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Investigação do perfil de expressão gênica e protéica de componentes do microambiente tumoral / Investigation of gene and protein expression profile of tumor microenvironment elementsBianca Rodrigues da Cunha 26 September 2011 (has links)
Tem se tornado evidente que a iniciação e a progressão do câncer depende de vários componentes do microambiente tumoral, incluindo células inflamatórias e imunes (linfócitos, macrófagos e mastócitos), fibroblastos, células endoteliais, adipócitos e matriz extracelular. De maneira geral, esses componentes são conhecidos como estroma. Tanto interações pró- como anti-tumor ocorrem entre um câncer e suas células vizinhas. Em um estudo prévio, avaliamos dados de bibliotecas SAGE de carcinoma epidermóide de cabeça e pescoço (CECP) usando ferramentas estatísticas e de bioinformática e pudemos identificar os genes mais e menos expressos em tumores metastáticos versus não metastáticos e em tumores versus tecidos normais. Em outro estudo, avaliamos fatores parácrinos solúveis produzidos por células do estroma e por células neoplásicas que poderiam influenciar proliferação e expressão gênica e protéica. Ambos os estudos identificaram marcadores potenciais associados a respostas inflamatórias ou imunes. Dezenove desses genes foram selecionados por PCR em tempo real e o estudo foi realizado nas linhagens SCC-9 de células epiteliais neoplásicas e de fibroblastos isolados de um câncer oral, e em 40 amostras de carcinomas primários de cabeça e pescoço (6 amostras micro e 34 macrodissecadas). Nós também utilizamos eletroforese unidimensional para analisar a expressão protéica nesse conjunto de amostras. Como a microdissecção produziu baixas concentrações de RNA e proteínas, ciclos extras de amplificação de mRNA foram necessários para obter material suficiente para experimentos de PCR. Nossos dados mostraram que os perfis de expressão foram provavelmente pouco preservados durante os ciclos extras de amplificação. Em amostras macrodissecadas, nós consistentemente observamos que o nível de transcritos de MGLL, COX2, EP3, EP4 e LTAH4 estava reduzido, porém presente nas suas margens cirúrgicas. Os dados não confirmaram, em células de CECP, a hipótese de que MGLL produz menssageiros lipídicos oncogênicos, esta via pode não atuar nesses pacientes. Nós também observamos que metaloproteinases são expressas em níveis elevados em CECP e devem estar envolvidas na degradação da matriz extracelular. Células neoplásicas e do estroma desses carcinomas exibem uma ampla variedade de proteínas com níveis muito diferentes de expressão. Este resultado abre perspectivas para realização de experimentos de validação / It has become evident that cancer initiation and progression depends on several components of the tumor microenvironment, including inflammatory and immune cells (lymphocytes, macrophages and mastocytes), fibroblasts, endothelial cells, adipocytes, and extracellular matrix. Collectively, these components are known as the stroma. Both pro- and anti-tumor interactions occur between a tumor and its surrounding cells. In a previous study, we evaluated data from SAGE libraries of head and neck squamous cell carcinoma (HNSCC) using statistical and bioinformatic tools and we could identify top-up and top-downregulated genes in metastatic versus non-metastatic tumors and in tumors versus normal tissues. In another study, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression. Both studies identified potential markers associated with immune or inflammatory response in head and neck tumorigenesis. Nineteen of these genes were selected for real-time polymerase chain reaction (PCR) and the study was carried out on the epithelial cancer cell line SCC-9 and on fibroblasts isolated from an oral cancer, and in 40 samples from primary HNSCC (6 micro and 34 macrodissected samples). We also used one-dimensional gel electrophoresis and mass spectrometry to analyze protein expression in this set of samples. As microdissection yielded low RNA and protein concentrations, extra rounds of mRNA amplification were necessary to obtain sufficient material for PCR experiments. Our data showed that expression profiles were probably scantily preserved during the extra rounds of amplification. In macrodissected samples, we consistently observed that the level of MGLL, COX2, EP3, EP4 e LTAH4 transcripts was low in most tumors but present in their surgical margins. The data do not confirm in HNSCC the hypothesis that MGLL produces oncogenic lipid messengers, this pathway may not act in these patients. We also observed that metalloproteinases are overexpressed in HNSCC and should be involved in extracellular matix degradation. Neoplastic and stromal cells from HNSCC exhibit a wide variety of proteins with very different levels of expression. This result opens the perspective to perform validation experiments.
