10 e expressão de HSP27 nas DCs de pacientes, comparadas com as DCs de voluntárias saudáveis que produzem mais Interferon-gama. A via p38MAPK parece ser importante na diferenciação de PBMCs em DCs, entretanto, estímulos estressantes podem ativar esta via e induzir a síntese de HSP27 inibindo este processo. O tratamento com tamoxifeno parece modular a expressão de algumas moléculas de membrana. Desta forma, os resultados sugerem que as DCs diferenciadas de pacientes com câncer de mama apresentam alterações fenotípicas e funcionais causadas pelo microambiente tumoral.
In breast cancer, the tumor metabolism, the action of estrogens antagonists can influence dendritic cells (DC) generation. The aim of this work was to evaluate the frequency of DCs in tumor tissue and the differentiation of DCs derived from peripheral blood mononuclear cells (PBMCs) and compared the phenotypic and functionally of these cells from healthy individuals and breast cancer patients. The results showed that patients PBMCs were unable to generate phenotypicaly, functionally mature DCs and presented larger production of interleukin 10 and higher expression of HSP27 when compared with healthy volunteers\' DCs, presented higher production of Interferon-gamma. The p38 MAPK signaling pathway seems to be important in PBMCs differentiation into DCs, and its activation by stress can induce the synthesis of HSP27, that inhibits DC generation. The tamoxifen treatment caused modulation of membrane DC markers expression. Therefore, these results show that patients\' DCs present phenotypic and functional alterations which can be caused by tumor microenvironment.

O carcinoma espinocelular (CEC) é a segunda forma de neoplasia cutânea mais prevalente. Os mecanismos exatos envolvidos na progressão desse tipo de tumor ainda não estão elucidados. Estudos recentes têm mostrado que a citocina IL33 é uma citocina reguladora da resposta imune adaptativa, principalmente como potente indutor do perfil Th2. Juntamente com seu receptor ST2, apresenta-se com os níveis elevados em alguns tipos de câncer, corroborando para a evidência de que essa citocina contribui para a carcinogênese. Baseado nessas informações, testamos a hipótese de que a presença de IL33 em carcinoma espinocelular, poderia estar relacionada a um melhor prognóstico. Neste estudo foram utilizadas amostras de carcinoma espinocelular, em diferentes gradações de malignidade tumoral (Grau I, Grau II e Grau III). Os resultados mostraram um infiltrado inflamatório mais intenso em tumores com Grau I e II. Imunorreatividade para IL33 foi observada em tumores de Grau I e II tanto por células epiteliais como por células do infiltrado inflamatório. A análise por microscopia confocal evidenciou que um grande número de células TCD4+ e TCD8+ que expressavam IL33 foi observado em tumores de Grau II. Esses resultados indicam a presença de um intenso infiltrado inflamatório e expressão de IL33 em amostras de carcinoma espinocelular com níveis menores de malignidade tumoral.
Squamous cell carcinoma (SCC) is the second most common form of cutaneous neoplasm. The exact mechanisms involved in the progression of this type of tumor have not yet been elucidated. Recent studies have shown that the cytokine IL33 is a cytokine regulating the adaptive immune response, mainly as a potent inducer of Th2 profile. Together with its ST2 receptor, its presents with elevated levels in some types of cancer, corroborating to evidence that this cytokine contributes to carcinogenesis. Based on this information, we tested the hypothesis that the presence of IL33 in squamous cell carcinoma could be related to a better prognosis. In this study, squamous cell carcinoma samples were used in three different gradations of tumor malignancy (Grade I, Grade II and Grade III). The results showed that a more intense inflammatory infiltrate in Grade I and II tumors. Immunoreactivity for IL33 was observed in Grade I and Grade II tumor, by epithelial cells and by inflammatory infiltrate cells. The analysis by confocal microscopy evidenced that a great number of TCD8+ and TCD4+ cells expressing IL33 was observed in grade II tumors. These results indicate the presence of an intense inflammatory infiltrate and expression of IL33 in samples of squamous cell carcinoma with lower levels of tumor malignancy.

The first chapter of this thesis describes the study of intestinal stem cells (ISCs) and their relationship to colon cancer. We describe the transcriptional landscape of intestinal epithelial populations through the use of the surface marker EphB2 which allowed us to build an ISC-specific gene signature. Elevated levels of this signature characterize CRC patients with high risk of disease relapse after therapy. Furthermore, our studies led us to discover that colon cancers are organized according to a hierarchical structure similar to that present in the normal colonic epithelium. EphB2- high cells retain an ISC-like phenotype and behave as cancer stem cells (CSCs). The second chapter of this thesis focuses on the description of the function of Mex3a, a novel ISC-specific gene. We generated genetic mouse models to disrupt Mex3a function in vivo. We show that this RNA binding protein is required for embryonic development, but dispensable in adult intestines. By generating a Mex3a reporter mouse we have been able to identify intestinal cells that resemble label-retaining cells. This mouse strain has also allowed us to purify stem/progenitor cells from other tissues. We demonstrate that Mex3a is a translational activator that interacts with the protein HuR. Our data suggest that Mex3a may be involved in the response of stem cells to stress.
El primer capítulo de esta tesis describe el estudio de células madre intestinales (CMIs) y su relación con el cáncer de colon. Describimos los perfiles de expresión génica de las distintas poblaciones intestinales utilizando la expresión del receptor EphB2. Esto nos permitió describir la firma genética de las CMIs. Observamos que los tumores de colon con altos niveles de expresión de la firma genética de CMI tienen mayor probabilidad de recaer después de terapia. Además de estas observaciones, describimos que los tumores de colon se organizan con una estructura similar a la del colon normal. Finalmente observamos que las células tumorales con altos niveles de EphB2 retienen una identidad de célula madre intestinal y se comportan como células madre tumorales. El segundo capítulo de esta tesis se centra en el estudio del gen Mex3a, el cual es un nuevo gen específico de células madre intestinal. Generamos ratones modificados genéticamente para eliminar la función de Mex3a in vivo. Observamos que este gen es esencial para el desarrollo embrionario, pero que es dispensable para la homeostasis del intestino adulto de ratón. También generamos un ratón reportero de Mex3a que nos permitió purificar una población de células intestinales que se asemejan a “células retenedoras de marcaje”. Además, con este ratón reportero pudimos purificar células madre de otros tejidos. Demostramos que Mex3a es un activador traduccional que interacciona con la proteína HuR. Nuestros datos sugieren que Mex3a podría estar involucrado en como las células madre responden a estrés.

Les tumeurs sont composées de cellules malignes et d'une grande variété de cellules non-tumorales, en particulier des cellules immunitaires qui forment le micro-environnement tumoral (MET). Il a été démontré que la composition du MET était associée au devenir clinique des patients, en termes de survie et de réponses thérapeutiques. Avec le développement récent des immunothérapies qui ciblent des éléments spécifiques du MET, l'immunité anti-tumorale a soulevé un intérêt majeur. Plusieurs méthodologies ont été mises au point afin d'étudier la composition du MET, avec une précision toujours plus grande. En particulier, des méthodes comme MCP-counter permettent d'exploiter les données transcriptomiques de la tumeur entière afin de quantifier les différentes populations qui composent le MET. Le volet méthodologique de ce travail de thèse a ainsi consisté à proposer une amélioration de MCP-counter, en particulier pour l'analyse de données RNA-Seq. Une adaptation de la méthode pour des données issues de modèles murins (mMCP-counter) est également proposée. MCP-counter permet d'analyser rapidement le MET de larges séries de tumeurs. Un second volet de cette thèse consiste en l'application de cette méthode pour établir une classification immunitaire des sarcomes des tissus mous, un type de cancer rare, hétérogène et agressif. Cette classification immunitaire a permis de mettre en évidence des groupes de tumeurs faiblement ou fortement infiltrés, ainsi qu'un groupe marqué par une forte vascularisation. De manière intéressante, la classification immunitaire permet de prédire la réponse des patients aux immunothérapies. Ce travail a aussi démontré un rôle important des structures lymphoïdes tertiaires (SLT). Les SLT sont des structures de type noeud lymphatique composées de lymphocytes B et T qui se forment dans la tumeur ou à proximité de celle-ci. Au sein des SLT, une réponse immunitaire anti-tumorale peut se former et maturer. L'intérêt porté aux SLT est de plus en plus important pour de nombreux types de cancers. Dans la plupart des types de cancer, une forte infiltration de la tumeur par des lymphocytes T, en particulier CD8+, est associée à une meilleure survie des patients. Cependant, le carcinome rénal à cellules claires et le cancer de la prostate sont des exceptions à cette règle. En effet, dans ces deux cancers urologiques, la présence dans la tumeur de lymphocytes T est associée à une survie plus courte des patients, ainsi qu'à une rechute et une progression plus précoce. Ces exceptions sont détaillées dans une troisième partie de cette thèse, par une description minutieuse du MET, ainsi que par l'analyse de l'implication du système du complément. Dans leur ensemble, les résultats présentés dans cette thèse démontrent qu'en combinant différentes méthodes d'analyse, in silico, in situ et in vivo, il est possible d'obtenir une vision extrêmement complète du MET. La connaissance des types cellulaires présents dans la tumeur ainsi que leur orientation fonctionnelle permet de guider le soin apporté aux patients et d'améliorer leur devenir clinique. La description complète du MET ouvre la voie à une médecine personnalisée pour les patients atteints de cancer
Tumors are composed not only of malignant cells but also contain a vast variety of non-malignant cells, notably immune cells forming the tumor microenvironment (TME). The composition of the TME was shown to be associated with clinical outcome for cancer patients, in terms of survival and therapeutic responses. With the relatively recent development of immunotherapies targeting specific elements of the TME, tumor immunology has risen a strong interest and holds a strong therapeutic potential. Several methodologies have been developed to study the composition of the TME with an increased precision. Notably, some methods such as MCP-counter enable the use of the tumor bulk transcriptome to quantify cell populations composing the TME. The methodological aspect of this PhD project consisted in setting up an enhanced version of MCP-counter that can be readily applied to RNA-Seq data, as well as propose an adaptation of the method for mouse models. Using MCP-counter, the TME of large series of tumors can be easily analyzed. The application part of this PhD work consisted of applying MCP-counter to establish an immune-based classification of soft-tissue sarcoma, a rare, aggressive and heterogeneous cancer type. The immune classification notably allowed to identify immune low and high groups, and a group characterized by a strong vasculature. Interestingly, the classification was notably found to be predictive of the patients' response to immunotherapies. It also highlighted an important role of tertiary lymphoid structures (TLS). TLS are lymph-node-like structures composed of T and B cells that form within the tumor or in close proximity. They are a site of formation and maturation of antitumoral immune responses. TLS are raising a growing interest in many malignancies. In most cancer types, a strong infiltration by T cells, in particular CD8+ T cells, is associated with a favorable clinical outcome. However, clear-cell renal cell carcinoma and prostate cancer are exceptions to this general rule. Indeed, in these urological cancers, an increased infiltration by T cells is associated with a decreased patient survival and with earlier relapse and disease progression. In a third part of this thesis, these exceptions are investigated with more details by scrutinizing the TME, and questioning the implication of the complement system. Overall, this thesis presents how the combination of several analysis methods, in silico, in situ and in vivo, can help achieve an extremely precise description of the TME. Knowing accurately what cell populations and what their functional orientation can help guide patients care and improve clinical outcome. Complete description of the TME opens the way towards personalized medicine for cancer patients

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
In the lesions carcinomatous, low oxygen tension plays a crucial step in the self-renewal, metastatic potential, and therapy resistance of cancers. To adapt to the hypoxic microenvironment, neoplastic cells activate hypoxia-induced factor-1 alpha (HIF-1 alpha), which may mediates invasion and metastasis. In addition, the human THY-1 (CD90) cell surface protein mediates cell adhesion expressed in stem cells, and seens to drive tumor development in some malignant tumors. The present study investigates HIF-1 alpha (n=98) and CD90 (n=97) expression in oral squamous cell carcinoma (OSCC) and metastatic lymph nodes (n=24), the intratumoral region and the invasive front, by immunohistochemistry. Furthermore, clinicopathological data revised from the medical records. In superficial OSCCs, most tumor cells overexpressed HIF-1 alpha, whereas was restricted in the intratumoral region in invasive conventional SCCs. Interestingly, metastatic lymph nodes (91.7%, p=0.001), and intratumoral regions of its corresponding primary tumors (83.3%, p<0.001) were invaded by HIF-1 alpha-positive neoplastic cells. Overall survival was poor in patients with nodal involvement. CD90 was expressed mostly in microvessels and granulocyte cells similar to mast cells. These cells expressed CD90 mostly in the peritumoral region of invasive SCC (p<0.001). Microvessels CD90 positive were higher in the intratumoral region (p=0.032). Interesting, mast cell and microvessels positively correlated in OSCC (p=0.006; r²=0.077). In conclusion, hypoxic environment may facilitate regional metastasis and serve as a potential diagnostic and prognostic marker in OSCC primary tumors. Microvessels CD90 positive seems to promote tumor growth except in BSCC. Mast cell may occur via CD90 for tumor progression.
Nas lesões carcinomatosas a baixa tensão de oxigênio desempenha um passo crucial para a auto-renovação, potencial metastático, e resistência à terapia no câncer. Para se adaptar ao ambiente hipóxico, células neoplásicas ativam o fator induzido por hipóxia-1 alfa (HIF-1 alfa), que pode facilitar a invasão e metástase. Além disso, o THY-1 (CD90) humano, uma proteína de superfície celular expressa em células estaminais, medeia a adesão celular, e parece promover o desenvolvimento em alguns tumores malignos. O presente estudo analisou a expressão das proteínas HIF-1 alfa (n = 98) e CD90 (n = 97) no carcinoma espinocelular de boca (CEC de boca) e linfonodos metastáticos (n=24), na região intratumoral e no fronte de invasão, por meio de imunoistoquímica. Além disso os dados clinicopatológicos foram revisados a partir dos prontuários médicos e a sobrevida foi analisada. No CEC microinvasivo, a maioria das células tumorais apresentaram superexpressão do HIF-1 alfa, enquanto que no CEC invasivo a superexpressão foi restrita na região intratumoral. Verificou-se que em linfonodos metastáticos (91,7%, p = 0,001), e regiões intratumorais dos seus tumores primários correspondentes (83,3%, p <0,001) houve forte expressão do HIF-1 alfa em células neoplásicas. A sobrevida global foi pior em pacientes com metástase regional. A proteína CD90 foi expressa principalmente em microvasos e células de granulócitos semelhantes aos mastócitos. Estas células expressaram CD90 principalmente na região fronte de invasão do CEC invasivo (p<0,001). A média de microvasos CD90 positivo foi maior na região intratumoral (p=0,032). Interessantemente, mastócitos e microvasos foram positivamente correlacionados no CEC de boca (p=0,006; r²=0,077). Em conclusão, o ambiente hipóxico pode facilitar metástases regionais e funcionar como um potencial marcador de diagnóstico e prognóstico em tumores primários do CEC de boca. Os microvasos CD90 positivo parecem promover o crescimento do tumor, exceto no carcinoma escamoso basalóide (CEB). Os mastócitos ativados via CD90 podem contribuir com a progressão do tumor.

