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.

Le cancer de l’ovaire, avec plus de 15.000 décès prévus en 2012, est le cancer gynécologique le plus meurtrier. Alors qu'environ 80% des patientes qui répondent à une chimiothérapie de première ligne, plus de 60% des patientes vont récidiver et seulement 44% seront encore en vie après 5 ans. Le rôle majeur du micro-environnement dans les processus de la carcinogénèse et la progression tumorale a été démontré par de nombreux travaux. Ce concept original de l’initiation et de la progression tumorale fait appel à des approches conceptuelles et expérimentales très diverses. Dans cette étude, nous avons pu démontrer le rôle important de la molécule de tolérance HLA-G (Human Leukocyte Antigene-G), ainsi que son expression et sa régulation par les cellules cancéreuses et les cellules du micro-environnement tumoral. Nous avons étudié les différents facteurs impliqués dans les mécanismes d’échappement tumoral et vérifié in vivo certains protocoles de chimiothérapie à base de médicaments immunomodulateurs. / With more than 15,000 deaths anticipated in 2012, ovarian cancer is the most deadly gynecologic malignancy. While approximately 80% of patients will respond to frontline chemotherapy, more than 60% of patients will experience disease recurrence and only 44% will be alive at 5 years. The role of the microenvironment in the process of carcinogenesis and tumor progression has been demonstrated in various studies. This original concept of initiation and tumor progression solicits a very varied conceptual and experimental approach. In this study, we demonstrate the important role of the immunosuppressive molecule HLA-G (Human Leukocyte Antigen-G), and its expression and regulation by cancer cells and tumor microenvironment cells. We studied the various factors involved in the mechanisms immune of tumor escape from the immune system and finally we analyse in vivo some chemotherapy protocols based on the immunomodulatory drugs.

Les lymphocytes « Natural Killer » (NK) sont des effecteurs de l’immunité innée, capables de lyser les cellules cancéreuses grâce au relargage de la protéase cytotoxique Granzyme B (GzmB). Récemment, de nouvelles stratégies anti-cancéreuses, basées sur l’utilisation des cellules NK, ont émergé et se sont révélées très prometteuses. Il est maintenant clairement établi que le microenvironnement tumoral hypoxique influence la réponse immunitaire et constitue, de ce fait, un obstacle majeur pour établir des protocoles d’immunothérapies efficaces. Des études récentes ont montré que l’autophagie est un régulateur important de l’immunité innée dans le microenvironnement tumoral, mais les mécanismes de régulation impliqués restent encore peu connus. Nous avons montré in vitro que l'hypoxie diminue la sensibilité des cellules de carcinome mammaire à la lyse dépendante des cellules NK par un mécanisme impliquant l'activation de l'autophagie. De manière intéressante, cette diminution de lyse est reversée par l’inhibition de l’autophagie. Nous avons démontré que la résistance des cellules tumorales hypoxiques à la lyse par les cellules NK n'est liée ni à un défaut de reconnaissance des cellules cibles, ni à une altération de l’activité cytotoxique des effecteurs. Nous avons mis en évidence que l'activation de l’autophagie conduit à la dégradation de GzmB dans les lysosomes des cellules hypoxiques. Ainsi, ces cellules deviennent résistantes à l’apoptose, qui est normalement induite par GzmB, transféré par les cellules NK. L’invalidation génétique et pharmacologique de l'autophagie permet de restaurer le niveau intracellulaire de GzmB et réduit la résistance des cellules cibles hypoxiques in vitro. Nos résultats mettent en évidence que l'autophagie est un régulateur primordial de la réponse immunitaire anti-tumorale dépendante des cellules NK. Nous avons validé ce concept in vivo en montrant que l’inhibition de l'autophagie favorise de manière significative la prise en charge de la tumeur par les cellules NK dans des modèles murins de mélanome et de carcinome mammaire. Cette étude contribue à l’avancé des connaissances sur la manière dont l'autophagie, induite par l'hypoxie, affecte la lyse dépendante des cellules NK et ouvre la voie à la formulation de nouvelles stratégies thérapeutiques anti-tumorales combinant l’utilisation des cellules NK à des inhibiteurs d'autophagie. / Natural killer (NK) cells are effectors of the antitumor immunity, able to kill cancer cells through the release of the cytotoxic protease granzyme B. NK-based therapies have recently emerged as promising anticancer strategies. However, it is well established that hypoxic microenvironment interferes with the function of antitumor immune cells and constitutes a major obstacle for cancer immunotherapies. Recent studies demonstrated that autophagy is an important regulator of innate immune response in this microenvironment, but the mechanism by which autophagy regulates NK cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis and provides a cutting-edge advance in our understanding of the underlying mechanism. This study might pave the way for the formulation of more effective NK cell-based antitumor therapies.