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A expressão de Angiopoetina 1 e Trombospondina 1 em desordens potencialmente malignas bucais e no carcinoma espinocelular / Angiopoetin 1 and Thrombospondin 1 expression in oral potentially malignant disorders and squamous cell carcinomaBernardes, Carlos Alberto Nascimento January 2015 (has links)
O carcinoma espinocelular é responsável por 90% das lesões malignas na cavidade bucal, sendo o oitavo tipo de câncer em todo o mundo e apresenta uma taxa de sobrevida de apenas 50% em 5 anos. Os carcinomas podem se desenvolver diretamente de células epiteliais normais ou a partir das desordens potencialmente malignas, as quais envolvem as hiperplasias e as displasias. Para que estas lesões se desenvolvam é necessário que nutrientes e oxigênio sejam disponibilizados, o que ocorre a partir da formação de uma nova vasculatura. Este processo, denominado angiogênese, é complexo e envolve a regulação de diferentes fatores indutores, como as moléculas Angiopoetina 1 (ANG1) e Trombospondina 1 (TSP1), cuja ação pode ser influenciada tanto pelas células de origem epitelial quanto pelos componentes do microambiente da lesão. O objetivo deste projeto foi descrever o perfil de expressão das moléculas angiopoetina 1 e trombospondina 1 em biópsias de desordens potencialmente malignas e em carcinoma espinocelular oral. A amostra foi composta por biópsias de pacientes que apresentaram diagnóstico de hiperplasia (n=6), displasia epitelial leve (n=7), displasia epitelial severa (n=15), carcinoma espinocelular oral grau 1 e 2 (n= 13) e 3 e 4 (n=13). As lâminas histológicas foram submetidas à reação de imunoistoquímica para ANG1 e TSP1, das quais foi realizada uma análise descritiva quanto ao perfil de marcação tanto em epitélio quanto em tecido conjuntivo subjacente à lesão. Angiopoetina apresentou uma marcação concentrada principalmente nas células endoteliais e com leve marcação nas células da camada basal do epitélio, sendo observada uma redução da intensidade de marcação de acordo com o grau de severidade da desordem epitelial, porém com um aumento de marcação em células do microambiente tumoral. Trombospondina apresentou marcação heterogênea tanto em tecido epitelial, endotelial e conjuntivo, com um aumento da marcação em macrófagos e fibroblastos de acordo com a severidade da lesão. Este perfil diferenciado de expressão destes marcadores do processo de angiogênese, tanto por células epiteliais modificadas quanto por células do microambiente celular, evidencia a necessidade de mais estudos que explorem a diversidade celular das lesões neoplásicas, com o objetivo de aprimorar as terapias anti-angiogênicas vigentes para o tratamento de desordens potencialmente malignas. / Oral squamous cell carcinoma is the eight most common cancer and it accounts for 90% of malignant lesions in the oral cavity, presenting a survival rate of only 50% after 5 years. Carcinomas may develop directly from normal epithelial cells or from the potentially malignant disorders, such as hyperplasia and dysplasia. During the carcinogenesis process, it is necessary an increase on nutrients and oxygen supply, which occurs through the formation of new vasculature. This process, known as angiogenesis, is complex and involves the regulation of various inducing factors such as angiopoietin molecules 1 (ANG1) and Thrombospondin 1 (TSP1), whose action can be influenced both by epithelial cells or cells from the lesion microenvironment . The objective of this project was to describe the expression profile of molecules angiopoietin 1 and thrombospondin 1 in biopsies of potentially malignant disorders and oral squamous cell carcinoma. The sample consisted of biopsies from patients who were diagnosed with hyperplasia (n = 6), mild dysplasia (n=7), severe dysplasia (n=15), squamous cell carcinoma grade 1 and 2 (n=13) and 3 and 4 (n=13). The histological slides were submitted to immunohistochemical reaction to ANG1 and TSP1, which underwent a descriptive analysis of the staining profile in both epithelium and in the underlying connective tissue to injury. Angiopoietin showed staining mainly in endothelial cells and a subtle staining on cells at the basal layer of the epithelium, and it was observed a reduction in the intensity of the staining in accordance with the severity of epithelial disorder, but with an increase of staining of cells from the microenvironment. Thrombospondin presented a heterogeneous staining among epithelial, endothelial, and connective tissue, with an increase in the staining on macrophages and fibroblasts according to the severity of injury. This differential expression profile of these markers of angiogenesis process, which was observed in both epithelial cells and cells from the microenvironment, highlights the need for more studies exploring the cellular diversity of neoplastic lesions, in order to improve the existing anti-angiogenic therapies for the treatment of malignant disorders.