Le système lymphatique est un réseau vasculaire unidirectionnel transportant la lymphe qui permet le drainage des fluides interstitiels, le transport des lipides intestinaux, ainsi que la surveillance et la tolérance immunitaire. Néanmoins, le système lymphatique est impliqué dans de nombreuses pathologies et particulièrement dans la progression tumorale. En effet, le système lymphatique favorise la dissémination des métastases qui sont acheminées par les vaisseaux lymphatiques jusqu'aux organes distants. Plus récemment, le système lymphatique a été identifié comme un régulateur clé des réponses immunitaires lors du développement tumoral. Le système immunitaire est indispensable à la détection des tumeurs et à l'établissement de réponses lymphocytaires anti-tumorales. Cependant, lors des stades de développement tumoral avancés, des mécanismes d'échappement immunitaire se mettent en place en faveur de la croissance des tumeurs. Ces mécanismes sont notamment médiés par les tumeurs elles-mêmes mais aussi par différents acteurs de l'environnement tumoral. Le système lymphatique est un de ces acteurs, particulièrement retrouvé dans l'environnement tumoral mammaire. Les adénocarcinomes mammaires à des stades avancés répondent aux immunothérapies qui ciblent les points de contrôle immunitaire responsables de l'échappement immunitaire. En effet, le système lymphatique potentialise la réponse des tumeurs à ces immunothérapies car il joue un rôle double dans l'immunomodulation en contexte tumoral. Les vaisseaux lymphatiques sont capables de recruter les lymphocytes T dans la tumeur pour stimuler la surveillance immune anti-tumorale mais sont aussi capables d'engendrer des mécanismes immunosuppresseurs des lymphocytes T par l'expression de points de contrôle immunitaire. Durant de ma thèse, j'ai donc étudié les mécanismes immunomodulateurs du système lymphatique lors du développement des tumeurs mammaires. J'ai observé que le système lymphatique contrôle le passage de la surveillance immune vers l'immunosuppression, particulièrement induite par les ligands du point de contrôle immunitaire TIGIT. J'ai ainsi montré que l'activation des vaisseaux lymphatiques tumoraux entraine la surexpression du ligand Nectine-2 qui va inhiber les lymphocytes T surexprimant TIGIT. Cela va alors réduire la réponse des lymphocytes T CD8+ cytotoxiques afin de favoriser la croissance tumorale
The lymphatic system is a unidirectional vascular network transporting lymph, enabling drainage of interstitial fluids, transport of intestinal lipids, and also immune monitoring and tolerance. Nevertheless, the lymphatic system is involved in many pathologies, and particularly in tumor progression. Indeed, the lymphatic system promotes the metastatic spread, carried by lymphatic vessels to distant organs. More recently, the lymphatic system has been identified as a key regulator of immune responses during tumor development. The immune system is essential for tumor detection and establishment of anti-tumor lymphocyte responses. However, at advanced stages of tumor development, immune escape mechanisms are established in favor of tumor growth. These mechanisms are mediated not only by tumors themselves, but also by various players in the tumor environment. The lymphatic system is one of these players, particularly found in breast tumor environment. Advanced-stage breast adenocarcinomas respond to immunotherapies that target immune checkpoints responsible for immune escape. Indeed, the lymphatic system potentiates tumor response to these immunotherapies, playing a dual role in immunomodulation in the tumor context. Lymphatic vessels are able to recruit T cells to the tumor site to stimulate anti-tumor immune surveillance, but are also able to generate T cell immunosuppressive mechanisms through the expression of immune checkpoints. During my thesis, I therefore studied immunomodulatory mechanisms of the lymphatic system during mammary tumor development. I observed that the lymphatic system controls a switch from immune surveillance to immunosuppression, particularly induced by ligands of TIGIT immune checkpoint. I have shown that activation of tumor lymphatic vessels leads to overexpression of Nectin-2, which inhibits T cells overexpressing TIGIT. This in turn reduces cytotoxic CD8+ T cell responses to promote tumor growth

Le cytomégalovirus (CMV), un β-herpes virus, est considéré comme un modèle d'immuno-évasion virale. Il s'agit d'un agent pathogène opportuniste fréquent chez les patients immunodéprimés et une cause majeure de malformations congénitales lors de l'acquisition in utero. Le CMV code pour des protéines (i) qui empêchent la présentation de l'antigène aux lymphocytes T αβ notamment par l'inhibition de l'expression des molécules HLA-I et (ii) qui suppriment les fonctions des cellules NK en imitant ou en régulant à la baisse les ligands des récepteurs NK (NKR). Ces mécanismes d'évasion ne devraient pas affecter les lymphocytes T γδ dont la reconnaissance antigénique est indépendante du HLA-I, et d’ailleurs leur réponse au CMV a été largement rapportée dans de nombreux contextes physiopathologiques. Notre objectif était de comprendre comment les mécanismes d’immuno-évasion du CMV affectent la réponse des lymphocytes T γδ. Nous avons utilisé des adénovirus recombinants exprimant chacun des quatre gènes du CMV impliqués dans l’inhibition de l’expression du HLA-I, et un mutant du HCMV déficient pour ces 4 gènes (CMV-∆US). Nous avons observé une induction de l'expression de HLA-I par l'adénovirus control, et une inhibition par US2, US3 et US11. Lors de l'utilisation de CMV-∆US, les cellules infectées exprimaient beaucoup plus de HLA-I que les cellules infectées par CMV-WT. De façon intéressante et à l’opposé des lymphocytes T αβ, les lymphocytes T γδ produisent plus d'IFNy en présence de fibroblastes infectés par le CMV-WT, qu’avec des fibroblastes infectés par CMV-∆US. Ces résultats indiquent que les molécules HLA-I régulent les lymphocytes T γδ grâce à des mécanismes qui sont en cours d'investigation dans notre équipe. Les processus d'échappement immunitaire développés par le CMV pourraient ainsi favoriser la réponse des lymphocytes T γδ par rapport à celle des lymphocytes T αβ et expliquer le rôle important des cellules T γδ dans le contrôle du virus chez les individus immunodéprimés. D'autre part, les acides nucléiques et les protéines du HCMV ont été trouvés dans les tissus tumoraux, mais la relation précise entre le HCMV et le cancer reste un sujet de débat. La plupart du temps, HCMV est décrit comme un virus oncomodulateur avec un rôle pro-tumoral. Notre objectif était d'utiliser le modèle de la souris pour tester in vivo l'impact de CMV de souris (MCMV) sur la croissance des cellules tumorales. Nous avons observé que MCMV pourrait inhiber la croissance de tumeurs sous-cutanées de côlon MC38 chez les souris immunodéficientes. Encore plus surprenant lorsque l'on considère la spécificité d’espèce des CMV, l'infection par le MCMV inhibe de la même façon la croissance des cellules cancéreuses du côlon humain HT29, qui n’est pas affectée par le HCMV. In vitro, les protéines MCMV précoces (IE-1) sont détectées dans des cellules cancéreuses humaines et murines après l'infection. Cependant, peu de cellules cancéreuses sont retrouvées positives pour le MCMV dans les tumeurs HT29 prélevées sur des souris infectées par le MCMV. De manière surprenante, le MCMV inhibe la prolifération des cellules cancéreuses de côlon humain contrairement au HCMV. De plus, la transcription de l'interféron β humain est induite après une infection par le MCMV. Cette induction n'a pas été observée après l'infection par le HCMV. En conclusion, nos données suggèrent un potentiel effet anti-tumoral de MCMV sur les cellules cancéreuses du côlon humain (HT29), qui pourrait être au moins partiellement médiée par l'interféron β. Ces résultats ouvrent la voie à l'utilisation potentielle du MCMV en tant que traitement du cancer du côlon humain
Cytomegalovirus (CMV), a Beta Herpes virus, is considered as a paradigm for viral evasion.It is an important opportunistic pathogen in immunocompromised patients and a major cause of congenital birth defects when acquired in utero. CMV encodes molecules to prevent antigen presentation to αβ T cells through inhibition of MHC Class I expression and to suppress NK cell functions by mimicking or down-regulating ligands of NK receptors (NKR). These evasion mechanisms are not expected to affect γδ T cells and, as a matter of fact, their response to CMV has been widely reported in many different physiopathological contexts as well as in CMV-seropositive healthy donors (Dechanet et al, 1999)( Scheper, 2013). Our aim was to understand how CMV induce γδ T cell response. We used recombinant adenoviruses expressing each of the four US genes, and a mutant HCMV deleted for these 4 genes (CMV-DUS). We observed an induction of HLA-I expression by the control adenovirus, and an inhibition by US2, US3 and US11. When using CMV-DUS, infected cells expressed much more native HLA-I than CMV-WT infected cells. Interestingly and in sharp contrast to αβ T cells, γδ T cell were activated to produce IFNg when cultured with fibroblasts infected with CMV-WT, but not when fibroblasts were infected with CMV-DUS. These results indicate that HLA-I molecules regulate γδ T cells through mechanisms that are under investigation in our team. The immune escape processes developed by CMV could thus promote γδ over αβ T cell response and explain the important response of γδ T cells to the virus in immunosuppressed individuals

O meduloblastoma é o tumor maligno do sistema nervoso central mais frequente na infância e adolescência. A expressão de genes tipicamente expressos em células-tronco está correlacionada com pior prognóstico em pacientes com meduloblastoma e a expressão de POU5F1 se mostrou capaz de distinguir pacientes com desfecho clínico desfavorável e pior sobrevida. Apesar do seu valor prognóstico, não há evidências diretas da contribuição de OCT4 para a aquisição de fenótipos mais agressivos em meduloblastoma. Nesse contexto, o presente trabalho investigou o papel da isoforma OCT4A em características pró-tumorigênicas de meduloblastoma in vitro e in vivo, e também avaliou as alterações moleculares que podem ser responsáveis pela aquisição de fenótipo mais agressivo em células de meduloblastoma humano. Para tanto, foi realizada a superexpressão de OCT4A mediada por retrovírus em três linhagens celulares de meduloblastoma (Daoy, D283Med e USP-13-Med). As células de meduloblastoma com superexpressão de OCT4A exibiram maior proliferação e alterações no ciclo celular. Foram observados também aumentos na atividade clonogênica, geração de esferas tumorais e desenvolvimento tumoral em modelo subcutâneo, sendo esses efeitos dependentes dos níveis de OCT4A. A avaliação da mobilidade celular in vitro demonstrou diminuição na adesão celular e aumento da invasão celular de esferoide 3D. Em modelo ortotópico de meduloblastoma, as células com superexpressão de OCT4A geraram tumores mais desenvolvidos, com fenótipos mais agressivos, infiltrativos e metastáticos. A superexpressão de OCT4A foi associada a maior instabilidade genômica, entretanto, as aberrações em números de cópias variaram em frequência e tipo de alteração dependendo da linhagem celular, e sendo pouco associada com os genes diferencialmente expressos. De forma interessante, uma relevante expressão diferencial de RNAs não-codificadores de proteínas foi observada em células de meduloblastoma com superexpressão de OCT4A, incluindo os recém descobertos e pouco caracterizados RNAs não codificadores longos, além de múltiplos RNAs pequenos nucleolares. Assim, os resultados aqui apresentados fundamentam a relevância de fatores envolvidos em pluripotência para o agravamento de traços associados com desfecho clínico desfavorável em meduloblastoma e destacam o valor prognóstico e terapêutico de OCT4A neste tumor pediátrico do sistema nervoso central
Medulloblastoma is the most common malignant brain tumor in infants. The expression of typical pluripotency genes is correlated with poor prognosis in medulloblastoma and POU5F1 expression was shown capable of discriminating patients with poor survival outcome. Despite this prognostic value, direct evidences of OCT4 contribution to more aggressive traits in medulloblastoma are missing. In this context, we investigated the role of OCT4A isoform on pro-tumorigenic features of medulloblastoma in vitro and in vivo and evaluated molecular alterations that could be responsible for acquisition of a more aggressive phenotype in medulloblastoma cells. Retroviral-mediated overexpression of OCT4A were performed in three medulloblastoma cell lines (Daoy, D283Med and USP-13-Med). Medulloblastoma cells overexpressing OCT4A displayed enhanced cell proliferation and cell cycle alterations. Increased clonogenic activity, tumorsphere generation capability and subcutaneous tumor development were also observed, and these effects were OCT4A expression level-dependent. Evaluation of cell mobility in vitro showed loss of cell adhesion and greater 3D-spheroid invasion. In an orthotopic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive, infiltrative and metastatic tumors. OCT4A overexpression was associated with chromosomal instability but copy number aberrations varied in frequency and type according to the cell line, with little association with differently expressed genes. Interestingly, marked differential expression of non-coding RNAs, including newly discovered, still poorly characterized, long non-coding RNAs and multiple small nucleolar RNAs were observed in medulloblastoma cells with OCT4A overexpression. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer

O desenvolvimento da imunoterapia do câncer baseada em células dendríticas (DCs) é alvo de vários estudos. Para tumores sólidos, a abordagem baseada no uso de DCs alogenêicas fundidas com células tumorais tem se mostrado relativamente eficaz. Por outro lado, esta estratégia necessita uma massa tumoral considerável de cada paciente para a geração das células híbridas. Para contornar este problema o uso de DCs transfectadas com mRNA tumoral, o qual pode ser amplificado in vitro a partir de uma pequena amostra inicial do tumor, tem sido investigado. Para isto, uma transfecção eficiente e tradução correta do mRNA tumoral nas DCs são etapas críticas. Sabendo-se que as DCs de pacientes com câncer possuem atividade aloestimuladora defeituosa, DCs derivadas de doadores saudáveis poderiam ser uma alternativa para induzir uma resposta imune mais eficiente. Assim, este trabalho pretendeu aprimorar a metodologia de transfecção de DCs alogenêicas, derivadas de monócitos de doadores saudáveis, com mRNA de antígenos tumorais (survivina e RPSA) super-expressos na leucemia linfóide crônica (LLC) e avaliar sua capacidade em estimular a resposta linfocitária. Ao mesmo tempo, foram estabelecidas as metodologias para amplificação e síntese do RNA destes antígenos tumorais específicos, assim como do RNA mensageiro total, contidos nas células tumorais de pacientes com LLC. Os resultados mostraram ser possível a amplificação do mRNA total extraído das células leucêmicas com manutenção da expressão dos antígenos tumorais. Ainda, várias condições de transfecção com mRNA da survivina, transcrito in vitro, foram testadas, encontrando-se na lipofecção, a melhor maneira de transfectar as DCs. A lipofecção mostrou-se com baixa toxicidade quando comparada à técnica de eletroporação. Observou-se uma eficiência em torno de 40% de células transfectadas num intervalo de tempo entre 12 e 48 horas. Estas células foram usadas como estimuladoras em ensaios de proliferação usando-se linfócitos T alogenêicos como células respondedoras. As células transfectadas com mRNA da survivina foram capazes de estimular resposta linfoproliferativa com maior produção de IFN-gama, avaliado por ELISA. Além disso, a transfecção não alterou o padrão de expressão dos marcadores de superfície característicos das DCs. Estes dados mostram que a transfecção das DCs com mRNA pode afetar a resposta imune induzida por estas APCs. Nossos resultados suportam o uso de DCs transfectadas com mRNA para produção de vacinas anti-tumorais e mostram a survivina como um potente antígeno indutor da resposta linfocitária.
The development of dendritic cell (DC)-based cancer immunotherapy has been the target of many studies. For solid tumors, a promising approach based in allogeneic DCs fused to tumor cells has been relatively effective. On the other hand, this approach needs large tumor samples to generate enough DC-tumor cell hybrids. To overcome this problem, tumor mRNA-transfected DCs can be used, since mRNA can be amplified in vitro and allow unlimited vaccine production. For this, efficient transfection and optimal translation of tumor mRNA in DCs are critical. Moreover, DC derived from cancer patients has defective alostimulatory activity. In this case, DC derived from healthy donors may be an alternative to induce immune response more efficiently. Here, we established the methodology of allogeneic DC transfection with mRNA for tumor antigens (survivin and RPSA) overexpressed in chronic lymphocytic leukemia (CLL), and evaluated their ability for T cell stimulation. At the same time, we established mRNA amplification and mRNA in vitro transcription methodology for specific tumor antigens and total messenger RNA, present in CLL tumor cells. Our results showed it to be possible to amplify total mRNA derived from leukemic cells maintaining tumor antigen expression. Moreover, several transfection conditions using survivin mRNA obtained from in vitro transcript reactions were evaluated, defining lipofection as the better way to transfect DC. We obtained nearly 40% of transfected DCs between 12 and 48 hours. Transfected DCs were used as stimulator cells in proliferation assays using allogeneic T cells as responder cells. Survivin mRNA transfected DCs were able to stimulate T cell proliferation with increased IFN-gama production, measured by ELISA. Furthermore, the transfection did not change the pattern of surface molecules expression characteristic of DC. These data show that mRNA DC transfection can affect immune responses induced by these APCs. These findings support the use of tumor mRNA transfected DCs for anti-cancer vaccine production and show survivin as a potent antigen to induce T cell responses.