O melanoma é a forma mais letal entre os cânceres de pele. Essa neoplasia freqüentemente apresenta-se resistente a abordagens terapêuticas. A angiogênese associada ao tumor representa um crítico passo da tumorigênese, resultado da ação de diferentes citocinas e fatores de crescimento como VEGF produzidos no microambiente tumoral. As galectinas extracelulares participam de múltiplos processos biológicos incluindo angiogênese tumoral e metástases, sua interação com as células presentes no microambiente tumoral pode ocorrer via receptores toll-like sugerindo seu envolvimento nos processos pro-inflamatórios e na secreção de citocinas. Recentemente mostramos que a ausência de gal-3 no estroma e parênquima tumoral diminui a angiogênese por interferir na resposta de macrófagos via VEGF e/ou TGFbeta1. Entretanto, o envolvimento de galectinas extracelulares na angiogênese e na modulação do sistema imune no microambiente tumoral ainda não está esclarecido. Assim, este estudo visa buscar respostas ao envolvimento das galectinas no crescimento tumoral e angiogênese contribuindo ao combate do melanoma maligno. Nossos resultados mostram a participação das galectinas 1 e 3 no crescimento tumoral e seu envolvimento com macrófagos via receptores toll-like, além de coordenarem a modulação do perfil de polarização de macrófagos derivados da medula óssea de camundongos wild-type. Dessa forma, podemos inferir que essas galectinas agem como coordenadoras de mudança de perfil dos macrófagos, uma vez que inibidas extracelularmente promovem uma diminuição do crescimento tumoral em camundongos wild-type, inoculados com células de melanoma murino e uma manutenção do perfil de macrófagos M1 in vitro. Assim, concluimos que as galectinas 1 e 3 extracelulares são importantes para o crescimento tumoral de melanomas murinos pois promovem o crescimento tumoral e são coordenadoras da mudança do perfil de macrófagos / Melanoma is the most aggressive form of skin cancer. This tumor often presents itself resistant to therapeutic approaches. The tumor-associated angiogenesis is a critical step in tumorigenesis and the result of the action of several cytokines and growth factors such as VEGF produced in the tumor microenvironment. The extracellular galectins participate in multiple biological processes including tumor angiogenesis and metastasis, their interaction with cells present in the tumor microenvironment may occur via toll-like receptors suggesting their involvement in pro-inflammatory processes and the secretion of cytokines. We have recently shown that the absence of Gal-3 the stroma and tumor parenchyma decreases angiogenesis by interfering with the macrophage response by VEGF and / or TGFbeta1. However, the involvement of extracellular galectins on angiogenesis modulation of the immune system in the tumor microenvironment is not yet clear. This study aims is to find answers to the involvement of galectins on tumor growth and angiogenesis contributing to the study of the malignant melanoma. Our results demonstrate the involvement of galectin 1 and 3 on tumor growth and its involvement in macrophage by toll-like receptors pathway, and coordinating the modulation of the polarization profile in wild-type mice bone marrow derived macrophages. Therefore, we show these galectins act as coordinators of macrophages profile change, since inhibited extracellularly promote a reduction in tumor growth in wild-type mice inoculated with murine melanoma cells and macrophages M1 maintenance of profile in vitro. Thus, we conclude that galectins 1 and 3 extracellular are important for tumor growth of murine melanomas because they promote tumor growth and are coordinators of change macrophages profile