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Interações entre células dendríticas, mastócitos e células tumorais. / Interactions between dendritic cells, mast cells and tumor cells.Cecília Pessoa Rodrigues 31 March 2015 (has links)
Os mastócitos (MC) são células teciduais, ricas em mediadoras inflamatórias, envolvidos na resposta alérgica, com papel imunomodulador cada vez mais reconhecido. Células Dendríticas (DCs), por sua vez, são sabidamente necessárias para a resposta imune, sendo as células apresentadoras de antígenos mais eficientes, capazes de responder prontamente frente à sinais de perigo. Neste trabalho, demonstramos que o contanto celular de DCs com MCs (iDC-MC) induz a geração de DCs com imunofenotipo tolerogênico. As iDC-MC exibiram menor expressão de HLA-DR e maior expressão de PD-L1, assim, não foram capazes de manter uma proliferação alogeneica. Ainda, os linfócitos expostos as iDC-MC induziram maior expressão de FoxP3+, IL-10 e TGF-β, capazes de suprimir a proliferação de linfócitos T naïve estimulados com mitógeno. Além disso, o contato resultou na maior produção de IDO, fenômeno este, bloqueado quando MCs foram tratados com anti-PD-1 ou iDCs tratadas com PD-L1 ou PD-L2, mas a produção se manteve inalterada após o tratamento das iDCs com anti-histamínicos. / Mast cells (MC) are tissue resident cells, rich in inflammatory mediators, involved in allergic reactions, and with an increasingly recognized role in immunomodulation. Dendritic cells (DCs), on the other hand, are central to the determination of immune response patterns, being highly efficient antigen-presenting cells that respond promptly to changes in their microenvironment. Here, we show that direct cell contact between iDCs and MC bends DCs towards tolerance induction. These MC-exposed DCs decreased HLA-DR but increased PD-L1 expression and stimulated T lymphocytes to express FoxP3+, to secrete TGF-β and IL-10, and to suppress the proliferation of mitogen-stimulated naïve T lymphocytes. Furthermore, contact with MC induced DCs to express higher levels of indoleamine-2,3-deoxigenase (IDO), a phenomenon that was blocked by treatment of MC with anti-PD-1 or by the treatment of DCs with anti-PD-L1 or PD-L2, but not by blocking of H1 and H2 histamine receptors on DCs.
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Avaliação do papel de galectina-3 no recrutamento de macrófagos e sua participação na angiogênese em modelo de fibrossarcoma / Evaluation of the role of galectin-3 in macrophage recruitment and its participation in angiogenesis in a fibrosarcoma modelKarina Mie Furuzawa 04 November 2016 (has links)
Assim como tecidos normais, tumores possuem uma demanda de nutrientes e oxigênio, suprida através da vasculatura a eles associada que resulta do processo de angiogênese. Fatores pró-angiogênicos são capazes de atrair monócitos, os quais se diferenciam em macrófagos associados a tumores (TAMs). TAMs comumente apresentam fenótipo M2, cujas características são consideradas pró-tumorais, como a promoção da angiogênese e a degradação de matriz extracelular. Estudos indicam que galectina-3 (gal-3), uma proteína pleiotrópica que se liga a ?-galactosídeos, participa do controle da angiogênese e da infiltração de macrófagos M2 na massa tumoral, mas pouco se sabe sobre os mecanismos envolvidos. No presente estudo, utilizamos um modelo de sarcoma induzido por carcinógeno em camundongos selvagens (WT) e knockout para gal-3 (Gal- 3 KO). Comparando os tumores de animais WT e Gal-3 KO, não observamos diferenças no padrão de crescimento tumoral, na área necrótica relativa, na proliferação celular e na quantificação de fibras de colágeno. Demonstramos que, embora ambos os grupos desenvolvam tumores, a angiogênese foi inibida em um microambiente desprovido de gal-3. Entretanto, não houve diferença na produção do fator de crescimento endotelial vascular (VEGF). As imagens obtidas in vivo indicaram que gal- 3 também influencia na formação estrutural de vasos adjacentes ao tumor. Além de mediar aspectos morfológicos relacionados à angiogênese, demonstramos que gal-3 também contribuiu para a funcionalidade vascular, pois houve uma redução na velocidade de fluxo sanguíneo nos vasos intratumorais de animais Gal-3 KO. Nossos dados sugeriram que há menos macrófagos no tumor que não expressa gal-3 e, dentre os TAMs, há mais M2 em comparação ao tumor gal-3-positivo. A análise do tecido onde o tumor se desenvolve, na fase inicial da tumorigênese, indicou que a ausência de gal-3 está relacionada a uma maior densidade de macrófagos M2. Considerando que a presença maior de macrófagos M2 nos sarcomas gal-3-negativos não resultou em maior produção de VEGF, mas sim na inibição da angiogênese, nossos resultados apontam para uma participação significativa de gal-3 na mediação da angiogênese pelos macrófagos / As well as normal tissues, tumors require nutrients and oxygen, which are supplied by the associated vasculature that results from the process of angiogenesis. Pro-angiogenic factors are able to attract monocytes and they differentiate into tumor-associated macrophages (TAMs). TAMs commonly exhibit M2 phenotype, which has characteristics considered pro-tumoral, such as angiogenesis promotion and degradation of extracellular matrix. Studies show that galectin-3 (gal-3), a pleiotropic ?