Los inhibidores de PARP han surgido como nuevas terapias basadas en el papel essencial de las Poly (ADP-ribosa) polimerasas PARP-1 y PARP-2 en la respuesta a daño en el DNA, explotando el efecto de las mismas en la célula tumoral. Sin embargo, la progresión de un tumor está muy determinada por su compleja interacción con otros múltiples tipos celulares, particularmente las células T, en las que no se está considerando la actividad de PARP. Superando la letalidad embrionaria de los ratones doble deficientes en PARP-1 y PARP-2, en el presente trabajo investigamos el papel de estas PARPs en la modulación de las respuestas ejercidas por las células T contra tumores de mama inducidos por la línea AT-3; utilizando para ello ratones deficientes en PARP-1 con una supresión de PARP-2 controlada bajo el promotor de CD4. Descubrimos efectos opuestos de las deficiencias únicas y dobles, donde la doble supresión de PARP-1 y PARP-2 promueve el crecimiento tumoral mientras que la supresión individual de cada proteína limita la progresión del tumor. El análisis de las células infiltrantes de tumor en ratones con deficiencia doble de PARP-1 y PARP-2 reveló un cambio global en el perfil inmunológico y alteraciones en el reclutamiento y la activación de las células T. Por el contrario, la deficiencia única de PARP-1 o PARP-2 tiende a generar un microambiente con una respuesta inmune activa y parcialmente elevada.
Based on the essential role of Poly(ADP-ribose)-polymerases (PARP)-1 and PARP-2 in the DNA damage response, PARP inhibitors have emerged as novel therapeutic tools exploiting the effect of PARPs in the tumor cell itself. However, tumor progression is heavily determined by its complex interaction with multiple other cell types, particularly T cells, in which the activity of PARP is not being considered. Here, we bypassed the embryonic lethality of dually PARP-1/PARP-2-deficient mice by using a PARP-1- deficient mouse with a Cd4-promoter-driven deletion of PARP-2 in T cells to investigate the understudied role of these PARPs in the modulation of T cell responses against AT-3-induced breast tumors. We found opposite effects of single and dual deficiency in modulating the anti-tumor response; where dual PARP-1/PARP-2-deficiency in T cells promote tumor growth while single deficiency of each protein limited tumor progression. Analysis of tumor-infiltrating cells in dually PARP-1/PARP-2-deficiency host-mice revealed a global change in immunological profile and impaired recruitment and activation of T cells. Conversely, single PARP-1 and PARP-2-deficiency tends to produce an environment with an active and partially upregulated immune response.

Orientador: Selma Giorgio
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-05T22:06:43Z (GMT). No. of bitstreams: 1 Albuquerque_MarcosEduardoLamasde_M.pdf: 618346 bytes, checksum: d8a7d01dacd5919516d8000db3c0e660 (MD5) Previous issue date: 2006
Resumo: A leishmaniose tegumentar é uma parasitose causada por protozoários do gênero Leishmania que acomete preferencialmente a pele e/ou as mucosas e caracteriza-se pelo aparecimento de lesões ulcerosas. Histologicamente, esta lesão caracteriza-se pela desintegração da epiderme e da membrana basal, com grande incidência de histiócitos, linfócitos, granulomas e proliferação de parasitos. Como conseqüência, a região lesada apresenta redistribuição de vasos sanguíneos, e dificuldades na difusão de oxigênio, resultando em microambiente hipóxico. Macrófagos se adaptam a hipóxia, alterando seu metabolismo, produção de linfocinas e atividade fagocítica. No presente trabalho comparamos os efeitos da hipóxia (6% O2) e da normóxia (21% O2) na infecção in vitro de macrófagos humanos obtidos de monócitos de sangue periférico com o parasito Leishmania amazonensis. Culturas de macrófagos humanos expostos a hipóxia antes da infecção com L. amazonensis mostraram redução na porcentagem de células infectadas quando comparadas com culturas de macrófagos humanos que permaneceram em normóxia. Nós também investigamos se a hipóxia estaria inviabilizando a sobrevivência dos macrófagos (teste do MTT) e induzindo a produção da linfocina TNF-a (boiensaio com células L929). Macrófagos humanos submetidos à hipóxia não mostraram diferenças significativas em relação viabilidade quando comparados aos macrófagos que permaneceram em ambiente normóxico culturas de macrófagos humanos estimulados com LPS e submetidos a períodos de hipóxia produziram mais TNF- a do que culturas de macrófagos estimulados com LPS que permaneceram em normóxia. Porém quando infectados com L. amazonensis, macrófagos estimulados com LPS em hipóxia mostraram produção significativamente menor de TNF- a quando comparados a macrófagos não infectados e estimulados por LPS, em ambiente hipóxico. Verificamos também que a produção de TNF- a nas culturas de macrófagos infectados com L. amazonensis, estimuladas com LPS, em hipóxia foi similar a produção de TNF- a dos macrófagos infectados com L. amazonensis, estimulados com LPS, mas que permaneceram em normóxia. Nossos resultados indicam que hipóxia altera a susceptibilidade de macrófagos humanos obtidos de monócitos de sangue periférico para a infecção com L. amazonensis, e que a produção de TNF- a não está envolvida no mecanismo pelo qual estas células controlam a carga parasitária
Abstract: The tegumentary leishmaniasis is a parasitic disease caused by protozoa Leishmania which attacks skin and/or mucosal tissues. The lesions are histologically characterized by degeneration of epidermal and the basal membranes with infiltration of histiocytes, lymphocytes, granuloma and parasite proliferation. Cutaneous lesions are associated with rearrangement of the blood vessels and decreased oxygen diffusion, resulting in a hypoxic microenvironment. Macrophages adapt to hypoxia altering their metabolism, lymphocytes production and phagocytosis activity. In the present study we have compared the effect of hypoxia (6% O2) with normoxia (21% O2) in human macrophages, derived from peripheral blood monocytes, infected with Leishmania amazonensis. Human macrophages exposed to hypoxia before infection with L. amazonensis showed a reduction of the percentage of infected cells. Cell viability was tested by MTT and TNF- a production was detected by bioassay using cell line L929. Human macrophages exposed to hypoxia showed similar viability when compared with human macrophages in normoxia. Human macrophages stimulated with LPS and exposed to hypoxia increased TNF- a production when compared with macrophage timulated by LPS cultured in normoxia. However, macrophage, infected with L. amazonensis stimulated with LPS and exposed to hypoxia showed a reduction in TNF- a production when compared with non infected macrophage, stimulated with LPS and exposed to hypoxia. We also observed that TNF- a production in L. amazonensis infected macrophages stimulated with LPS and exposed to hypoxia was similar to TNF- a production of infected macrophages stimulated with LPS, in normoxia. Our data indicated that hypoxia cam alter human macrophages susceptibility to L. amazonensis infection and no correlation between TNF-a production and control of parasite infection in this cells under hypoxia conditions
Mestrado
Mestre em Parasitologia

Les tumeurs épithéliales thymiques (TET) humaines sont rares (250 - 300 cas/an en France). On distingue les thymomes de type A, AB ou B d'évolution lente avec une survie actuarielle > 95% à 5 ans pour les stades précoces et les carcinomes thymiques d'évolution plus sévère avec une survie actuarielle à 5 ans < 20% pour les stades IV. La pierre angulaire du traitement des TET est l'exérèse chirurgicale complète, facteur pronostique le plus significatif identifié à ce jour. Les récidives des TET, essentiellement pleurales pour les thymomes et générales pour les carcinomes thymiques, sont de prise en charge complexe. Les avancées thérapeutiques sont limitées notamment par l'absence de modèles d'étude de la cellule épithéliale thymique tumorale. Dans le cadre d'une prise en charge multidisciplinaire des récidives pleurales métastatiques, nous avons développé la pleurectomie de cytoréduction associée à une chimio hyperthermie (cisplatine/ mitomycine ; 42°C) intra thoracique (CHIT) pour la prise en charge des métastases pleurales de thymome. Chez des patients sélectionnés (n=19), la médiane de survie sans récidive était de 53 mois et les survies actuarielles à 1 an et 5 ans étaient respectivement de 93% et 86%. Cette technique chirurgicale innovante a permis de développer une alternative à la morbide pleuro pneumonectomie. L'efficacité de la CHIT pose des questions sur le rôle de l'hyperthermie et sur le type de chimiothérapie à associer. Avec pour objectif d'améliorer la prise en charge des patients, la connaissance de la biologie tumorale thymique et l'identification de potentielles cibles thérapeutiques sont des voies de recherche importantes pour améliorer la survie des patients. Nous avons développé des cultures de cellules épithéliales thymiques dérivées in vitro de 12 TET (11 thymomes A, AB ou B et un carcinome thymique), caractérisées par leur potentiel prolifératif et leur expression de cytokératine. La voie PI3K / Akt / mTOR joue un rôle clé dans de nombreux cancers ; plusieurs études de phases I / II ont rapporté un effet positif des inhibiteurs de mTOR pour le contrôle de l'évolution du thymome chez les patients. Nous avons mis en évidence l'expression et l'activation des effecteurs mTOR, Akt et P70S6K dans les thymomes et dans les cellules épithéliales thymiques dérivées in vitro. Nous avons montré l'efficacité de la rapamycine, inhibiteur de mTOR, à réduire la prolifération cellulaire (30%) sans induire de mort cellulaire. Nos résultats suggèrent que l'activation de la voie Akt / mTOR participe à la prolifération cellulaire associée à la croissance tumorale. Nous avons établi un nouvel outil permettant l'étude de la dérégulation cellulaire au cours des thymomes. Dans un contexte de tumeurs rares, ces cellules permettront d'aborder des études mécanistiques in vitro et de tester l'efficacité de drogues anti tumorales
Human thymic epithelial tumors (TETs) are rare (250 – 300 cases/ year in France). We distinguish thymomas (A, AB and B subtypes) with indolent evolution (5 years actuarial survival in early stages >95%) and more aggressive thymic carcinomas (5 years actuarial survival <20% in stage IV). Surgical complete resection when feasible is the corner stone of a multimodal therapy and the most significant factor on survival. Relapse of TETs principally in pleura for thymomas (75%) and general for thymic carcinomas are difficult to treat. Therapeutics advances are limited given the lack of studies models of tumoral thymic epithelial cell. In a multidisciplinary approach for the treatment of metastatic pleural relapse of thymomas we developed an innovative surgical technique: cytoreductive pleurectomy associated with hyper thermic intra pleural chemotherapy (Cysplatin/ Mitomycin; 42°C) (ITCH). In selected patient (n=19), ITCH provides an efficient alternative to the morbid pleuro pneumonectomy. The median of free disease survival was 53 months, one year and five years actuarial survival were respectively 93% and 86%. However, the effectiveness of ITCH procedure questions on the played role ok hyperthermia, on the choice of chemotherapy association. With the aim to improve TETs therapies, the knowledge of TETs biology to identify potential target therapies is currently challenging. We developed an in vitro study model of tumoral thymic epithelial cells derived from 12 TETs (11 A, AB and B thymomas and one thymic carcinoma) characterized by their proliferative abilities and the cytokeratin expression. The PIK3 / Akt / mTOR pathway is implicated in numerous cancers. Several phase I, II studies advocate the potential role of mTOR inhibitors in the control of the metastatic disease. We highlighted the expression and the activation of mTOR, Akt and P70S6K effectors in TETs and in thymic epithelial cells in vitro derived. We showed the efficacy of rapamycin (mTOR inhibitor) in the inhibition (-30%) of in vitro cell proliferation without cell death induction. Our results suggest the implication of the PIK3 / Akt / mTOR pathway in the tumoral cell growth. We established a new tool to study cell dysregulation in TETs. In the context of rare tumors, these cells could allow in vitro mechanistic studies and test the efficacy of new anti tumoral therapies

A terapia antiangiogênica surgiu como uma alternativa promissora para o tratamento do câncer. No entanto, evidências recentes mostram que as células endoteliais isoladas diretamente de um tumor maligno são mais resistentes a diferentes drogas do que as células endoteliais presentes no mesmo tecido normal. Essas diferenças podem ser atribuídas em parte à adaptação das células endoteliais ao microambiente tumoral. Uma característica singular do microambiente tumoral é a consistente acidificação do meio extracelular, cujos efeitos nas células endoteliais não são conhecidos. Acidez extracelular pode alterar múltiplas funções biológicas, causar estresse do retículo endoplasmático (RE) e ativação da Unfolded Protein Response (UPR). Células endoteliais humanas primárias de derme (HDMEC) cultivadas em pH 6.4, ajustado tanto com ácido lático tanto com ácido clorídrico, apresentaram aumento da expressão de proteínas relacionadas à UPR, como GRP78, ATF4, elf2α fosforilada e aumento na clivagem do mRNA de XBP1. Nessas condições massiva morte celular ocorreu após 48 horas. Em contrapartida, quando as células endoteliais eram expostas à acidez crônica não-letal com pH 7.0 durante sete dias, essas foram capazes de se adaptar coincidentemente com um aumento da expressão da proteína GRP78 Após sete dias sob pH 7.0, as células HDMEC apresentaram maior resistência à morte celular quando tratadas com as drogas Etoposide, Adriamicina e Sunitinib em doses que variavam entre 0.0025μM a 100μM. O silenciamento do gene GRP78 com ShRNA reverteu esse fenótipo resistente. Para determinar os níveis de UPR in vivo utilizou-se captura por microdissecção à laser de células endoteliais em lâminas histológicas de 14 carcinomas espinocelulares bucais. Observou-se um aumento significativo dos níveis de mRNA de GRP78, ATF4 e CHOP em células endoteliais dos tumores quando comparadas a células endoteliais primárias (HDMEC). Além do mais, células endoteliais tumorais apresentaram intensa imunomarcação para GRP78 comparativamente a células endoteliais de mucosa bucal normal. A acidez, uma importante fonte de estresse no microambiente tumoral, pode ativar uma UPR adaptativa em células endoteliais. Aumento da expressão de GRP78 em células endoteliais é associado com maior resistência a drogas quimioterápicas. Os resultados sugerem que a resistência mediada pela UPR pode contribuir com o insucesso terapêutico na resposta a drogas antitumorais.
Antiangiogenic therapy has emerged as a promising alternative for cancer treatment. However, growing evidence has shown that endothelial cells isolated directly from malignant tumors are more resistant to different drugs than endothelial cells from normal tissues. These differences may due to the adaptation of endothelial cells to the tumor microenvironment. A unique feature of tumor microenvironment is the consistent acidification of the extracellular environment, whose effects on endothelial cells are not known. Extracellular acidity can alter multiple biological functions, including endoplasmic reticulum stress and activation of the Unfolded Protein Response (UPR). Primary human dermal microvascular endothelial cells (HDMEC) cultured at medium pH 6.4, adjusted with either lactic acid or either hydrochloric acid, showed strong up-regulation of the UPR-related proteins: GRP78, ATF4, phospho-elf2α and increased XBP1 mRNA splicing. However massive cell death occurred after 48 hours. In contrast, when endothelial cells were exposed to chronic nonlethal acidic stress at pH 7.0 for up to seven days, cells were able to adapt, coincidental with a marked increase in GRP78 protein expression. After 7 days at pH 7.0, HDMEC cells showed increased resistance to cell death when exposed to Etoposide, Adriamycin and Sunitinib at doses ranging from 0.0025μM to 100μM. Knockdown of GRP78 by shRNA reversed the resistance phenotype. To determine the levels of UPR in vivo, laser capture microdissection of endothelial cells from oral squamous cell carcinomas biopsies was done. There is a significant increase in mRNA levels of GRP78, ATF4 and CHOP on endothelial cells of tumors compared to untreated primary endothelial cells (HDMEC). Moreover, tumor 16 endothelial cells showed strong GRP78 immunostaining compared to endothelial cells from normal oral mucosa. Low pH, an important source of cellular stress in the tumor microenvironment, can activate an adaptive UPR response in endothelial cells. Increased expression of GRP78 in endothelial cells is associated with chemoresistance. The results suggest that UPR-mediated resistance may contribute to therapeutic failures in response to anticancer drugs.