O transplante de células-tronco hematopoéticas (CTHs) é o tipo mais bem-sucedido de terapia celular realizado até os dias atuais. No entanto, apesar do sucesso e da relevância clínica das CTHs isoladas a partir de fontes adultas, o uso destas células tem algumas limitações em relação à sua disponibilidade, compatibilidade imunológica e risco de contaminação. Desse modo, busca-se o desenvolvimento de soluções para as dificuldades apontadas para suprir a demanda de transplantes. Uma abordagem emergente para superar este problema é baseada na cultura e diferenciação de células-tronco embrionárias humanas (CTEhs). Estas são célulastronco pluripotentes e indiferenciadas com elevada capacidade de auto-renovação e diferenciação em todas as células derivadas dos três folhetos germinativos. No entanto, os métodos de diferenciação utilizados para a produção de CTHs a partir de células pluripotentes ainda não são eficientes. Os protocolos descritos até o momento têm gerado números variados e populações de células heterogêneas, e produz apenas CTHs muito primitivas e imaturas com baixa capacidade funcional in vivo. Parte desta dificuldade pode decorrer da ineficiência do microambiente de cultura para a diferenciação. Neste trabalho, nós demonstramos um eficiente protocolo de diferenciação hematopoética baseado em cocultivo de CTEhs com fibroblastos embrionários murinos com alto rendimento na geração de célulastronco/progenitoras hematopoéticas (CTPHs) que expressam os antígenos CD45, CD43, CD31 e CD34, e apresentam potencial clonogênico in vitro equivalente ao de células mononucleares isoladas de sangue de cordão umbilical. Nós fomos capazes de produzir todas as células das linhagens eritróide e mielóide em diferentes estágios de maturação, como também células positivas para marcadores linfóides. Demonstramos ainda que as células hematopoéticas surgem no sistema de cultura a partir de um endotélio-hemogênico constituído por células CD34+CD31+. No entanto, apesar das características maduras das CTPHs obtidas por tal método, os ensaios de reconstituição hematopoiética mostraram que estas células ainda possuem limitada capacidade funcional de enxertamento em camundongos imunocomprometidos quando transplantadas por via retro-orbital. / Hematopoietic stem cells (HSC) transplant is the most successful type of cell therapy carried out to date. However, despite the success and the clinical relevance of HSC isolated from adult sources, these cells have some limitations regarding its availability, immunological compatibility and risk of contamination. Thus, we seek to develop solutions to overcome these difficulties to supply the demand for transplants. An emerging approach to overcome this problem is based on human embryonic stem cells (hESCs) culture and differentiation. These are pluripotent and undifferentiated stem cells with high capacity for self-renewal and differentiation in all cells derived from the three embryonic germ layers. However, differentiation methods used for HSC production from pluripotent cells are not efficient yet. Protocols described so far have generated varying numbers and heterogeneous cell populations, and produce only very primitive and immature HSC with low in vivo functional capacity. Part of this difficulty may result from the inefficiency of the microenvironment of culture for differentiation. Here, we demonstrate an efficient protocol based on co-culture of hESCs with mouse embryonic fibroblasts for hematopoietic differentiation with high performance to generate in vitro hematopoietic stem/progenitor cells (HSPCs) that express CD45, CD43, CD31 and CD34 antigens with high purity of positive cells. We were able to produce all cells of erythroid and myeloid lineages at different stages of maturation. Lymphoid potential of hematopoietic cells was also evidenced. We demonstrated the primitive origin of hematopoietic cells through capillary-like structures constituted by hemogenic CD34+CD31+ cells. However, despite mature features of HSPCs obtained by our protocol, hematopoietic reconstitution assays showed that these cells have yet limited functional capacity for grafting into immunocompromised mice when exogenously transplanted by retro-orbital route.