-galactosidebinding protein, participates in angiogenesis control and M2 macrophage infiltration into the tumor mass, but little is known about the mechanisms involved. In this work, we established a model of carcinogen-induced sarcoma in wild-type (WT) and gal-3 knockout (Gal-3 KO) mice. Comparing tumors from WT and Gal-3 KO animals, there were no differences in the pattern of tumor growth, relative necrotic area, cell proliferation and collagenous fibers. We demonstrated that, although both groups develop tumors, angiogenesis was inhibited in a microenvironment devoid of gal-3. However, there was no difference in the production of vascular endothelial growth factor (VEGF). The images obtained in vivo indicated that gal-3 also influenced the structural formation of vessels adjacent to the tumor. In addition to mediating morphological aspects related to angiogenesis, we demonstrated that gal-3 also contributes to vascular functionality, since there was a reduction in blood flow velocity in intratumoral vessels from Gal-3 KO animals. Our data suggested that there are fewer macrophages in tumors without gal-3 and, among TAMs, there are more M2 compared to gal-3-positive tumors. Analysis of the tissue where the tumor develops, in early stages of tumorigenesis, indicated that the lack of gal-3 is related to an increased density of M2 macrophages. Since the greater number of M2 macrophages in gal-3-negative fibrosarcomas did not result in increased VEGF production, but inhibited angiogenesis, our results suggest a significant role of gal-3 in regulation of angiogenesis by macrophages
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Interplay between cancer cells and cancer-associated fibroblasts in tumor invasion and metastasis formation / Rôle des fibroblastes associés au cancer dans l'invasion tumoraleAtieh, Youmna Marie Lyne 04 July 2017 (has links)
Les carcinomes sont des cancers touchant plusieurs organes du corps humain, notamment les seins, le pancréas, les poumons, l'intestin… et sont issus de la transformation de cellules épithéliales en cellules tumorales. Au cours du développement d'une tumeur, les cellules cancéreuses, contrairement aux cellules normales, acquièrent la capacité de se déplacer dans le corps humain, jusqu'à coloniser des organes voisins. Ces colonies sont appelées métastases. Le processus métastatique est responsable de 90% des décès dans le cadre des carcinomes. Ce processus n'est pas dû à l'action isolée des cellules cancéreuses mais est aussi le résultat d'une coopération entre la tumeur et son voisinage – le microenvironnement tumoral – favorisant la survie et la migration des cellules cancéreuses. Les fibroblastes sont une population cellulaire du microenvironnement tumoral. Il a été démontré que les fibroblastes sont activés à proximité des cellules cancéreuses ; on les qualifie de fibroblastes associés au cancer ou CAFs. Dans des tissus de patients, les tumeurs les plus agressives corrèlent avec un enrichissement en fibroblastes et une matrice plus dense. Mon projet de thèse illustre un nouveau mécanisme de coopération entre CAFs et cellules cancéreuses. Cibler l’action des fibroblastes pourrait ralentir la progression tumorale, voire bloquer la formation de métastases. / Cancer-associated fibroblasts (CAFs) are the most abundant cells of the tumor stroma. Their capacity to contract the matrix and induce invasion of cancer cells has been well-documented. However, it is not clear if CAFs remodel the matrix by other means (degradation, matrix deposition or stiffening). This project demonstrates that CAFs induce cancer cell invasion through assembly of FN into the matrix. CAFs assembled fibronectin (FN) mainly via integrin α5 but integrin αvβ3 was necessary for initial mechanosensing and fibrillar adhesion formation. In the absence of FN, contractility of the matrix by CAFs is preserved. When degradation is impaired, CAFs retain the capacity to induce invasion in a FN-dependent manner. In all cases, the levels of expression of integrin β3 and the amount of assembled FN was directly proportional to the invasion induced by fibroblast populations. Our results highlight FN assembly and integrin β3 as new hallmarks of CAFs. We also noticed that cancer cells migrate towards CAFs suggesting a possible chemotactic response. Using Dunn’s chemotaxis chamber, we found that cancer cells migrate along a gradient of CAF-conditioned media and a gradient of fibronectin. Finally, orthotopic injections of cancer cells and CAFs in the colon wall of mice revealed that CAFs stimulate metastasis of cancer cells to the liver. In conclusion, our data show that CAFs promote cancer cell invasion by depositing fibronectin that can guide cancer cells favoring metastasis formation.