Le cancer broncho-pulmonaire (CBP) le 4ème cancer le plus répandu au niveau mondial après les cancers de la prostate, du sein et du côlon. Diagnostiqués à des stades tardifs, il est la première cause de cancer par décès. Cependant, une meilleure compréhension des mécanismes moléculaires sous-jacents au cancer a permis de développer des thérapies personnalisées pour chaque patient. L’émergence des thérapies ciblées et de l’immunothérapie a révolutionné la prise en charge thérapeutique, permettant d’améliorer la survie globale, la survie sans progression et les effets secondaires des patients en comparaison avec les traitements de chimiothérapies conventionnelles. La prescription des thérapies personnalisées est basée sur les caractéristiques moléculaires de la tumeur et, par conséquent, nécessite des analyses moléculaires innovantes. Néanmoins, entre 10 et 30% des analyses moléculaires des patients atteints de CBNPC sont non contributives et l’accès aux thérapies ciblées est compromis. De plus, même si l’analyse anatomo-pathologique reste utile pour l’évaluation du stade ou encore de l’histologie, elle reste peu adaptée en cas d’actes répétitifs, tout au long de la maladie. La « biopsie liquide », est un concept émergeant, correspondant à l’analyse des acides nucléiques circulants mais aussi des cellules tumorales circulantes (CTC), issus de la tumeur. Cette méthode faiblement invasive, basée sur un prélèvement sanguin, permet d’analyser le patrimoine tumoral circulant et donne accès aux informations moléculaires de la tumeur primaire. Le développement de nouvelles activités de diagnostic est donc primordial pour répondre à ses nouvelles demandes cliniques. Depuis 2015, les Hospices Civils de Lyon (HCL) ont déployé un programme de recherche translationnelle, appelé CIRCAN « CIRculating Cancer » dans lequel s’inscrit ce travail de thèse. De nombreuses méthodes de détection des biomarqueurs pertinents en oncologie thoracique dans l’ADNcir ont été développées et été validées au laboratoire pour plus de 1500 patients actuellement, leur permettant de bénéficier de thérapies ciblées. L’optimisation et la validation des technologies de biologie moléculaire telles que le séquençage à haut débit et la PCR digitale ultra-sensible ont été réalisées durant ce travail de thèse et valorisées dans des journaux internationaux. Au-delà des thérapies ciblées, les immunothérapies représentent les nouveaux traitements prometteurs pour ces patients dont le taux d’expression PD-L1 sur biopsie tissulaire est le biomarqueur de choix. Etant donné les contraintes qu’occasionne la biopsie tissulaire, nous avons développé un protocole de caractérisation phénotypique de PD-L1 dans les CTC. De plus, de nombreuses études montrent la pertinence clinique de l’utilisation de la charge mutationnelle (TMB) comme marqueur prédictif de réponse à l’immunothérapie. En parallèle, nous avons développé des outils moléculaires en cours de validation pour le calcul du TMB dans l’ADNcir et dans les CTC en comparaison avec celui calculé dans le tissu, et le taux de PD-L1 évalué par immunohistochimie. Toutefois, pour environ 50% des patients atteints de CBP, aucun biomarqueur n’est retrouvé, bloquant l’accès à des thérapies personnalisées et réduisant les chances de survie du patient. A la recherche de nouveaux biomarqueurs, nous avons développé un protocole permettant d’accéder à la signature transcriptomique des CTC à un niveau « single-cell » afin de caractériser l’hétérogénéité tumorale de ces cellules et de mieux comprendre les mécanismes de résistance mis en place. Les échantillons cliniques de patients sont en cours d’analyse, avec ce protocole validé avec une lignée cellulaire modèle. En effet, les résultats de la validation de méthode mettent en exergue la possibilité d’évaluer l’hétérogénéité tumorale et les voies de signalisation impliquées dans la dissémination métastasique, telles que la transition épithélio-mésenchymateuse
Broncho-pulmonary cancer (PBC) is the 4th most common cancer worldwide after prostate, breast and colon cancer. Diagnosed at late stages, it is the leading cause of cancer death. However, a better understanding of the molecular mechanisms underlying cancer has led to the development of personalized therapies for each patient. The emergence of targeted therapies and immunotherapy has revolutionized the therapeutic management, improving the overall survival, progression-free survival and side effects of patients compared to conventional chemotherapy treatments. The prescription of personalized therapies is based on the molecular characteristics of the tumor and, therefore, requires innovative molecular analyzes. Nevertheless, between 10 and 30% of the molecular analyzes of NSCLC patients are non-contributory and access to targeted therapies is compromised. Moreover, even if the pathological analysis remains useful for stadium evaluation or histology, it remains unsuitable for repetitive actions throughout the illness. The "liquid biopsy", is an emerging concept, corresponding to the analysis of circulating nucleic acids but also circulating tumor cells (CTC), derived from the primary tumor. This low-invasive method, based on a blood sample, makes it possible to analyze the circulating tumor inheritance and gives access to the molecular information of the primary tumor. The development of new diagnostic activities is therefore essential to meet its new clinical demands. Since 2015, Hospices Civils de Lyon (HCL) has deployed a translational research program, called CIRCAN "CIRculating Cancer" in which this thesis. Many methods for detecting relevant biomarkers in thoracic oncology in circulating free DNA (cfDNA) have been developed and validated in the laboratory for more than 1500 patients currently, allowing them to benefit from targeted therapies. The optimization and validation of molecular biology technologies such as high-throughput sequencing and ultra-sensitive digital PCR were performed during this thesis work and published in international journals. Beyond targeted therapies, immunotherapies represent promising new treatments for these patients whose PD-L1 expression level on tissue biopsy is the biomarker of choice. Given the constraints of tissue biopsy, we have developed a phenotypic characterization protocol for PD-L1 in CTCs. In addition, numerous studies show the clinical relevance of the use of mutational load (TMB) as a predictive marker of response to immunotherapy. In parallel, we have developed molecular tools undergoing validation for the calculation of TMB in cfDNA and in CTC compared with the value calculated in tissue, and the PD-L1 level evaluated by immunohistochemistry. However, for about 50% of patients with CBP, no biomarker is found, blocking access to personalized therapies and reducing the patient's chances of survival. In search of new biomarkers, we have developed a protocol allowing access to the transcriptomic signature of CTCs at a "single-cell" level in order to characterize the tumor heterogeneity of these cells and to better understand the resistance mechanisms implemented. The clinical samples of patients are being analyzed, with this protocol validated with a model cell line. Indeed, the results of the method validation highlight the possibility of evaluating tumor heterogeneity and the signaling pathways involved in metastatic spread, such as the epithelial-mesenchymal transition

Le microenvironnement tumoral via les macromolécules matricielles est connu pour jouer un rôle clé dans la réponse des cellules cancéreuses à la chimiothérapie en favorisant leur survie et leur prolifération. L'impact du collagène de type I, protéine matricielle majeure du microenvironnement, a été évalué au niveau des capacités migratoires des cellules tumorales et de leur réponse aux agents anticancéreux, doxorubicine et metformine. Cette approche a étémenée chez des cellules humaines HT1080 hautement invasives au moyen de systèmes de culture par coating 2D ou en matrice 3D. Les effets de modifications post-traductionnelles du collagène comme la carbamylation et de la glycation ont été également étudiées. Les résultats montrent que le collagène 3D inhibe l'activité anti-migratoire de la doxorubicine. Cette protection met en jeuune préservation des niveaux d'activation de FAK et RhoA impliquées dans la formation des fibres de stress d'actine et des plaques d'adhésion focales. Le collagène glyqué 2D et dans une moindre mesure le carbamylé inhibent l'adhésion, la migration des cellules tumorales et désorganisent leur cytosquelette d'actine via des modifications de distribution de la vinculine, de FAK et des intégrines 1. Cet impact de la glycation a été aussi mis en évidence en matrice 3D après modification du processus de glycation. Enfin, la glycation exerce un effet protecteur vis-àvis des capacités anti-prolifératives et anti-migratoires de la doxorubicine et de la metformine. En conclusion, nous mettons en évidence une nouvelle forme de résistance CAM-DR dirigée contre l'activité anti-invasive de médicaments ; cet effet pouvant être généré par une protéine matricielle native ou modifiée lors de situations physiopathologiques associées au cancer
The tumor microenvironment via the extracellular matrix plays an important role in cancer cell response to chemotherapy by promoting their survival and proliferation. In this work, we studied the impact of collagen type I, a major matrix protein of tumor microenvironment, on the migration capacities of tumor cells and on their response to anticancer drugs such as doxorubicin and metformin. This approach was performed with the highly invasive human cell line HT1080,by means of 2D coating or 3D matrix cell culture systems. The effects of collagen posttranslational modifications such as carbamylation and glycation were also assessed. The results show that the 3D collagen inhibits the anti-migratory effect of doxorubicin. This protection is carried out through the preservation of the activation states of FAK and RhoA, which are involved in the formation of actin stress fibers and focal adhesions. On 2D coating, the glycated collagen and at a lesser extent the carbamylated one decrease the adhesion, the migration oftumor cells and, disorganize the actin cytoskeleton via a modified distribution of vinculine, FAK and beta1 integrins. This impact is also demonstrated by using 3D matrices, after adaptation of the glycation process. In addition, we reported that the glycated collagen protects against the antiproliferative and the anti-migratory effects of doxorubicin and metformin. In conclusion, we highlighted a new form of CAM-DR resistance that targets the drugs anti-invasive activity. This impact could be induced by the native form of matrix proteins or the modified one found inpathological situations which are associated to cancer

Les tumeurs rhabdoïdes (TR) constituent un rare cancer indifférencié du jeune enfant et du nourrisson, avec un âge médian au diagnostic de 20 mois. Ces tumeurs sont caractérisées par une inactivation biallélique du gène suppresseur de tumeur SMARCB1, un des membres du complexe SWI/SNF, acteur majeur du remodelage de la chromatine, sans autre altération génomique récurrente. Le pronostic des TR est péjoratif, le taux de survie globale atteignant 30% dans la plupart des séries, malgré des approches thérapeutiques conventionnelles particulièrement agressives. Les approches d’immunothérapies ont obtenu un succès certain dans certains cancers de l’adulte, et récentes analyses de l’infiltrat immun des cancers pédiatriques ne montrent pas un fort taux de tumeurs infiltrées à l’exception de rare types de cancers dont les TR intracrâniennes. Nous avons donc procédé à une analyse multimodale de l’infiltrat immun de cohortes de patients ainsi que d’un modèle de TR murines établi dans notre laboratoire. Nous avons identifié une forte proportion de tumeurs infiltrées dans certains sous-groupes de TR. Cet infiltrat était composé à la fois de cellules myéloïdes incluant des populations au phénotype immunosuppresseur, et lymphocytaires T notamment de phénotype résident mémoire caractérisées par une forte expansion clonale probablement spécifique d’un antigène tumoral. Nous avons identifié des cibles thérapeutiques communes aux tumeurs humaines et au modèle murin syngénique, et trouvé que cibler l’infiltrat lymphocytaire T ou myéloïde était susceptible d’induire une réponse tumorale complète avec induction d’une mémoire immunitaire, confirmant le caractère immunogénique des TR, et apportant de nouvelles stratégies thérapeutiques utiles en clinique. Enfin, nous avons identifié que les TR étaient le site d’une réexpression de rétrovirus endogènes, dépendante de celle de SMARCB1, avec activation des voies de l’interféron, apportant une base à une immunogénicité des TR issue du génome non codant
Rhabdoid tumors (RT) are highly undifferentiated cancers occurring in infancy and early childhood, with a median age at diagnosis about 20 months. These tumors are characterized by the biallelic inactivation of SMARCB1 tumor suppressor gene, core member of the SWI/SNF complex, one major chromatin remodeling actor, in an otherwise highly stable genome. The prognosis of RT is dismal with overall survival hardly reaching 30% in most series, despite particularly aggressive conventional treatment. Immunotherapy approaches has gained a striking success within some adult cancer types and recent analyses of immune cell content of pediatric cancers don’t reveal a high rate of infiltrated tumors, except in few tumor types such as intracranial rhabdoid tumors. Then, we conducted a comprehensive analysis of the immune context of both human RT cohorts and a mouse RT model, including at single cell level. We identified a high recurrence of infiltrated tumors, in a RT-subgroup related manner, composed of both myeloid cells including cells with immune suppressive phenotypes, and T cells with notably a tissue resident memory phenotype demonstrating a high clonal expansion highly suggestive of immunogenicity. We identified common targetable immune populations between human and mouse RTs, and found that targeting both T and myeloid infiltrating cells was able to induce complete anti-tumor response with induced memory, confirming the immunogenic properties of RTs, and identifying new therapeutic strategies of clinical relevance. We finally identified that RTs were the site of SMARCB1-dependent endogenous retroviruses reexpression, with subsequent activation of interferon signaling, likely triggering the immune response in the context of RT, and providing a basis of non-coding genome-driven immunogenicity for these tumors