Les nucléotides jouent un rôle majeur dans une pléiade de processus biologiques comme la composition des acides nucléiques, la signalisation, ou la régulation de la balance énergétique. Les nucléotides extracellulaires exercent également des fonctions biologiques. Par conséquent, des dérégulations des pools de nucléotides impactent l’homéostasie de multiples façons, par exemple en promouvant l’instabilité génétique ou un environnement immunosuppresseur. Or, ces paramètres font partie des « Hallmarks du Cancer » décrits par Hanahan et Weinberg. Ces observations confirment l’éventualité d’un rôle clé des nucléotides dans le cadre du cancer.cN-II et CD73 sont des 5’-nucléotidases impliquées respectivement dans les métabolismes nucléotidiques intra- et extracellulaire. Elles sont de nouvelles cibles thérapeutiques en oncologie. Cependant, leurs rôles dans la biologie de la cellule cancéreuse, ou le possible impact de leur utilisation en tant que cible thérapeutique sur le comportement des cellules tumorales sont peu connus. Considérant l’implication de ces enzymes dans les métabolismes nucléotidiques, nous avons enquêté sur les modifications de l’agressivité de la cellule cancéreuse ou sur sa capacité à interagir avec son microenvironnement, dans le cas d’une invalidation ou une diminution d’expression de cN-II et/ou CD73. cN-II semble donc impliquée dans l’adaptabilité métabolique et la combinaison des invalidations de cN-II et CD73 est associée à une modification d’expression d’enzymes du métabolisme du glucose. CD73 peut aussi moduler l’expression de gènes de la migration cellulaire. cN-II est impliquée dans la migration cellulaire, via l’axe COX-2/PGE2, et dans la sensibilité à des agents modulant ce paramètre. Ces caractéristiques sont plus marquées en association avec une invalidation de CD73. Ici, cN-II et CD73 ne semblent pas jouer de rôle dans la prolifération ou le dialogue avec une sous-population de cellule de l’immunité innée / Nucleotides play a major role in nucleic acids constitution and are involved in various cell phenomena. Indeed, intracellular ATP, GTP, AMP, GMP and their cyclic forms are components of cell signaling and define the energetic balance. Extracellularly, they also play multiple roles. Thus, when nucleotide pools are deregulated various processes are impacted. For example, a low availability of nucleotides supports genetic instability and aberrant levels of extracellular adenosine can lead to an immunosuppressive microenvironment. Interestingly, the cited parameters are among the Cancer Hallmarks described by Hanahan and Weinberg. These observations confirm the possibility of a key role of these molecules in this pathology. cN-II and CD73 are 5’-nucleotidases, involved in intra- and extracellular nucleotide metabolism respectively and have been identified as possible targets for new anti-cancer therapies. Nevertheless, very little is known about their biological roles on cancer cells and what parameters of cell biology could be impacted by such strategies. Considering the involvement of these purines in cell metabolism, we wondered what changes a decrease in cN-II and/orCD73 expressions or their silencing could trigger in cancer cells as well as in the interplay with their microenvironment.We studied cancer cell aggressiveness and the interplay with innate immune cells under cN-II and CD73 modulations. We observed that cN-II is involved in metabolic adaptability. The association of cN-II and CD73 invalidations results in glucose-metabolism-related gene modifications. CD73 can regulate migration-related genes expression but does not affect the process. cN-II is also involved in cell migration, via the COX-2/PGE2 axis. Again, these characteristics are accentuated when associated with CD73 deficiency. Here, cN-II and CD73 do not seem to be involved in cancer cell proliferation or in their interplay with a subset of innate immune cells

O melanoma é o mais agressivo tumor da pele, cuja resistência aos tratamentos quimioterápicos tem promovido a crescente utilização de imunoquimioterapia, como é o caso da utilização de agonistas dos receptores Toll-Like (TLRs). Nesse contexto, os compostos abreviados por P-MAPA e seu sintético estrutural MRB-CFI-1 com reconhecidas propriedades antitumorais e imunológicas, são fortes candidatos na terapia e prevenção desse tipo de câncer. Esse estudo visa determinar o potencial anticâncer do P-MAPA e de MRB-CFI-1 contra o melanoma murino em consequência ao padrão de resposta microambiental semelhante ao de morte imunogênica, em regimes de tratamento terapêutico ou vacinal, na vigência de quimioterapia com cisplatina e/ou em associação