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Etude de l’impact de la vitronectine et de la fibronectine ascitiques sur la récidive des carcinomes ovariens / Study of the impact of ascites derived fibronectin and vitronectin on the recurrence of ovarian carcinomasBlay, Lyvia 29 June 2016 (has links)
Les cancers de l’ovaire représentent la première cause de mortalité due aux cancers gynécologiques dans les pays développés. La plupart de ces cancers sont diagnostiqués à des stades tardifs quand les cellules cancéreuses ovariennes ont disséminé et colonisé les parois de la cavité abdominale. En conséquence, ces cancers sont associés à un sombre pronostic.Plus d’un tiers des patientes présentent une accumulation de liquide d’ascite au moment du diagnostic. L’ascite est un fluide exsudatif ayant une composante cellulaire et également une composante liquide acellulaire, constituant un réservoir dynamique de molécules bioactives. Malgré une réponse efficace aux traitements standards, les taux de récidive de cette pathologie restent élevés. L’acquisition d’une chimiorésistance et le mode de propagation atypique des cellules cancéreuses sont deux éléments importants impliqués dans la récidive des cancers ovariens.Le rôle de l’ascite et plus particulièrement de la composante matricielle ascitique, dans le processus de dissémination et dans la chimiorésistance des cellules cancéreuses ovariennes reste peu étudié et constitue le cœur de ce travail de thèse.L’objectif des travaux a été d’étudier l’influence de l’ascite et de deux glycoprotéines matricielles purifiées à partir d’échantillons d’ascites sur les comportements cellulaires propices à la dissémination et à la récidive des cancers ovariens. De plus, l’influence de l’ascite sur la réponse des cellules aux traitements à base de platine a également été recherchée et estimée.L’étude démontre que l’ascite est un microenvironnement propice à la dissémination des cellules cancéreuses ovariennes et que la fibronectine et la vitronectine ascitiques ainsi que leurs récepteurs d’adhérence spécifiques sont des éléments qui participent à la régulation de cette dissémination. L’ascite exerce également un effet protecteur contre l’effet cytotoxique des agents chimiothérapeutiques.Ces résultats mettent en perspectives l’intérêt potentiel de la fibronectine et de la vitronectine ascitiques comme outils diagnostiques et/ou cibles thérapeutiques des cancers ovariens et nous engagent à approfondir l’étude de ces deux molécules et de leurs conséquences sur la progression de la maladie. / Ovarian cancers are the leading cause of death among gynaecological cancers in western countries. Most of these cancers are diagnosed at a late stage, when ovarian cancer cells have spread and colonized the walls of the abdominal cavity. Therefore, these cancers are associated to a poor prognosis.More than one third of the patients show an accumulation of ascites at the time of the diagnosis. Ascites are exudative fluids composed of a cellular and also an acellular fraction. In fact, ascites constitute a dynamic reservoir of bioactive molecules. Even if the response of ovarian cancers to the current first-line therapy, that consit in debulking surgery followed by chemotherapy, is satisfactory, the rate of recurrence remains important. The gain of a chemoresistance and the atypical widespread of cancer cells are two important factors involved in the recurence of the ovarian cancers.The role of ascites and more particularly, of the ascitic matrix componants on the dissemination process and on the chemoresitance of the ovarian cancer cells remain poorly studied and is the aim of this work.The objective of this study was to investiguate the influence of ascites and of two matrix glycoproteins purified from samples of ascites i) on the cells behavior convenient to the dissemination and the recurrence of the ovarian cancers and ii) on the response of cells to therapeutics treatments with platinum.This study suggests that ascites are a permissive microenvironment to the dissemination of ovarian cancer cells and that ascitic fibronectin and vitronectin as well as their specific receptors are actors which participate to the regulation of this dissemination. Ascites also protect ovarian cancer cells against the cytotoxicity of chemotherapeutic drugs.These results illustrate the potential interest of ascites derived fibronectin and vitronectin as diagnosis tools and/or therapeutic targets for ovarian cancers and encourage us to deepen the study of these two molecules and their consequences in the progress of the disease.