Human breast cancer (HBC), canine (CMT), and feline mammary tumors (FMT) are extremely common and are characterized by a remarkable both inter- and intra-tumor heterogeneity. Intra-tumor heterogeneity is due to the coexistence of cancer cells that differ between each other in terms of phenotypic, genetic, behavioral characteristics, and metastatic potential. Cancer stem cells (CSCs) are thought to be responsible for such heterogeneity, resistance to therapy, and metastasis development. Several pathways are altered in CSCs, such as the oncogenic Wnt/-catenin and Hippo pathways, and CSCs are associated to the epithelial-to-mesenchymal transition (EMT) process. The aims of this study were to i) isolate and characterized mammary CSCs; ii) investigate EMT process and Wnt/-catenin and Hippo pathways in mammary cancer of the three species; iii) establish a metastatic mouse model of breast cancer seeking for genes responsible of metastatic dissemination; iv) isolate and characterize extracellular vesicles (EVs), which is one of the main forms of intercellular communication, from canine and feline mammary tumors as well as study the role that EVs play during tumor development. CSC-like cells were isolated from established canine and feline mammary tumor cell lines (CYPp and FMCp, respectively) and phenotypically and molecularly characterized for common CSC markers: CD44, CD24, CD133, SOX2, OCT4. Moreover, gene (qPCR) and protein (IHC and WB) expression of Wnt/-catenin and Hippo pathways-related molecules (-catenin, CCND1, YAP, TAZ, CTGF, ANKRD1) as well as protein expression (IHC) of EMT-related molecules (E-cadherin, SNAIL, TWIST, ZEB) were evaluated in a subset of human, canine, and feline mammary cancer tissues, that were also phenotypically characterized for the following markers: CK8/18, CK5/6, CK14, CD44, and vimentin. Additionally, triple negative breast cancer (TNBC) cell line MDA-MB-231 was used to establish a clinically relevant in vivo metastatic model. Finally, EVs were isolated and characterized from CYPp and FMCp and human glioblastoma-derived EVs were used to study tumor angiogenesis. We found that CD44, CD133, SOX2, and OCT4 expression increase in CSC-like cells (mammospheres) compared to parental adherent cells, therefore they could be used as useful markers in CMTs and FMTs. Wnt/-catenin and Hippo pathways seem to be deregulated at a post-transcriptional level in HBCs, CMTs, and FMTs. Interesting similarities were confirmed between TNBCs and FMTs, as well as between ER+ HBC and CMTs. In our metastatic model, mice developed distant metastases and we found a few genes that might play a role during metastatic dissemination. Among these, caspase 3 seems to be involved in brain metastases. Additionally, EVs were isolated from CYPp and FMCp, visualized by transmissible electron microscopy, counted using nanoparticle tracking analysis, and characterized by immunogold and WB (Alix, CD63, TSG101). Finally, using a human glioblastoma cell line (GBM8) we demonstrated that EVs are directly involved in tumor angiogenesis. Overall, this study confirms the use of dogs and cats as spontaneous models of mammary cancer development due to highly interesting biological similarities among the three species. Also, identification of EVs in CMTs and FMTs opens an interesting unexplored field in veterinary medicine.
Human breast cancer (HBC), canine (CMT), and feline mammary tumors (FMT) are extremely common and are characterized by a remarkable both inter- and intra-tumor heterogeneity. Intra-tumor heterogeneity is due to the coexistence of cancer cells that differ between each other in terms of phenotypic, genetic, behavioral characteristics, and metastatic potential. Cancer stem cells (CSCs) are thought to be responsible for such heterogeneity, resistance to therapy, and metastasis development. Several pathways are altered in CSCs, such as the oncogenic Wnt/-catenin and Hippo pathways, and CSCs are associated to the epithelial-to-mesenchymal transition (EMT) process. The aims of this study were to i) isolate and characterized mammary CSCs; ii) investigate EMT process and Wnt/-catenin and Hippo pathways in mammary cancer of the three species; iii) establish a metastatic mouse model of breast cancer seeking for genes responsible of metastatic dissemination; iv) isolate and characterize extracellular vesicles (EVs), which is one of the main forms of intercellular communication, from canine and feline mammary tumors as well as study the role that EVs play during tumor development. CSC-like cells were isolated from established canine and feline mammary tumor cell lines (CYPp and FMCp, respectively) and phenotypically and molecularly characterized for common CSC markers: CD44, CD24, CD133, SOX2, OCT4. Moreover, gene (qPCR) and protein (IHC and WB) expression of Wnt/-catenin and Hippo pathways-related molecules (-catenin, CCND1, YAP, TAZ, CTGF, ANKRD1) as well as protein expression (IHC) of EMT-related molecules (E-cadherin, SNAIL, TWIST, ZEB) were evaluated in a subset of human, canine, and feline mammary cancer tissues, that were also phenotypically characterized for the following markers: CK8/18, CK5/6, CK14, CD44, and vimentin. Additionally, triple negative breast cancer (TNBC) cell line MDA-MB-231 was used to establish a clinically relevant in vivo metastatic model. Finally, EVs were isolated and characterized from CYPp and FMCp and human glioblastoma-derived EVs were used to study tumor angiogenesis. We found that CD44, CD133, SOX2, and OCT4 expression increase in CSC-like cells (mammospheres) compared to parental adherent cells, therefore they could be used as useful markers in CMTs and FMTs. Wnt/-catenin and Hippo pathways seem to be deregulated at a post-transcriptional level in HBCs, CMTs, and FMTs. Interesting similarities were confirmed between TNBCs and FMTs, as well as between ER+ HBC and CMTs. In our metastatic model, mice developed distant metastases and we found a few genes that might play a role during metastatic dissemination. Among these, caspase 3 seems to be involved in brain metastases. Additionally, EVs were isolated from CYPp and FMCp, visualized by transmissible electron microscopy, counted using nanoparticle tracking analysis, and characterized by immunogold and WB (Alix, CD63, TSG101). Finally, using a human glioblastoma cell line (GBM8) we demonstrated that EVs are directly involved in tumor angiogenesis. Overall, this study confirms the use of dogs and cats as spontaneous models of mammary cancer development due to highly interesting biological similarities among the three species. Also, identification of EVs in CMTs and FMTs opens an interesting unexplored field in veterinary medicine.

L'expression de récepteurs inhibiteurs tels PD-1, TIM-3, LAG-3, TIGIT sur les lymphocytes T participe à l'immunosuppression dans l'environnement tumoral. Le ciblage de PD-1 par les immunothérapies anti-PD-1/PD-L1 notamment révolutionne depuis peu la prise en charge de nombreux types de cancers en particulier dans le mélanome, cancer du poumon et aussi du rein. Dans la plupart des cancers comme dans celui du poumon et le mélanome, l'infiltrat CD8 et la réponse Th-1/IFN-gamma sont associés à un meilleur pronostic, contrairement aux tumeurs du rein et aux hémopathies. Les travaux de ma thèse s'intéressent à la caractérisation de l'expression des récepteurs inhibiteurs des lymphocytes, notamment PD-1 et TIM-3 dans le carcinome rénal et dans le lymphome. Mes travaux de thèse ont été réalisés avec des outils d'exploration du microenvironnement tumoral permettant une analyse multiplexe de nombreux paramètres in situ. En théorie jusqu'à 7 protéines peuvent être mises en évidence à la fois, j'ai pu mettre au point des comarquages de 4 protéines membranaires et/ ou nucléaires + Dapi ainsi que la détection d'ARN in situ dans les tumeurs. L'utilisation d'un outil technologique de microscopie à fluorescence multispectrale a permis une étude fine de la coexpression de PD-1 et Tim-3 sur les lymphocytes T CD8 (LT-CD8) grâce à la visualisation aisée de la colocalisation de ces 3 marqueurs. De la même façon, j'ai mis en évidence de l'expression de leurs ligands PD-L1 et Galectin-9 (Gal-9) dans l'environnement des tumeurs du rein. J'ai démontré que la co-expression de Tim-3 et PD-1 sur les CD8 dans le carcinome rénal avait un rôle délétère aussi bien sur le plan (i) fonctionnel puisque les LT-CD8 sécrétaient moins d'IFN-gamma, (ii) et clinique puisque les patients présentant un infiltrat de LT-CD8 double positifs pour PD-1 et TIM-3 récidivaient plus fréquemment. La présence des ligands PD-L1 et Gal-9 a été mise en évidence dans l'environnement tumoral laissant suggérer des interactions possibles avec les récepteurs inhibiteurs exprimés par les LT. J'ai également caractérisé les LT-CD8 PD-1+ TIM-3+ dans les lymphomes en combinant un marquage CD20 (quadruple + Dapi). Selon le type de lymphome, TIM-3 était coexprimé avec PD-1 à la surface des CD8 et plus ou moins au contact avec les cellules lymphomateuses CD20+. D'autre part, lorsque TIM-3 était coexprimé, les LT-CD8 étaient plus volontiers proliférant (comarquage Ki-67) en comparaison aux PD-1+ TIM-3-. Afin de poursuivre dans la caractérisation Th-1/IFN-gamma, j'ai élaboré la mise au point de la détection d'ARN dans les lymphocytes T in situ dans la tumeur, permettant de faire des liens avec leur fonctionnalité. Au total mes travaux de thèse ont permis de mettre en évidence des biomarqueurs immunologiques composites en lien avec l'expression des récepteurs inhibiteurs PD-1 et/ou TIM-3 et la perte de fonctionnalité (IFN-gamma) des lymphocytes T intratumoraux
It has been mainly described that the inhibitory receptors coexpression (PD-1, TIM-3, LAG-3, TIGIT) by lymphocytes in the tumor microenvironment (TME) induces a local immunosuppression. Targeting these receptors particularly PD-1 and its ligand PD-L1 is of great clinical benefit in cancer many types treatment (melanoma, renal and lung cancer in particular). In the most cases of cancer, like melanoma and lung cancer, a CD8-T cell and Th-1/IFN-gamma response is of good prognosis. But this is not the case in renal cancer and in hemopathies. My PhD work attempts to characterize clinical and biological implication of PD-1 and TIM-3 expression by intra-tumor lymphocytes in the setting of renal cancer and lymphoma. My PhD work has been conducted thanks to new methods of multiplexed characterization of the TME. Multispectral immunofluorescence lead to identify 7 parameters at the same time, and in this study I elaborated the identifications of lymphocytes markers in situ within the tumor: 4 membrane and/or nuclear proteins + nuclei (Dapi counterstain) and also coupled with the RNA detection. This tool allows me to accurately study the coexpression of PD-1 and TIM-3 at the CD8-T cell surface thanks to colocalisation identification and counting of these 3 markers. With the same method, I found that PD-L1 and Gal-9, which are PD-1 and TIM-3 ligands, were also expressed in the TME of renal carcinoma. I found that the coexpression of TIM-3 together with PD-1 in the CD8-T cells had a double relevance (i) at functional level, CD8-T cells were less able to secrete gamma-IFN (ii) at clinical level, patients harboring a higher infiltrate were more likely to relapse. The presence of PD-L1 and Gal-9 suggested interactions with inhibitory receptors of T cells. I also characterized CD8-T cells expressing PD-1 and TIM-3 in lymphomas, combining a CD20 staining (quadruple staining + Dapi). TIM-3 was more or less expressed depending of the lymphoma type near to CD20+ cells. TIM-3 PD-1 CD8-T cells were more likely Ki-67+ compared to TIM-3- cells, suggesting a more proliferative capacity. In order to continue the characterization of the Th-1/gamma-IFN-gamma immune response, I elaborate a technic to detect the gamma-IFN RNA in situ, together with lymphocytes staining, allowing the exploration of functionality within the tumor. To summarize, during my PhD work I could characterize composite immune biomarkers linked to the functionality of CD8-T cell and gamma-IFN Th-1 response

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The cancer encompasses a huge range of different types, requiring several therapeutic approaches for control and/or remission. An approach that has been considered quite promising is the inhibition of the formation of the MDM2/p53 complex. The formation of this complex hinders the triggering of the apoptotic process. The example of the Nutlins, inhibitors of this complex, have stood out as promising prototype drugs and are already in the clinical phase of investigation. The compound LQFM030 originated from the molecular simplification of Nutlin-1, which has already demonstrated antitumor activity against the Ehrlich Ascites Tumor model. In this work, we investigated the cytotoxic effect of compound LQFM030 against the K562 leukemic line, as well as some mechanisms of cytotoxicity. In the evaluation of cytotoxicity using the trypan blue exclusion method, we obtained an IC 50 value of 0.56 mM, similar to results obtained previously. For the other mechanics assays, IC25 (0.283 mM) of compound LQFM030 and 0.01 mM of compound Nutlin-3a were used for treatment for 24 hours. The compound Nutlin-3a was used as the standard for comparison. Morphological analyzes of K562 cells after treatment with compound LQFM030 suggest core fragmentation and chromatin condensation, indicating apoptosis triggered cell death, which was confirmed by flow cytometric analyzes. In cell cycle investigation, LQFM030 treated K562 cells demonstrated increased sub-G1 phase, phosphatidylserine exposure, decreased membrane potential, increased expression of TNF-R1 and caspase -3/7 caspase -9; However, there was a decrease in caspase -8. In turn, the treatment of leukemic cells with Nutlin-3a did not promote changes in the cell cycle, exposure of phosphatidylserine corroborating with data from the literature. In conclusion the studied compound, LQFM030, presented important biological activity with the capacity to induce death in leukemic cells by apoptotic mechanisms and interaction with MDM2 and MDX. The compound presented mechanisms in some aspects similarities to Nutlin-3a, but also showed different mechanisms evidencing its versatility in its biological performance. The discovery of new agents with multiple mechanisms is of great importance to increase the therapeutic armamentarium of cancer in a complementary way and, in addition, it can enhance the effectiveness of the treatment.
O câncer engloba uma variedade enorme de tipos diferentes, sendo necessárias diversas abordagens terapêuticas para controle e/ou remissão. Uma abordagem considerada bastante promissora é a inibição da formação do complexo MDM2/p53. A formação deste complexo dificulta o desencadeamento do processo apoptótico. Já se encontra em fase clínica de investigação um exemplo promissor inibidor deste complexo, os Nutlins. O composto LQFM030 foi originado a partir da simplificação molecular do Nutlin-1, o qual já demonstrou atividade antitumoral contra o modelo do Tumor Ascitico de Ehrlich. Neste trabalho, investigamos mecanismos de citotoxicidade do composto LQFM030 na linhagem leucêmica K562. Na avaliação da citotoxicidade utilizando o método de exclusão do azul de tripano, obtivemos um valor de IC50 de 0,56 mM, similar a resultados obtidos anteriormente. Para os demais ensaios mecanisticos, foi utilizado o IC25 (0,283 mM) do composto LQFM030 e 0,01 mM do composto Nutlin-3a, para tratamento por 24 horas. O composto Nutlin-3a foi utilizado como padrão de comparação. As análises morfológicas das células K562 após o tratamento com o composto LQFM030 demonstrou fragmentação do núcleo e a condensação da cromatina, indicando morte celular desencadeada por apoptose, onde juntamente com citometria de fluxo indicam um processo de morte celular. Na investigação do ciclo celular, as células K562 tratadas com LQFM030 demonstraram um aumento da fase sub-G1, exposição da fosfatidilserina, diminuição do potencial de membrana mitocondrial, aumento da expressão de TNF-R1, caspases -3/7 e caspase -9; contudo, houve diminuição da caspase -8. Por sua vez, o tratamento das células leucêmicas com o Nutlin-3a não promoveu alterações no ciclo celular, exposição de fosfatidilserina corroborando com dados da literatura. Em conclusão o composto estudado, LQFM030, apresentou atividade biológica importante com capacidade de induzir morte em células leucêmica por mecanismos apoptoticos e interação com MDM2 e MDMX. O composto apresentou mecanismos em alguns aspectos semelhanças ao Nutlin-3a, porém também mostrou mecanismos distintos evidenciando a sua versatilidade em sua atuação biológica. A descoberta de novos agentes com múltiplos mecanismos é de grande importância para aumentar o arsenal terapêutico de combate ao câncer de forma complementar e, podendo ainda potencializar eficácia do tratamento.