com antígenos de células tumorais totais. Após avaliação In vivo do crescimento de tumores B16F10 implantados em modelos murinos selvagem e nocaute para o gene Myd88, na vigência ou não do tratamento com cisplatina e/ou P-MAPA, nossos resultados mostraram que o P-MAPA apresentou atividade pró-tumoral e antagonizou a ação da cisplatina em inibir o crescimento dos tumores, de forma dependente de Myd88. Além disso, através de análises qualitativa e quantitativa pelo software ImageJ em fotomicrografias de secções tumorais coradas histologicamente, observamos que o P-MAPA promoveu mudanças microambientais nos tumores que podem impactar negativamente em seu desempenho. Como monoterapia em esquema de vacinação com lisado tumoral total em combinação com quimioterapia, o P-MAPA em dose baixa falhou em suprimir o crescimento de tumores B16F10, mas o seu sintético MRB-CFI-1 foi capaz de prevenir o crescimento desse tipo de melanoma num regime de vacinação profilática. Apesar do sucesso terapêutico desse imunomodulador em diversos modelos de câncer e de doenças infecciosas, o P-MAPA não foi eficaz em produz respostas microambientais contra o melanoma murino, dados esses que limitam a aplicabilidade clínica do composto. De outro modo, o composto fosfato inorgânico MRB-CFI-1 foi protetivo em retardar o aparecimento desse tipo de doença. Assim, o presente estudo foi importante por ampliar o entendimento funcional do P-MAPA numa abordagem imunoquimioterápica em modelos biológicos de tumores de melanoma, e representa uma importante mudança na utilização de constituintes individuais similares ao P-MAPA que sejam mais eficazes, de fácil obtenção, e de produção controlada e garantida / Melanoma is the most aggressive skin cancer, whose resistance to chemotherapeutic treatments has promoted the increasing use of immunochemotherapy, as is the case for the use of Toll-Like receptor agonists (TLRs). In this context, the compounds abbreviated by P-MAPA and its structural synthetic MRB-CFI-1 with recognized antitumor and immunological properties are strong candidates in the therapy and prevention of this type of cancer. This study aims to determine the anti-cancer potential of P-MAPA and MRB-CFI-1 against murine melanoma as a consequence of the microenvironmental response pattern similar to that of immunogenic death in therapeutic or vaccine treatment regimens when using chemotherapy with cisplatin alone or in combination with whole tumor cell antigens. After In vivo evaluation of the growth of B16F10 tumors implanted in wild-type and Myd88 gene knockout mice, under treatment or not with cisplatin and / or P-MAPA, our results showed that P-MAPA showed pro-tumor activity and antagonized the action of cisplatin in inhibiting the growth of tumors in a Myd88-dependent manner. In addition, using qualitative and quantitative analysis by ImageJ software in histological images of tumor sections, we observed that P-MAPA promoted microenvironmental changes in tumors that may negatively impact its performance. As monotherapy in vaccination schedule with total tumor lysate in combination with chemotherapy, low dose P-MAPA failed to suppress the growth of B16F10 tumors, but its synthetic MRB-CFI-1 was able to prevent the growth of this type of melanoma in prophylactic vaccination regimen. Despite the therapeutic success of this immunomodulator in various cancer models and infectious diseases, P-MAPA has not been effective in producing microenvironmental responses against murine melanoma, data that limit the clinical applicability of the compound. Otherwise, the inorganic phosphate compound MRB-CFI-1 was protective in delaying the onset of this type of disease. Thus, the present study was important because it broadened the functional understanding of P-MAPA in an immuno-chemotherapeutic approach in biological models of melanoma tumors and represents an important change in the use of individual constituents similar to P-MAPA that are more efficient, easily obtainable, and controlled and guaranteed production

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