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Approche immunologie des systèmes pour l'étude du microenvironnement tumoral et de l'interface foeto-maternelle / Systems immunology to the study of the tumour microenvironment and the fetomaternal interfaceNehar-Belaid, Djamel-Eddine 22 September 2014 (has links)
Il existe de nombreuses similarités entre le développement du foetus et celui des cellules tumorales. En effet, dans les deux cas ils requièrent une division cellulaire intense, une invasion des tissues de l’hôte ainsi qu’une vascularisation soutenue. De plus, malgré le fait que le foetus et les cellules tumorales expriment à la fois des antigènes étrangers (les antigènes paternels pour le foetus et les antigènes du soi modifiés au niveau des cellules tumorales) ils ne sont pas rejetés par le système immunitaire. Parmi plusieurs populations cellulaires impliquées dans ce phénomène de non rejet ou de tolérance, les cellules T régulatrices (Tregs) jouent un rôle primordial dans les deux processus. En effet, notre laboratoire a démontré que l’émergence des cellules tumorales ainsi que l’implantation fœtale s’accompagnent d’une activation forte et rapide des Tregs. Cette observation prend tout son sens quand l’élimination de ces Tregs a conduit à un rejet immunitaire de la tumeur ou du foetus. Afin de valider ces observations, nous avons mis au point une étude transcriptomique comparative entre le microenvironnement tumoral et l’interface foeto-maternelle. Cela a révélé une forte similarité et une importante diminution des voies de signalisation immunologiques associées à la présentation antigénique et à l’activation des cellules T. De plus, des analyses non supervisées ont mis en évidence une coévolution des signatures immunitaires inhibées et cela dés les premiers jours suivant l’implantation des tumeurs ou du foetus. Par ailleurs, l’élimination des Tregs (qui a conduit un rejet de la tumeur ou du foetus) a permis de faire basculer les mêmes signatures immunitaires d’un état d’inhibition à un état d’activation. En définitive, nous pensons que les mécanismes déployés au cours de l’évolution pour protéger les foetus du rejet immunitaire sont détournés afin de favoriser le développement des cellules tumorales. / There are striking similarities between fetus and tumor development. They both require intense cell division, invasion of host tissues and sustained vascularization. Moreover, despite that fetus and tumor express foreign antigens - paternal allo-antigens for fetuses and modified auto-antigens for tumors, they are not rejected by the immune system. Among others, regulatory T cells (Tregs), which are key players in tolerance, appear to play a significant role in both processes. We showed that tumor emergence as well as embryo implantation elicit a strikingly similar brisk Treg response, which functional relevance is supported by the fact that and Treg depletion leads to fetus or tumor immune rejection. Comparison of fetal and tumor microenvironments through transcriptomics revealed strikingly similar and dramatic decrease in expression of multiple immune-related pathways, including antigen presentation and T cell response. Unsupervised analyses highlighted the co-evolution in time of downregulated immune signatures, from the very first days after tumor or embryo implantation. Treg depletion, which leads to fetus or tumor rejection, converted the very same down-modulated immune signatures to up-regulated ones. We propose that means selected during evolution to protect mammalian fetuses are hijacked to license tumor development.