Câncer é a principal causa de morte nos países desenvolvidos e a segunda causa de morte nos países em desenvolvimento. O trabalho de diversos grupos de pesquisa tem sugerido que os tumores estão organizados em uma hierarquia de células, na qual uma pequena fração apresenta propriedades de células-tronco. Essas células tem se mostrado resistentes à quimioterapia convencional, dependentes da sinalização do microambiente tumoral e responsivas à terapia diferenciativa. Aqui nós mostramos que butirato sódico (NaB), um inibidor de desacetilase de histonas, diminui a proliferação celular e a formação de colônias em linhagens celulares de meduoblastoma humano. Estes efeitos foram acompanhados de um aumento da expressão de RNAm Gria2, um marcador de diferenciação neuronal, em duas das três linhagens celulares testadas. A formação de neuroesferas também foi impedida com a exposição de uma linhagem crescida em meio de cultura apropriado para células-tronco e NaB. NaB se mostrou capaz de potencializar os efeitos do quimioterápico etoposídeo e do fator neurotrófico derivado de cérebro (BDNF) na inibição da viabilidade celular de meduloblastoma. Além disso, nós observamos que o tratamento com cisplatina aumenta a proporção de células-tronco tumorais (CTT), identificadas por ALDH+CD44+, em células de carcinoma de cabeça e pescoço, quando tratadas também com interleucina 6 (IL-6), uma citocina liberada pelo nicho perivascular. O mesmo tratamento promoveu a proliferação, sobrevivência e auto-renovação de CTTs in vitro ao aumentar o número de esferas em placas de baixa aderência e a expressão de Bmi-1 em western blots. A fosforilação do transdutor de sinal e ativador de transcrição 3 (STAT3), um indicativo de propriedades de CTTs, induzida por IL-6 não foi afetada pelo tratamento com cisplatina em células de HNSCC, enquanto que a indução de fosforilação de quinases reguladas por sinais extracelulares (ERK), indicativo de processo de diferenciação, por IL-6 foi parcialmente inibido por cisplatina. Células resistentes a baixas doses de cisplatina também expressaram mais Bmi-1 do que células nunca expostas ao quimioterápico. Experimentos in vivo corroboram os achados in vitro, uma vez que tumores humanos induzidos em camundongos imunocomprometidos apresentaram aumentada proporção de células ALDH+CD44+ após tratamento dos animais com cisplatina. O anticorpo contra o receptor de IL-6 foi capaz de reverter a indução de expressão de Bmi-1 por cisplatina e IL-6. Em conjunto, esses resultados sugerem que a modulação de mecanismos epigenéticos das células tumorais e de sinais provenientes do nicho tumoral são novos alvos promissores para o desenvolvimento de terapias adjuvantes contra o câncer.
Cancer is the leading cause of death in economically developed countries and the second cause of death in developing countries. Work from a number of laboratories strongly suggests that tumors are organized as a hierarchy based on a subset of cancer cells that have stem-cell properties. These cells have been shown to be resistant to conventional therapy, dependent on contextual signals within the tumor microenvironment and, to be responsive to differentiation therapy. Here we show that sodium butyrate (NaB), a histone deacetylase inhibitor, decreases cell proliferation and colony formation in human medulloblastoma cell lines. These effects were accompanied by an increased mRNA expression of Gria2, a neuronal differentiation marker, in two out of three cell lines tested. In addition, neurosphere formation was impaired by NaB exposure in a cell line submitted to stem cells proper media. NaB also may potentiate the effect of etoposide chemotherapy and BDNF (Brain-derived neurotrophic factor) on the inhibition of medulloblastoma cells viability. Moreover, we observed that cisplatin treatment increased the proportion of cancer stem cells (CSC), identified by ALDHhighCD44high cells, in head and neck squamous cell carcinoma (HNSCC), when treated together with recombinant human IL-6 (rhIL-6). The same regimen promoted proliferation, self-renewal and survival of CSC in vitro as seen by the increase in orosphere number formed in ultra-low attachment plates, and Bmi-1 expression induction in western blots. IL-6–induced signal transducer and activator of transcription 3 (STAT3) phosphorylation (indicative of stemness) was unaffected by treatment with cisplatin in HNSCC cells, whereas IL-6–induced extracellular signal-transducer kinases (ERK) phosphorylation (indicative of differentiation processes) was partially inhibited by cisplatin. Cells resistant to lower doses of cisplatin also expressed more Bmi-1. In vivo experiments corroborated in vitro findings by showing increased proportion of ALDH highCD44high cells in xenograft tumors of mice treated with cisplatin. An antibody against the receptor of IL-6 was able to revert the induction of Bmi-1 expression seen in cells treated with cisplatin plus IL-6. Taken together, these results suggest that the modulation of the epigenetic states of the cancer cell and modulation of signals provided by the niche are promising new molecular targets for the development of adjuvant therapy for cancer.

Vitamin D3 must be metabolically activated by the liver to 25-hydroxyvitamin D3 (25OHD3) and then by the kidney 1alphahydroxylase (1alphaOHase) to become 1,25dihydroxyvitamin D 3 (1,25(OH)2D3). 1,25(OH)2 D3 is a potent inhibitor of tumor growth in vitro and in vivo. Recent studies indicate that metabolic activation of 1,25(OH) 2D3 also occurs in cancer cells such as breast cancer. Consequently, the major objective of this project was to determine if tumoral 25OHD 3-1alphahydroxylase modulates any or all of the stages of breast tumor progression without inducing the hypercalcemic side effects of 1,25(OH) 2D3. For this purpose we used the PyVMT breast cancer mouse model in which the oncoprotein, polyomamiddle T antigen (PyMT) is under the control of mouse mammary tumor virus LTR (MMTV LTR). Mice exhibited tumors restricted to the mammary epithelium progressing to the various stages of breast cancer. Animals were treated with either vehicle, 25OHD3 (2000 pM/24h) or 1,25(OH)2D3 (12pM/24h). Mice treated with the vitamin D precursor, 25OHD3, exhibited a marked reduction in tumor onset and growth comparable to the 1,25(OH)2D3 treated group. Furthermore, biomarkers of tumor progression were markedly reduced in 25OHD3 and 1,25(OH)2D3 animals as compared to vehicle-treated animals. However, mean circulating calcium concentrations remained unchanged in 25OHD3 treated animals but increased significantly in 1,25(OH)2D3 treated animals as compared to controls. Tumoral levels of 1,25(OH)2D3 in mice treated with 25OHD3 were increased 79% in comparison to vehicle control mice. Additionally, 25OHD3 and 1,25(OH)2D 3 treated animals had a significant decrease in the mean number of lung metastases per animal as compared to vehicle treated control animals. This study therefore suggests an important autocrine role of 1alphaOHase expression in breast tumor cells. Furthermore, accumulation of intra-tumoral 1,25(OH) 2D3 in response to 25OHD3 administration strongly suggests that locally produced 1,25(OH)2D3 plays a significant role in restraining tumor growth without inducing the hypercalcemic side effects associated with 1,25(OH)2D3.

Les médulloblastomes (MB) sont les tumeurs cérébrales les plus fréquentes en pédiatrie. Ils apparaissent le plus souvent au niveau du cervelet. Ils peuvent être stratifiés en quatre groupes : les groupes WNT et SHH, où ces voies de signalisation sont altérées, et les groupes 3 et 4, présentant des anomalies chromosomiques et amplifications multiples, dont c-Myc (groupe 3) et N-Myc (groupe 4). L’une des altérations génétiques les plus retrouvées dans les MB est la surexpression du facteur de transcription OTX2. Ce facteur est exprimé dans les précurseurs des cellules granulaires (GCP) du cervelet, cellules d’origine de la majorité des MB. Pendant la période périnatale, les GCP subissent une phase de prolifération très intense en réponse au mitogène Sonic Hedgehog (SHH), ce qui les rendrait particulièrement sensibles à la tumorigenèse. Au cours de cette thèse, nous nous sommes intéressé à la fonction d’Otx2 dans ces GCP. Nous avons montré que l’ablation conditionnelle d’Otx2 conduit à un défaut de prolifération des ces cellules. L’analyse approfondie de ce phénotype a permis de révéler qu’Otx2 stimule la prolifération des GCP parallèlement à la voie de signalisation Shh. Par ailleurs, l’ablation d’Otx2 dans un modèle murin mimant la formation de MB Shh-dépendants a montré qu’Otx2 s’avère indispensable pour leur maintien à long terme. En parallèle, nous avons tenté de créer un nouveau modèle murin mimant la formation de MB de groupe 3 en induisant l’expression, pendant la période postnatale, d’un dominant actif de c-Myc dans les cellules exprimant Otx2. Cette approche a donné des résultats inattendus : des carcinomes de plexus choroïdes, et non des MB, ont été obtenus
Medulloblastomas (MB) are the most common brain tumors in paediatrics. They appear during development in the posterior part of the brain, mainly in cerebellum. MB can be stratified in four groups: the WNT and SHH groups, where these signalling pathways are aberrantly activated, and the groups 3 and 4, which display chromosomal abnormalities and multiple amplifications, including c-Myc (group 3) and N-Myc (group 4). One of the most frequent genetic alterations in MB is the overexpression of the Otx2 transcription factor (in 75% of cases). This factor, which is essential for central nervous system development, is expressed in granule cell precursors (GCP) of the cerebellum, which represent the cell of origin of the majority of MB. During the perinatal period, GCPs undergo intense proliferation in response to Sonic Hedgegog (SHH), making them particularly susceptible to tumorigenesis. During this thesis, we were interested in examining the function of Otx2 in GCPs. We have shown that conditional ablation of Otx2 leads to a GCP proliferation defect and that Otx2 stimulates the proliferation of these cells independently of the Shh signaling pathway. Moreover, ablation of Otx2 in a mice model of Shh-dependent medulloblastomas yielded very interesting results: while Otx2 does not seem to be required for the initiation of these tumors, it is essential for their long-term maintenance. In parallel, we tried to create a new murine model for the MB group 3 by inducing the expression, during the postnatal period, of an active dominant of c-Myc in cells expressing Otx2. This approach yielded unexpected results: choroid plexus carcinomas, instead of MB, were obtained

Le transport de substances dotées de propriétés pharmacologiques au travers de la membrane plasmique ainsi que leur accès aux divers compartiments intracellulaires, en particulier le compartiment cytoplasmique et nucléaire, demeure un obstacle pour la recherche biotechnologique et biomédicale et pour l'industrie pharmaceutique. Parmi les moyens actuellement connus pour introduire des substances dans les cellules, les peptides de translocation également dénommés CPPs (Cell- Penetrating-Peptides) qui représentent des vecteurs particulièrement intéressants. Dans le présent travail, nous avons utilisé trois CPPs, Tat, penétratine et un analogue de la maurocalcine (MCaAbu) pour la délivrance de la doxorubicine (Dox), une drogue utilisée en chimiothérapie anticancéreuse et dont son effet est limité par la résistance des cellules tumorales. Afin d'évaluer l'efficacité des trois complexes formés (Dox-CPPs), deux modèles cellulaires du cancer mammaire ont été utilisés; les cellules MDA-MB 231 et les cellules MCF7 qui présentent une sensibilité différente à la drogue seule. Notre étude nous a permis de monter en premier lieu que les trois CPPs utilisés représentent de puissants vecteurs pour l'entrée de la Dox à l'intérieur des cellules et que la conjugaison de la drogue a contourné la résistance des cellules MDA-MB 231 à la Dox. Nous avons également montré que la distribution de la Dox est plutôt nucléaire à l'état libre et cytoplasmique lorsqu'elle est couplée aux CPPs. Dans la deuxième partie de notre travail, nous avons montré que la Dox ainsi que les Dox-CPPs induisent l'apoptose des cellules MDA-MB 231 et que cet effet est observé en traitant les cellules avec une dose cinq fois plus faible de Dox-CPPs par rapport à la Dox. Cette mort est dépendante des caspases et implique la voie mitochondriale. De plus, l'apoptose induite par la Dox est médiée par les radicaux oxygénés (ROS). Ceux-ci sont en partie impliqués lors de l'apoptose induite par les Dox-CPPs puisque l'utilisation d'un inhibiteur de ROS inhibe partiellement l'apoptose induite par ces composés. Nous avons montré également que la surexpression de Bcl-2 protège l'apoptose induite par la Dox et partiellement par les Dox-CPPs, ce qui suggère qu'une autre voie est impliquée dans l'apoptose induite par les Dox-CPPs et qui expliquerait la plus forte toxicité de ces composés. Concernant cette deuxième voie nous avons pu montré que les récepteurs de mort TRAIL sont bien impliqués dans l'apoptose induite par les Dox-CPPs via la clustérisation membranaire des récepteurs de mort DR4 et DR5. Cette clustérisation modifie le taux d'expression de ces récepteurs membranaires et à l'origine d'une sensibilisation des cellules MDA-MB 231 au TRAIL endogène au cours de l'apoptose induite par les Dox-CPPs. Une telle sensibilisation au TRAIL est à l'origine de la génération de céramide, qui constitue une autre voie d'induction d'apoptose par les Dox-CPPs en plus de la voie mitochondriale. L'ensemble de ces résultats devraient nous permettre à valoriser la stratégie de couplage des CPPs à des agents antitumoraux afin d'améliorer leur effet et de mieux comprendre les voies de signalisation de la mort induite suite au couplage. Ceci pourrait à terme conduire à la conception de nouveaux analogues d'index thérapeutique plus élevé. Mots clés : Cell- Penetrating-Peptides, Doxorubicine, résistance cellulaire, apoptose, ROS, récepteurs de mort, céramide.