Tout épithélium glandulaire sécrétoire est constitué d'une unité structurale et fonctionnelle commune, l'acinus. C'est une architecture sphérique pluricellulaire parfaitement différentiée et polarisée qui, reconstruite en culture 3D, mime l'organisation réelle du tissu. Etudier les déterminants environnementaux et génétiques qui gouvernent la transformation d'un acinus en sphéroïde s'apparentant à une tumeur est l'un des enjeux majeurs des modèles in vitro. Un des défis actuels est d'adapter ces modèles in vitro à des conditions de culture 3D qui soient compatibles avec la réalisation de cribles génétiques en 3D, basés par exemple sur l'ARN interférence (RNAi). Cependant, les formats standards de culture 3D et les méthodes analytiques ne sont pas compatibles aux cribles haut-débit. Ils ne permettent pas de contrôler la taille et la distribution des acini, sont dépendants d'immuno-marquages et les acquisitions sont longues. Par ailleurs, la microscopie confocale et vidéomicroscopie offrent un champ d'observation restreint qui ne permet pas d'observer un grand nombre de structures 3D en même temps, pour permettre une analyse statistique. Ainsi, dans le but i) de développer des modèles cellulaires appropriés en 3D, ii) d'adresser des questions fondamentales relatives au cancer de la prostate et iii) de réaliser des cribles RNAi dans un contexte plus pertinent que la culture 2D, j'ai développé des outils innovants au format microsystèmes adaptés à l'analyse haut-débit d'un grand nombre d'objets 3D. En optimisant les conditions de culture cellulaire 3D sur le modèle de la lignée cellulaire RWPE1, j'ai pu récapituler les étapes de formation des acini prostatiques et montrer que la formation du lumen est indépendante de la polarité et est gouvernée par deux mécanismes, « hollowing » et cavitation. / In all secretory epithelia from glandular tissues, there is a common structural and functional unit, the acinus. It is a well polarized and organized pluricellular structure that is spontaneously reconstructed in 3D culture, therefore closely mimics the real structure we find in vivo. For my purpose, acini are used as models for tumor initiation and cancer development. One of the objectives of Biomics laboratory is to identify the genetic and microenvironmental determinants of prostate acini morphogenesis and polarity. The strategy is based on High-Throughput (HT) RNA interference (RNAi)-based screening. To meet this objective, my project was to develop appropriate 3D cell models which closely mimic the cyst-like and duct-like structure of prostate. By optimizing conventional 3D culture in Matrigel, I could recapitulate prostate acini morphogenesis and showed that lumen formation is independent to the polarity, which appears later. However, the conventional 3D cell culture formats and analytical tools are not suited for HT Screening (HTS). They lack control over acini size, are label-dependant and therefore time-consuming and labor intensive. Also, classical microscopy offers a very limited field of view and hence does not allow observing a large amount of 3D structures for statistical analysis.

Trop souvent diagnostiqués à des stades tardifs du fait de leur quasi asymptomatie, les adénocarcinomes séreux ovariens posent un véritable problème de santé publique. Malgré les progrès récents de prise en charge chirurgicale, l’émergence d’une maladie résiduelle microscopique chimiorésistante impacte grandement le pronostic des patientes.Le microenvironnement tumoral est un acteur clé de la progression tumorale et de l’émergence de résistances aux traitements anticancéreux. Durant ces travaux de thèse, nous nous sommes intéressés à deux composants majeurs du stroma tumoral, d’une part les cellules souches mésenchymateuses, d’autre part les cellules endothéliales.Nous avons pu démontrer que les cellules souches mésenchymateuses participent à la progression tumorale et l’émergence de résistances. Enfin nous avons démontré que les cellules endothéliales, via la production de facteurs angiocrines, participent à la chimiorésistance des cellules tumorales ovariennes.Dans ce travail, nous avons pu définir de nouvelles cibles thérapeutiques mettant en jeu la relation entre les cellules tumorales ovariennes et l’hôte. / Ovarian cancers constitute a poor prognosis disease. Due to their absence of symptoms, ovarian cancers are generally diagnosed at late stages. Despite major breakthrough regarding ovarian cancer surgery, minimal residual disease-induced relapse is still a hurdle for clinicians.Tumor microenvironment is a key actor on disease progression and resistance to therapy. In this study, we have focused on two major components of the tumor stroma, on one hand, the mesenchymal stem cells, and the endothelial cells on the other hand.We were able to demonstrate that mesenchymal stem cells are critically involved in ovarian cancer progression and resistance to therapy, while the endothelium, through production of angiocrine factors, is deeply involved in resistance of ovarian cancer cells to platinum and taxane based therapy.Here, we set forth the idea that disrupting the relationship between ovarian cancer cells and their host stroma constitute a new therapeutic window.