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Polynucléaires neutrophiles, cellules stromales, lymphocytes B : interaction tripartite dans la niche des lymphomes B / Neutrophils, stromal cells, B cells : tripartite interaction in B-cell lymphoma nichesGrégoire, Murielle 15 December 2014 (has links)
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.
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Contribuição das células-tronco mesenquimais para as propriedades tumorigênicas de células de glioblastoma humano / Contribution of mesenchymal stem cells to the tumorigenic properties of human glioblastoma cellsCarolina de Oliveira Rodini 11 March 2016 (has links)
Células-tronco mesenquimais (CTM) apresentam tropismo a tumores, sendo importantes componentes do estroma tumoral. No cérebro, o nicho perivascular é uma importante fonte de CTM, as quais podem contribuir direta e/ou indiretamente para o desenvolvimento de tumores, embora os mecanismos envolvidos sejam pouco conhecidos. No presente trabalho, investigou-se a influência de CTM sobre a proliferação, capacidade invasiva e tumorigenicidade de células de Glioblastoma (GBM) humano. Sabe-se que CTM produzem TGFB1, uma citocina multifuncional envolvida em imunomodulação, proliferação, migração e transição epitelial-mesenquimal de células tumorais. Experimentos in vitro, realizados com meios condicionados de CTM de cordão umbilical humano com silenciamento permanente do gene TGFB1, demonstraram que o TGFB1 secretado por CTM é capaz de aumentar significativamente a proliferação e viabilidade de células de GBM humano da linhagem U87FP635. Esses resultados revelam uma importante ação parácrina dessa citocina regulatória, quando produzida por outros tipos celulares contidos no microambiente tumoral. Entretanto, sob condições experimentais que melhor mimetizam o microambiente tumoral, detectou-se que CTM também afetam o comportamento de células tumorais por um mecanismo alternativo, dependente de contato celular, mas independente dos níveis de TGFB1 secretados pelas CTM. Sob condições de cocultivo celular, envolvendo contato físico entre CTM e células de GBM U87FP635, detectou-se um aumento significativo na quantidade de células tumorais viáveis. Quando cultivadas na forma de esferoides tumorais, o contato com CTM aumentou a capacidade invasiva das células U87FP635. Finalmente, em modelo in vivo ectópico de GBM, células U87FP635 geraram tumores mais desenvolvidos quando coinjetadas com CTM. Esses efeitos pró-tumorigênicos foram observados tanto em contato com CTM controles, quanto com CTM contendo o gene TGFB1 permanentemente silenciado. Assim, esses achados indicam que CTM podem exercer efeitos pró-tumorigênicos por dois mecanismos alternativos e independentes: ação parácrina de TGFB1 secretado por CTM e ação mediada por contato célula-célula. Nas condições experimentais testadas, o mecanismo dependente de contato célula-célula demonstrou ser predominante. O estudo proteômico do secretoma dessas células identificou 126 proteínas diferencialmente expressas além de 10 proteínas exclusivamente detectadas em meios condicionados de cocultivos de CTM com células de GBM U87FP635. Cerca de 80% dessas proteínas exclusivamente secretadas pelo contato célula-célula são componentes de exossomos e estão envolvidas em proliferação celular e desenvolvimento tecidual. Esses resultados apontam uma interação dinâmica de comunicação entre CTM e células tumorais, e revelam algumas proteínas interessantes potencialmente envolvidas em uma ação pró-tumorigênica de CTM mediada por contato celular / Mesenchymal stem cells (MSC) display tropism to tumors, being recruited to its microenvironment where they comprise the tumor stroma. In brain, perivascular niche is a substantial source of MSC. Although mechanisms involved are poorly understood, MSC may directly and/or indirectly contribute to tumor development. Herein, the influence of MSC on the proliferation, invasiveness and tumorigenicity of human glioblastoma cells (GBM) was investigated. Moreover, since MSC releases TGFB1, a multifunctional cytokine with roles in immunomodulation, proliferation, migration and epithelial-mesenchymal transition of tumor cells, we analyzed if MSC-secreted TGFB1 affects GBM behavior. In vitro studies performed in the presence of conditioned media from human umbilical cord MSC with a stable TGFB1 gene expression knockdown showed that MSC-secreted TGFB1 is able to significantly increase the proliferation and viability of a GBM cell line (U87FP635). These results revealed an important paracrine effect of this regulatory cytokine when secreted by other cell types in tumor microenvironment. However, under