Normal tissues are organized in a hierarchical fashion, where rare somatic cells endowed with stem-like properties give origin to a population of differentiated cells forming the bulk of tissue. These self-maintaining cells, called stem cells, are characterized by 4 fundamental properties: longevity, multipotency, quiescence, and asymmetric cell division. We decided to develop a new in vitro system for separation of stem-like epithelial cell based not on surface marker-driven selection, but by simply taking advantage of their slow proliferation rate in the absence of serum. Dissociated colonic epithelial cells were stained with PKH26, which allows identification of distinct populations based on their proliferation rate, and cultured in vitro in the absence of serum. The cytofluorimetric expression of CK20, Msi-1 and Lgr5 was studied. The mRNA levels of several stemness-associated genes were also compared in cultured cell populations and in three colon crypt populations isolated by microdissection. A PKHpos population survived in culture and formed spheroids; this population included subsets with slow (PKHhigh) and rapid (PKHlow) replicative rates. Molecular analysis revealed higher mRNA levels of both Msi-1 and Lgr5 in PKHhigh cells; by cytofluorimetric analysis, Msi-1+/Lgr5+ cells were only found within PKHhigh cells, whereas Msi-1+/Lgr5- cells were also observed in the PKHlow population. As judged by qRT-PCR analysis, the expression of several stemness-associated markers (Bmi-1, EphB2, EpCAM, ALDH1) was highly enriched in Msi-1+/Lgr5+ cells. While CK20 expression was mainly found in PKHlow and PKHneg cells, a small PKHhigh subset co-expressed both CK20 and Msi-1, but not Lgr5; cells with these properties also expressed Mucin-1, and could be identified in vivo in colon crypts. These results mirrored those found in cells isolated from different crypt portions by microdissection, and based on proliferation rates and marker expression they allowed to define a hierarchy of cellular subsets at different maturation stages: PKHhigh/Lgr5+/Msi-1+/CK20-, PKHhigh/Lgr5-/Msi-1+/CK20+, PKHlow/Lgr5-/Msi-1+/Ck20-, and PKHlow/ Lgr5-/Msi-1-/ CK20+ cells. We next addressed the issue of cancer stem cells in colorectal cancer (CRC). Isolated cells from five primary colon tumors and 15 CRC metastatic samples were stained with PKH26 and cultured in PhEMA-treated plate in the absence of serum. As in normal samples, in tumors we identified a slowly cycling population able to persist in vitro in serum-free condition. On the contrary, in metastatic samples we could not document an in vitro selection of a PKHhigh population; actually PKHlow cells seemed to remain constant over several weeks. Cytofluorimetric analysis revealed that, at variance with normal samples, in metastatic samples Lgr5+/Msi-1+ cells were not only present in the slowly cycling PKHhigh population but also in PKHlow cells. No Lgr5 or Msi-1 expression was recorded in PKHneg cells. Both PKHhigh and PKHlow cells were able to form spheroids; in the presence of 10% FCS in uncoated plates they adhered to the plate and differentiated assuming an epithelial morphology. They eventually lost Msi-1 and Lgr5 expression after 14 days of culture in the presence of serum and adhesion conditions, and they also acquired CK20 expression. Finally, we investigated the possible involvement of Notch signaling on regulation of Msi-1 levels in the metastatic cell line MICOL-14tum and primary metastatic samples. Seventy two hours of treatment with the Notch ligand DLL4 increased Msi-1 mRNA and protein levels. This phenomenon was largely prevented by the addition of an antibody neutralizing Notch2/3, whereas no reduction was detected after incubation with anti-Notch1. Since Msi-1 regulates Numb levels, its reduction led to increased Numb protein, as demonstrated by Western Blot analysis of MICOL-14tum and primary metastatic cells. Furthermore, after four days of treatment, formation of spheroids was significantly reduced by incubation with anti-Notch2/3 antibody in primary samples as well as in MICOL-14tum cells. In conclusion, our data showed the possibility of deriving in vitro from normal colon tissue, without any selection strategy, an array of cellular subsets differentially expressing the most common stemness and differentiation markers of human colon epithelial cells, which recapitulate the phenotypic and molecular profile of cells in an anatomical position compatible with stem-like cells. Furthermore in metastatic cell line and primary samples we described a novel feed-forward circuit involving Msi-1, Numb, Notch3 and Notch1. In particular, we demonstrated that Msi-1 could be considered a target of Notch3, and Notch3 could act as a positive regulator of Notch1 levels through Msi-1/Numb circuit.
I tessuti normali non patologici sono organizzati in una struttura gerarchica all'interno della quale un numero esiguo di cellule origina l'intera totalità della popolazione. Queste cellule presentano longevità, multipotenza, quiescenza e capacità di dividersi in maniera asimmetrica, tutte caratteristiche di staminalità. Abbiamo deciso di sviluppare un sistema in vitro per separare le cellule staminali epiteliali del colon, non basandoci sull'espressione di specifici marcatori di superficie, ma semplicemente traendo vantaggio dal ridotto tasso di proliferazione che queste cellule presentano quando tenute in assenza di siero. Da campioni primari di colon le cellule epiteliali sono state dissociate, marcate con PKH26, che permette l'identificazione di distinte sottopopolazioni basandosi sul tasso di proliferazione, e coltivate in vitro in assenza di siero. Sono stati analizzati i livelli di espressione proteica di CK20, Msi-1 e Lgr5 e di mRNA di alcuni geni associati alla staminalità. L'espressione di questi geni è stata confrontata tra la popolazione cellulare in cultura e tre sottopopolazioni isolate dalle cripte di colon attraverso microdissezione laser. In coltura abbiamo identificato una popolazione PKHpos, in grado di sopravvivere e formare sferoidi, comprendente cellule con un ridotto tasso di proliferazione (PKHhigh) e cellule con rapido tasso (PKHlow). L'analisi citofluorimetrica ha dimostrato che cellule Msi-1+/Lgr5+ si trovano solo nella popolazione PKHhigh, mentre cellule Msi-1+/Lgr5- sono presenti anche nella popolazione PKHlow. Questi risultati sono stati supportati dall'analisi molecolare nelle diverse sottopopolazioni. Nelle cellule Msi-1+/Lgr5+ abbiamo, infatti, riscontrato livelli più alti di espressione di geni associati con la staminalità (Bmi-1, EphB2, EpCAM, ALDH1). Invece l'espressione di citocheratina (CK20) è quasi totalmente ristretta alle cellule PKHlow e PKHneg, le quali esprimono anche Mucin-1 ma non Lgr5. Questi risultati rispecchiano quelli trovati analizzando le popolazioni isolate dalle diverse porzioni delle cripte attraverso microdissezione, e ci hanno permesso di definire una gerarchia di sottopopolazioni cellulari diverse in differenti fasi di maturazione: PKHhigh/Lgr5+/Msi-1+/CK20-, PKHhigh/Lgr5-/Msi-1+/CK20+, PKHlow/Lgr5-/Msi-1+/Ck20-, e PKHlow/ Lgr5-/Msi-1-/ CK20+. Per valutare la presenza di diverse sottopopolazioni con caratteristiche di staminalità anche nel tessuto tumorale, da 5 campioni di tumore primario colorettale e da 15 di metastasi, abbiamo isolato le cellule, marcate con PKH26 e mantenute in assenza di siero. Come per i campioni normali, nei tumori abbiamo identificato una popolazione cellulare poco proliferante e in grado di sopravvivere in vitro in assenza di siero. Invece, nei campioni metastatici non siamo stati in grado di definire chiaramente una popolazione PKHhigh; sembra, infatti, che siano le cellule PKHlow in grado di sopravvivere più a lungo in cultura e quindi selezionarsi. L'analisi citofluorimetrica ha dimostrato che, a differenza dei campioni normali, in quelli metastatici cellule Lgr5+/Msi-1+ non sono presenti solo nella popolazione PKHhigh, ma anche all'interno della sottopopolazione PKHlow. Nelle cellule PKHneg invece non si riscontra l'espressione nè di Lgr5 nè di Msi-1. Entrambe le popolazioni PKHhigh e PKHlow sono state in grado di formare sferoidi e, quando mantenute in presenza del 10% di siero, hanno aderito alle piastre differenziandosi ed assumendo una morfologia epiteliale. Queste cellule perdono conseguentemente l'espressione di Msi-1 e Lgr5 solo dopo 14 giorni in queste condizioni, mentre acquistano l'espressione di CK20. Vista la ridotta presenza di dati inerenti la regolazione di Msi-1, abbiamo infine indagato il possibile coinvolgimento del pathway di Notch sulla regolazione di Msi-1 nelle cellule di tumore del coloretto. Dopo 72 ore di trattamento con il ligando di Notch (DLL4) i livelli di proteina e mRNA di Msi-1 aumentano. Questo fenomeno è bloccato dall'aggiunta di un anticorpo neutralizzante Notch2/3, mentre nessuna variazione è stata riscontrata dopo il trattamento con anti-Notch1. Poiché Msi-1 regola i livelli di Numb, la sua diminuzione comporta un aumento dei livelli proteici di Numb, come riscontrato dall'analisi Western Blot effettuata nei campioni tumorali primari e nella linea cellulare MICOL-14tum. Inoltre, dopo quattro giorni di trattamento, la formazione di sferoidi appare significativamente ridotta dal trattamento con anti-Notch2/3. In conclusione, i nostri dati dimostrano la possibilità di derivare in vitro da campioni di colon normale, senza alcuna strategia selettiva, delle sottopopolazioni in differenti fasi di maturazione. Inoltre, nel tessuto metastatico siamo stati in grado di descrivere un possibile circuito di regolazione che coinvolge Msi-1, Numb, Notch3 e Notch1. In particolare, abbiamo dimostrato che Msi-1 può esser considerato un target di Notch3, e Notch3 può agire come un regolatore positivo di Notch1 attraverso il circuito Msi-1/Numb.

Les Cancers Epidermoïdes de la Tête et du Cou (CETC) représentent les septièmes tumeurs les plus fréquentes au niveau mondial en 2020. Si les perspectives thérapeutiques ont évolué favorablement ces dix dernières années, la recherche de biomarqueurs utiles pour la stratification thérapeutique reste un enjeu de taille pour ces tumeurs. En outre, l'induction d'une immunité adaptative antitumorale apparait comme un mécanisme de plus en plus important pour l'efficacité des thérapeutiques approuvées dans les CETC. Dans cette perspective, l'étude des interactions entre cellules tumorales et cellules immunitaires du microenvironnement tumoral (MT) est incontournable. Plusieurs thérapies anticancéreuses ont été rapportées pour induire la mort des cellules tumorales par ferroptose, une nécrose régulée dépendante du fer survenant lors d'un stress oxydant non résolu par la cellule. Nous nous sommes intéressés dans la première partie de nos travaux à la possibilité d'exploiter la ferroptose dans les CETC positifs au Papillomavirus Humain (CETC HPV+ve), les CETC HPV+ve montrant habituellement un état de stress oxydant supérieur aux CETC négatifs pour l'HPV (CETC HPV-ve). Nous avons mis en évidence une expression faible de SLC7A11, transporteur essentiel de la cellule pour la synthèse du glutathion inhibiteur du stress oxydant, dans les CETC HPV+ve par rapport aux CETC HPV-ve in silico. In vitro, nous avons mis en évidence une vulnérabilité supérieure à la ferroptose induite par l'érastine dans les lignées de CETC exprimant les protéines transformantes E6/E7 de l’HPV par rapport à leurs lignées parentales. Dans la deuxième partie de nos travaux, nous avons étudié la régulation de l'expression du ligand PD-L1 (Programmed Death-Ligand 1) par différentes thérapies approuvées dans les CETC. L'expression de PD-L1 par les cellules tumorales et inflammatoires du MT est en effet responsable en partie de l'inactivation fréquente des cellules T cytotoxiques du MT, et représente donc un obstacle pour l'induction de l'immunité adaptative antitumorale. Nous avons mis en évidence une régulation spécifique de PD-L1 par le 5-fluorouracile (5-FU) dépendante de la réponse aux dommages de l'ADN ainsi que de la voie JAK / STAT
Head and Neck Squamous Cell Carcinomas (HNSCC) were the seventh most frequent tumors worldwide in 2020. Although therapeutic perspectives have evolved favorably over the last ten years, the search for useful biomarkers for therapeutic stratification remains a major challenge for these tumors. In addition, the induction of antitumor adaptive immunity seems to be an increasingly important mechanism for the efficacy of approved therapies in HNSCC. In this perspective, the study of the interactions between tumor cells and immune cells of the tumor microenvironment (TM) is essential. Several anticancer therapies have been reported for the induction of tumor cell death by ferroptosis, an iron-dependent regulated necrosis that occurs during unresolved oxidative stress in the cell. In the first part of our work we focused on the possibility of exploiting ferroptosis in Human Papillomavirus positive HNSCC (HPV+ve HNSCC), which usually show higher oxidative stress than HPV negative HNSCC (HPV-ve HNSCC). Using in silico analyses, we observed low expression of SLC7A11, an essential transporter in the cell for the synthesis of glutathione, an inhibitor of oxidative stress, in HPV+ve HNSCC compared to HPV-ve HNSCC. In vitro, HNSCC cell lines expressing HPV E6/E7 transforming proteins showed higher sensitivity to erastin-induced ferroptosis compared to their parental cell lines. In the second part of our work, we studied the regulation of PD-L1 (Programmed Death-Ligand 1) expression by different approved therapies in HNSCC. The expression of PD-L1 by tumor and inflammatory cells of the TM is indeed partly responsible for the frequent inactivation of cytotoxic T cells of the TM, and thus represents an obstacle for the induction of antitumor adaptive immunity. We observed a specific regulation of PD-L1 by 5-fluorouracil (5-FU) that was dependent on the DNA damage response and the JAK / STAT pathway

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Universidade Federal de Minas Gerais
Tumor spreading occurs mainly by two pathways: through blood vessels and by lymphatic vessels, but the last is preferred by breast tumor cells. Some proteins are involved in cell adhesion and proteolysis, causing metastasis, such as ADAMs, a family of multi-domain and multi- functional proteins that contribute in these processes. ADAM9, a member of this family, has been increased in a large number of human carcinomas, including, breast cancer. In this context, the aim of this study was to evaluate the role of ADAM9 in tumor spreading via blood and lymphatic systems, in the search for new targets and focusing the development of new therapeutical tools. Therefore, MDA-MB-231 breast tumor cells were silenced for ADAM9 and tested with respect to their adhesive and invasive activity against blood and lymphatic endothelium. Our results showed that ADAM9 silencing in MDA-MB-231 breast cancer cells inhibited the invasion of this cells in matrigel (71.51 ± 8.02%) when compared to control cells, without affecting cell adhesion, proliferation, migration, and gene expression of the ADAM10, ADAM12, ADAM-17, cMyc, MMP9, VEGF-A, VEGF-C, Osteopontin and Collagen XVII, however, there was a decrease in the expression of the ADAM15 and increased expression of MMP2 when compared to controls. Furthermore, ADAM9 silencing did not affect the adhesion under flow to these vascular endothelial cells (HMEC-1 and HUVEC) and lymphatic (HMVEC-dLyNeo-Der). However, there was a decrease in the rate of trans-endothelial migration through the monolayer endothelial cells (HUVEC, HMEC-1 and HMVEC-dLyNeo-Der) by approximately 50%, 40% and 32%, respectively. In conclusion, ADAM9 showed to be essential in invasion and extravasation of MDA-MB-231 breast cancer cells through the blood and lymphatic vessels in vitro.
A disseminação tumoral ocorre principalmente por duas vias: por vasos sanguíneos e por vasos linfáticos, sendo esta última preferida pelos tumores mamários. Algumas proteínas estão envolvidas na proteólise e na adesão celular, ocasionando metástase, tais como as ADAMs, uma família de proteínas multi-domínios e multi- funcionais que contribuem nesses processos. A ADAM9, um membro desta família, apresenta expressão aumentada em um grande número de carcinomas humanos, entre eles, mama. Nesse contexto, o objetivo desse estudo foi avaliar o papel da ADAM9 na disseminação tumoral via sistema sanguíneo e linfático, visando o desenvolvimento de novas ferramentas terapêuticas. Para tanto, células de tumor de mama MDA-MB-231 foram silenciadas para a ADAM9 e testadas com relação às suas atividades adesivas e invasivas frente ao endotélio sanguíneo e linfático. Nossos resultados mostraram que o siADAM9 inibiu a invasão das células de câncer de mama MDAMB- 231 em matrigel (71,51 ± 8,02%) quando comparado com os controles, sem afetar a adesão celular, proliferação, migração, e expressão gênica da ADAM10, ADAM12, ADAM17, cMYC, MMP9, VEGF-A, VEGF-C, Osteopontina e Colágeno XVII, entretanto houve uma diminuição da expressão da ADAM15 e um aumento da expressão da MMP2 quando comparado com as controles: meio e negativo. O siADAM9 nas células MDA-MB- 231 não afetou sua adesão sob fluxo às endoteliais vasculares (HMEC-1 e HUVEC) e linfáticas (HMVEC-dLyNeo-Der). Entretanto, houve uma diminuição na taxa de transmigração através da monocamada das células endoteliais (HUVEC, HMEC-1 e HMVEC-dLyNeo-Der) em aproximadamente 50%, 40% e 32%, respectivamente. Assim, conclui- se que a ADAM9 mostrou-se essencial no processo de invasão e extravasamento das células de câncer de mama MDA-MB-231 pelos vasos sanguíneos e linfáticos in vitro.

O carcinoma espinocelular (CEC) é um dos cânceres humanos mais incidentes. A despeito do entendimento da fisiopatologia do CEC, as opções terapêuticas ainda são limitadas e o(s) exato(s) mecanismo(s) envolvido(s) na progressão deste tipo de tumor ainda não foi descrito. Estudos recentes mostram a existência de uma associação direta entre a resposta imune TH1 e um melhor prognóstico em pacientes com CEC. Aumento da expressão de componentes do eixo IL-33/ST2 foi demonstrado contribuir para transformação neoplásica em diversos modelos tumorais, incluindo cânceres de estômago e de mama. Trabalho recente do nosso e de outros laboratórios indicam que IL-33 pode impedir a resposta imune TH1 . Baseado nessas observações, a hipótese testada foi que o impedimento da resposta imune pela interação IL-33/ST2 pode contribuir para iniciação e progressão do CEC. Utilizando modelo de carcinogênese química em camundongos WT e deficientes de ST2 (ST2KO), os resultados mostram que a deficiência de ST2 leva a uma notável redução da severidade das lesões 20 semanas após a carcinogênese química, sugerindo que a sinalização ST2 é necessária para o desenvolvimento tumoral neste modelo. Análises do infiltrado inflamatório presente nas lesões em camundongos WT e ST2KO revelaram redução significativa nas percentagens de macrófagos, células T CD4+ e células dendríticas, mas não em células T CD8+, células B e células natural killer (NK) no microambiente tumoral de camundongos ST2KO. Além disso, células NK esplênicas isoladas de camundongos ST2KO exibiram atividade citotóxica aumentada contra células YAC quando comparado com células de camundongos do grupo controle (WT). Os resultados indicam que a via IL-33/ST2 contribui para o desenvolvimento de carcinoma espinocelular recrutando células T CD4+, macrófagos e células dendríticas e reduzindo a citotoxicidade de células NK.
Squamous cell carcinoma (SCC) is the second most common form of skin cancer and is most commonly observed in photo-exposed areas of the body. The mechanism(s) involved in the progression of this tumor are unknown. Recent studies have shown that there is a direct association between a TH1-related immune response and a better prognosis in patients with SCC. Increased expression of the IL33/ST2 axis components has been demonstrated to contribute to neoplastic transformation in several tumor models, including gastric and breast cancer. Recent work from ours and other laboratories indicate that can IL-33 impair TH1-type immune responses. Based on these observations, we hypothesized that TH1-type immune response impairment by IL33/ST2 could contribute to the initiation and progress SCC. We found that ST2 deficiency led to a marked decreased in severity of skin lesions at 20 weeks post-DMBA, suggesting that ST2 signaling is necessary for tumor development in this model. Analysis of tumor lesions in WT and ST2KO mice revealed that lack of ST2 led to a specific and significant reduction in the frequency of macrophages, T CD4+ and dendritic cells, but not CD8+, B and NK cells. In addition, splenic NK cells isolated from DMBA-treated ST2KO mice exhibited increased cytotoxicity activity against YAC cells targets when compared with WT splenic NK cells in the same cytotoxic assay. Altogether, our findings indicate that IL-33/ST2 pathway contributes to the SCC development by recruitment T CD4+ cells, macrophages, and dendritic cells and impairing NK cytotoxicity.

A proteína moesina, uma das proteínas do complexo ERM (ezrina, radixina e moesina), participa do processo de migração de células tumorais controlando a ligação entre o citoesqueleto de actina e os receptores transmembrana. As proteínas ERM vêm sendo investigadas como ligantes de outras glicoproteínas, como a podoplanina, cuja expressão é encontrada em células malignas de diversas neoplasias, incluindo o carcinoma espinocelular (CEC) de boca. O objetivo desse estudo foi avaliar as expressões imuno-histoquímicas da moesina e da podoplanina pelas células malignas no front de invasão de 84 pacientes com CEC de boca e suas associações com a evolução clínica e com o prognóstico dos pacientes. A associação entre a expressão imuno-histoquímica da moesina e da podoplanina pelas células malignas e as variáveis demográficas, clínicas e microscópicas foi avaliada pelo teste qui-quadrado ou teste exato de Fisher. As análises de sobrevida global e livre de doença em 5 e 10 anos foram calculadas pelo estimulador produto-limite de Kaplan-Meier e a comparação das curvas de sobrevida realizada pelo teste de log-rank. Os resultados mostraram que houve expressão da moesina pelas células malignas na região do front de invasão tumoral, entretanto, nenhuma associação estatisticamente significativa foi encontrada entre esta proteína e as características clínicas, demográficas e microscópicas. A expressão da podoplanina, pelas células malignas, foi significativamente associada à radioterapia (p=0,004), à invasão muscular (p=0,006) e ao comprometimento linfonodal (p=0,013). Não houve associação significativa entre a expressão das duas proteínas nos CECs de boca (p=0,460). A forte expressão da moesina pelas células malignas constituiu um fator de prognóstico desfavorável para os pacientes com CEC de boca e estadiamento clínico II e III. O comprometimento linfonodal histopatológico também se mostrou fator de prognóstico significativo para a recidiva da doença (p=0,018). Estes resultados sugerem que a expressão de moesina, pelas células malignas juntamente com o comprometimento linfonodal pode contribuir para determinar os pacientes com CEC de boca que apresentam um pior prognóstico. Além disso, verificamos que as proteínas moesina e podoplanina se expressam pelas células neoplásicas nos CEC de boca mas não parecem estar associadas no processo de invasão tumoral.
The moesin protein, one of the proteins of the ERM complex (ezrin, radixin and moesin) takes part in the migration of tumor cells process by controlling the relation between actin cytoskeleton and transmembrane receptors. The ERM proteins have been investigated as ligants of other glycoproteins, such as podoplanin, which are found in malignant cells of malignant, including oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the immunohistochemical expressions of moesin and podoplanin by malignant cells in the invasive front of 84 patients with oral squamous carcinoma and its association with clinical outcome and patients\' prognosis. Chi- square or Fisher\'s exact test was used to analyze the association between the moesin and podoplanin expressions by malignant cells and demographic, clinical and microscopic variables in oral squamous cell carcinoma patients. The 5 and 10 years survival rates were calculated by Kaplan-Meier method and the comparison of survival curves were performed using log-rank test. The results showed that there was moesin expression by malignant cells in the invasive front, however, no statistically significant association was found between this protein and demographic, clinical and microscopic features. The expression of podoplanin by malignant cells was significantly associated with radiotherapy (p=0.004), with muscular invasion (p=0.006) and lymph node involvement (p=0.013). There was no significant association between the expression of two proteins in OSCC (p=0.460). The strong expression of moesin by malignant cells was a factor of unfavorable prognosis for patients with OSCC and clinical stage II and III. The histopathological lymph node involvement was also significant prognostic factor for disease recurrence (p=0.018). These results suggest that the expression of moesin by malignant cells and lymph node involvement may help to determine patients with squamous cell carcinoma who have a poor prognosis. Furthermore, we found that moesin and podoplanin proteins are expressed by neoplastic cells in oral squamous cell carcinoma but not appear to be associated in the process of tumor invasion.

Les médulloblastomes (MB) sont les tumeurs cérébrales les plus fréquentes en pédiatrie. Ils apparaissent le plus souvent au niveau du cervelet. Ils peuvent être stratifiés en quatre groupes : les groupes WNT et SHH, où ces voies de signalisation sont altérées, et les groupes 3 et 4, présentant des anomalies chromosomiques et amplifications multiples, dont c-Myc (groupe 3) et N-Myc (groupe 4). L’une des altérations génétiques les plus retrouvées dans les MB est la surexpression du facteur de transcription OTX2. Ce facteur est exprimé dans les précurseurs des cellules granulaires (GCP) du cervelet, cellules d’origine de la majorité des MB. Pendant la période périnatale, les GCP subissent une phase de prolifération très intense en réponse au mitogène Sonic Hedgehog (SHH), ce qui les rendrait particulièrement sensibles à la tumorigenèse. Au cours de cette thèse, nous nous sommes intéressé à la fonction d’Otx2 dans ces GCP. Nous avons montré que l’ablation conditionnelle d’Otx2 conduit à un défaut de prolifération des ces cellules. L’analyse approfondie de ce phénotype a permis de révéler qu’Otx2 stimule la prolifération des GCP parallèlement à la voie de signalisation Shh. Par ailleurs, l’ablation d’Otx2 dans un modèle murin mimant la formation de MB Shh-dépendants a montré qu’Otx2 s’avère indispensable pour leur maintien à long terme. En parallèle, nous avons tenté de créer un nouveau modèle murin mimant la formation de MB de groupe 3 en induisant l’expression, pendant la période postnatale, d’un dominant actif de c-Myc dans les cellules exprimant Otx2. Cette approche a donné des résultats inattendus : des carcinomes de plexus choroïdes, et non des MB, ont été obtenus
Medulloblastomas (MB) are the most common brain tumors in paediatrics. They appear during development in the posterior part of the brain, mainly in cerebellum. MB can be stratified in four groups: the WNT and SHH groups, where these signalling pathways are aberrantly activated, and the groups 3 and 4, which display chromosomal abnormalities and multiple amplifications, including c-Myc (group 3) and N-Myc (group 4). One of the most frequent genetic alterations in MB is the overexpression of the Otx2 transcription factor (in 75% of cases). This factor, which is essential for central nervous system development, is expressed in granule cell precursors (GCP) of the cerebellum, which represent the cell of origin of the majority of MB. During the perinatal period, GCPs undergo intense proliferation in response to Sonic Hedgegog (SHH), making them particularly susceptible to tumorigenesis. During this thesis, we were interested in examining the function of Otx2 in GCPs. We have shown that conditional ablation of Otx2 leads to a GCP proliferation defect and that Otx2 stimulates the proliferation of these cells independently of the Shh signaling pathway. Moreover, ablation of Otx2 in a mice model of Shh-dependent medulloblastomas yielded very interesting results: while Otx2 does not seem to be required for the initiation of these tumors, it is essential for their long-term maintenance. In parallel, we tried to create a new murine model for the MB group 3 by inducing the expression, during the postnatal period, of an active dominant of c-Myc in cells expressing Otx2. This approach yielded unexpected results: choroid plexus carcinomas, instead of MB, were obtained

Introdução: Um dos principais obstáculos para compreensão dos eventos biológicos envolvidos no câncer é a falta de modelos adequados para o estudo in vitro em especial em relação ao câncer de próstata (CaP) e ao câncer de bexiga (CaB). Há um número limitado de linhagens celulares de CaP e de CaB sendo a maioria proveniente de tumores invasivos e metastáticos. Sabe-se ainda, que existem diferenças étnicas entre as populações quanto ao comportamento de neoplasias. Desta forma, a pesquisa baseada em linhagens de uma população homogênea seria fonte de resultados limitados, não contemplando a diversidade que sabidamente ocorre entre os diferentes grupos. Além desse aspecto, as linhagens celulares comerciais são na sua maioria adquiridas na Coleção Americana de Culturas de Tecido (ATCC, do inglês American Tissue Cell Culture) que apesar de serem bem padronizadas, requerem processos de importação com aumento do custo e demandas burocráticas que dificultam a pesquisa. Portanto, consideramos vital para a compreensão dos fenômenos relacionados à carcinogênese, assim como estudos de resistência a drogas, quimioprevenção e novas estratégias terapêuticas, o desenvolvimento de linhagens tumorais derivadas de tumores primários que acometem a nossa população, peculiarmente miscigenada. No presente trabalho, fragmentos de carcinoma urotelial da bexiga e de adenocarcinoma da próstata foram obtidos durante cirurgia para remoção de tumores primários de pacientes tratados e acompanhados na Divisão de Urologia da Faculdade de Medicina da Universidade de São Paulo (FMUSP) e no Hospital Sírio Libanês. As linhagens estabelecidas a partir destes fragmentos foram caracterizadas através da análise da cinética de crescimento, análises imunocitoquímicas e anormalidades cromossômicas incluindo cariótipo e hibridização in situ por fluorescência (FISH). Além disso, as linhagens obtidas foram submetidas a estudos de quimiossensibilidade com o uso dos compostos curcumin e Prima-1. Avaliamos ainda, a tumorigenicidade de nossas linhagens em camundongos atímicos. Os resultados deste trabalho demonstram o desenvolvimento de três linhagens de CaB e três linhagens de CaP sendo as mesmas não tumorigênicas em camundongos atímicos. Além disso, demonstramos que o curcumin na concentração de 50 M induziu morte celular em todas as linhagens estudadas, sendo seu efeito mais evidente nas linhagens de CaP. Por fim, Prima-1 reduziu a viabilidade celular independente do status de p53 nas linhagens de CaB
Introduction: One of the main obstacles for understanding biological events involved in cancer is the lack of appropriated models for in vitro studies especially for prostate cancer (PC) and bladder cancer (BC). There are a limited number of PC and BC cell lines being the majority originated from metastatic and invasive tumors. Also it is well known that there are ethnic differences between populations concerning the behavior of tumors. In such a way, the research based on cell lines derived from a homogenous population should be source of limited results, not contemplating the diversity known to occur among different groups. In addition the commercial cell lines are generally acquired at American Tissue Cell Culture (ATCC) that although wellestablished requires importation processes with cost increase and bureaucratic demands that difficult the research. Therefore we consider vital to the comprehension of the carcinogenesis phenomena, as well as drug resistance studies, chemoprevention and new therapeutic strategies, the development of tumor lineages derived from primary tumors that assail our miscigenated population. At the present work, fragments of bladder urothelial carcinoma and prostate adenocarcinoma were obtained by surgical resection of primary tumors from patients treated and followed in the Division of Urology of the Clinical Hospital of the São Paulo University (FMUSP) and Syrian Lebanese Hospital. The cell lines established from these fragments were characterized through growth kinetic, immunocytochemistry and chromosome abnormalities including karyotyping and Fluorescence in situ hybridization (FISH). Moreover, the cell lines were submitted to chemosensitivity studies using curcumin and Prima-1 and analyzed regarding their tumorigenicity in athymic mice. The results of this work show the development of three BC and three PC cell lines that were not tumorigenic in athymic mice. Curcumin at 50 M concentration induced cell death in all studied lineages, being more effective in PC cell lines. Finally, PRIMA-1 reduced the cellular viability independent of the p53 status in BC cell lines

Les sarcomes pléomorphes sont caractérisés par un nombre important de réarrangements chromosomiques et un génome complexe dans lequel peu d'altérations récurrentes ont été identifiées. La plupart des études se sont intéressées aux conséquences de ces altérations sur la cellule et le patient, cependant leur origine reste majoritairement inconnue. La fusion cellulaire entre deux cellules cancéreuses est un mécanisme décrit comme étant à l'origine d'aneuploïdie et d'instabilité chromosomique. Ce processus a été avant tout identifié dans le cadre de la fécondation, de la formation des fibres musculaires et des ostéoclastes et de la régénération tissulaire. La fusion cellulaire, détournée au profit de l'oncogenèse, favorise la progression métastatique et la résistance aux traitements. Nous avons alors émis l'hypothèse que ce mécanisme puisse être à l'origine de la complexité génomique des sarcomes pléomorphes et favoriser le développement et la progression de ces tumeurs. Les résultats obtenus au cours de ma thèse montrent, tout d'abord, que la fusion cellulaire permet le regroupement des oncogènes présents dans deux cellules différentes, initiant ainsi développement de tumeurs. Les hybrides générés présentent de nombreux réarrangements génomiques, avec des pertes et des gains de chromosomes correspondant à ceux retrouvés dans les sarcomes pléomorphes humains. Nous avons aussi démontré que ce mécanisme induit la progression tumorale après fusion d'un fibroblaste immortalisé et d'un fibroblaste transformé. Enfin, nous avons démontré que la fusion non contrôlée de deux myoblastes immortalisés conduit au développement de rhabdomyosarcomes pléomorphes, présentant une délétion de DMD, retrouvée aussi dans les tumeurs humaines à différenciation myogénique. Cette délétion impacte seulement les isoformes les plus grandes de la dystrophine, déclenchant une relocalisation des plus petites dans le noyau. L'ensemble de mes travaux de thèse permettent alors de proposer un modèle selon lequel l'oncogenèse des sarcomes pléomorphes passe par des évènements de fusion cellulaire non-contrôlée, induisant le développement d'altérations génomiques spécifiques de la cellule d'origine et sélectionnées en fonction des pressions de sélection exercées par l'environnement. Ce processus oncogène conduit alors au développement de tumeurs et à la progression métastatique des sarcomes pléomorphes
Pleomorphic sarcomas are characterized by several chromosomic rearrangements and a complex genome with few recurrent alterations identified. Most studies have looked at the consequences of those alterations on the cell and the patient, however their origin remains largely unknown. Cell fusion between two cancer cells is a mechanism described as the origin of aneuploidy and chromosomal instability. This process is mainly involved during fecundation, formation of muscle fibers and osteoclasts and tissue regeneration. Cell fusion, hijacked by oncogenesis, drives to metastatic dissemination and drug resistance. That is why we hypothesize this mechanism could be the origin of genomic complexity of pleomorphic sarcomas and induces the development and progression of those tumors. The results obtained during my thesis show, first of all, cellular fusion allows merging of oncogenes present in two different cells, which initiates tumor development. Hybrid tumors harbor many genomic rearrangements, with chromosome losses and gains comparable to those find in human sarcomas. We also demonstrate that this mechanism leads to tumor progression after the fusion of an immortalized fibroblast and a transformed fibroblast. Finally, uncontrolled cell fusion of two immortalized myoblasts drives pleomorphic sarcoma development, with DMD deletion, also found in human myogenic sarcoma. This deletion only affects the largest isoforms of dystrophin, setting off the relocation of the smallest one in the nucleus. All of my thesis results allow me to propose a model where pleomorphic sarcoma oncogenesis is initiated by an uncontrolled cell fusion process, leading to genomic alterations, specific of cell origin and modeled by selective pressure waves which are controlled by the cell environment. This oncogenic process leads to the development of tumors and metastatic progression of pleomorphic sarcomas