experimental conditions that better mimic the tumor microenvironment, it was found that MSC also affect tumor cell behavior by an alternative mechanism dependent on cell-cell contact, but independent of TGFB1 levels secreted by MSC. The cell-cell contact between MSC and GBM U87FP635 significantly enhaced tumor viable cells. Additionally, the spheroid tumor cell culture with MSC cell contact increased the invasiveness of U87FP635 cells. Finally, in vivo ectopic implantation model showed more developed tumors when GBM U87FP635 cells were coinjected with MSC. These pro-tumorigenic effects were found both in cell-cell contact with control MSC, as with MSC containing TGFB1 gene expression knockdown. Thus, these findings indicate that MSC can exert pro-tumorigenic effects by two alternative and independent mechanisms: paracrine action of TGFB1 secreted by MSC and action mediated by cell-cell contact. In the present experimental conditions, the cell-cell contact-dependent mechanism was predominant. The secretome proteomic study of those cells identified 126 differentially expressed proteins as well as 10 proteins exclusively detected in conditioned media from GBM U87FP635 cell cocultures with MSC. About 80% of proteins uniquely secreted by cell-cell contact are exosomes components and are involved in cell proliferation and tissue development. These results indicate a dynamic interaction of communication between MSC and tumor cells and reveal some interesting proteins potentially involved in a MSC pro-tumorigenic action mediated by cell contact
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Characterizing the immune landscape of tumor draining lymph nodesMuscarella Jr., Ronald Anthony 24 November 2021 (has links)
BACKGROUND: Breast cancer is the most common cancer type in women, with 276,480 women diagnosed with breast cancer in the United States in 2020.1 Death from breast cancer is usually caused by metastasis to distant sites, rather than from the primary tumor itself.2 Regional spread of breast cancer to the tumor draining lymph nodes (TDLN) often precedes further dissemination to the rest of the body, and as such is an important prognostic tool during cancer diagnosis and staging.3 During tumor growth, multiple immune cells and stromal cells in both the primary tumor microenvironment (pTME) and the TDLN undergo changes that promote tumor growth, metastasis and immune evasion in the TDLN and to the rest of the body. Among breast cancer subtypes, triple negative breast cancer (TNBC) is one of the most aggressive, and is most likely to be resistant to traditional chemotherapies, has the worst 5-year survival percentage, regardless of stage.4 Additionally, human growth factor receptor-2 (Her2) positive breast cancers are aggressive breast cancers as well, and Her2+ breast cancers are most likely to metastasize to the TDLN.5
PROBLEM: Little is known about how breast cancer cells induce genetic or transcriptomic changes in immune cells and stromal cells in the lymph node microenvironment and pTME as cancer cells metastasize to the TDLN.
OBJECTIVE: To elucidate changes observed in immune and stromal cells in the TDLN via single cell sequencing, and to quantify changes in exhaustion status of lymphocytes in the metastatic TDLN microenvironment that may be causal of lymph node metastasis.
METHODS: With a bioinformatics approach on single cell RNA sequencing, and with flow cytometry, we will measure changes in the transcriptomes of cells in the primary tumor microenvironment and the TDLN and compare changes in the primary site, tumor draining lymph node, and a normal lymph node. We measure changes in the expression of canonical exhaustion markers and inhibitory receptors: Tim-3, Lag-3, Tigit, CTLA-4, PD-1, and CD160 in lymphocytes in the metastatic TDLN and nonmetastatic spleens in Balb/c mice with flow cytometry. Additionally, we measured changes in myeloid and lymphoid populations in the spleens and lymph nodes with flow cytometry.
RESULTS: B cell accumulation was observed in both the single cell RNA sequencing analysis and via flow cytometry in both the metastatic TDLN itself and in the spleens of mice with metastasis to the TDLN. Increased RNA of exhaustion markers in CD8+ T cells was observed in the primary tumor samples. Additionally, a trend of increased Natural Killer cells, B cells, naive and memory CD4+ and CD8+ T cells expressing canonical exhaustion markers was observed in both the metastatic TDLNs and the spleens of mice with cancer, indicating systemic immune suppression may occur as TNBCs and Her2+ breast cancers metastasize to the TDLN. / 2022-11-24T00:00:00Z
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