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The aim of this thesis was to study how to increase effectiveness of cancer immunotherapy based on synergy of compounds stimulating phagocytosis and TLR agonist. Tumor microenvironment was modified by enzymes, which catalised conversion of lactate to pyruvate or acetate. It was monitored effect of enzymes on tumor size, survival of experimental mice and cytotoxicity on tumor cells.

INTRODUÇÃO: A incidência de câncer da glândula tireoide é a que mais vem crescendo nas últimas décadas. Dentro desse grupo de diferentes neoplasias, o carcinoma papilífero, um dos carcinomas bem diferenciados, representa a maioria e tem prognóstico favorável, com sobrevida acima dos 90% em 5 anos. Embora sejam utilizadas diversas classificações de risco baseadas em diferentes fatores prognósticos, ainda não se consegue predizer quais pacientes terão maior chance de recorrência, metástases linfonodais e desfecho desfavorável, que se beneficiariam de um tratamento mais agressivo. Já foi demonstrado em diversos tipos de neoplasias que diferenças no perfil do infiltrado imune tumoral têm relação com prognóstico e resposta ao tratamento. Neste estudo caracterizou-se o microambiente imune do carcinoma papilífero através de marcadores imuno-histoquímicos de células inflamatórias e relacionou-se este perfil de infiltração com fatores prognósticos clínico-patológicos e com sobrevida livre de recorrência. MÉTODOS: Foram incluídos 151 casos selecionados com base em um banco de dados que incluiu todos os pacientes submetidos a tratamento cirúrgico para câncer de tireoide no A.C.Camargo Cancer Center entre 2008 e 2010. Casos com tireoidite significante foram excluídos. Estes tumores selecionados foram então submetidos a reação imuno-histoquímica com marcadores de células inflamatórias e foram analisados por dois patologistas experientes. As características clínicas e patológicas foram avaliadas, assim como as recorrências e sobrevida, relacionando-as com as leituras de células marcadas. As análises de sobrevida global e livre de doença foram realizadas pelo método de Kaplan-Meier, com comparação de curvas de sobrevida pelo teste de Logrank. RESULTADOS: Cento e cinquenta e um pacientes foram incluídos, sendo 130 (86,1%) mulheres e 21 (13.9%) homens. Multifocalidade foi encontrada em 41 (27.2%), extensão extratireoidiana em 43 (28.5%) e metástase linfonodal em 36 (23.8%) casos. Apenas dois pacientes apresentaram metástase a distância. O tempo de seguimento médio foi 65,1 meses e observou-se nove (6%) pacientes com recorrências de neoplasia. Os pacientes com tumores com metástase linfonodal e/ou extensão extratireoidiana apresentaram maior risco de recorrência. Dos marcadores analisados, uma maior densidade de CD8 (que marca linfócitos citotóxicos) na área peritumoral esteve associada a uma tendência a melhor sobrevida livre de recorrência: 97,1% versus 87,5% (p=0,057), além de significativos menores índices de multifocalidade tumoral e de metástase linfonodal. A maior infiltração de linfócitos T CD8+ no tumor também se relacionou com menor ocorrência de metástase linfonodal nesta amostra (18,4% versus 38,1%, p=0,011). Além disso, a densidade desta marcação, tanto no interior da lesão neoplásica como em área peritumoral foi significativamente maior nos casos de carcinomas papilíferos restritos à tireoide, ou seja, sem extensão extratireoidiana e sem metástases. Os demais marcadores analisados não apresentaram relação significativa e consistente com recorrência ou outros fatores prognósticos. CONCLUSÕES: Nas neoplasias malignas de tireoide, o microambiente imune parece ter uma relação com características patológicas de agressividade. Este estudo mostrou que em carcinoma papilífero de tireoide quando não associado à tireoidite significativa, a densidade do infiltrado tumoral e peritumoral por linfócitos T CD8+ está inversamente relacionada com chance de disseminação metastática linfonodal e, provavelmente, com recidiva da doença, sendo, portanto, um marcador de melhor prognóstico. Este dado sugere que estes linfócitos exercem efeito antitumoral no carcinoma papilífero de tireoide, corroborando a importância da resposta imune na evolução desta neoplasia / INTRODUCTION: Within the last few decades thyroid cancer has the fastest rising incidence rate among all malignancies. In this group of different neoplasms, the papillary carcinoma, one of the well-differentiated carcinomas, represents the great majority and has favorable prognosis, with overall survival rates above 90% in five years. Although several risk classifications based on different prognostic features have been used, they are not accurate to predict which patients will have higher chance of recurrence, lymphatic metastasis and worse outcome, benefiting from more aggressive treatment. It has been described in several kinds of malignancies that tumor related immune infiltration has relation with prognosis and response to treatment. In this study, we characterize the immune microenvironment in papillary thyroid carcinomas, using immunohistochemical markers to inflammatory cells, and relate it with clinical and pathological prognostic features and with recurrence free survival rates. METHODS: The 151 included cases were selected from a database that included all patients who underwent surgical treatment for thyroid cancer at A.C.Camargo Cancer Center between the years 2008 and 2010. Tumor with significant thyroiditis were excluded. The selected tumors were submitted to immunohistochemical reactions with markers of inflammatory cells and analysis in complete slides by two experienced pathologists. Clinical and pathological features were evaluated, as well as recurrence and survival, relating them with the reading of marked cells. Survival analysis were made using Kaplan-Meier method, comparing survival curves with the Logrank test. RESULTS: One hundred and fifty one patients were included, of which 130 (86.1%) were females and 21 (13.9%) males. Multifocal disease was found in 41 cases (27.2%), extrathyroidal extension in 43 (28.5%) and lymph node metastasis in 36 (23.8%). Only two patients had distant metastasis. The mean follow-up time was 65.1 months and we observed nine (6%) tumor recurrences. Tumors with lymph node metastasis and/or extrathyroidal extension showed significantly higher recurrence rates. Of the analyzed markers, the cases with a higher density of CD8 (which marks cytotoxic T lymphocytes) in peritumoral areas presented a trend to better recurrence-free survival: 97.1% versus 87.5% (p=0.057), in addition to lower rates of mutifocal tumors and lymph node metastasis. A higher infiltration rate of CD8+ T lymphocytes in the tumor also correlated with less risk of lymph node metastasis in this sample (18.4% versus 38.1%, p=0.011). Besides that, the density of this marking both in the tumor and in peritumoral areas, was significantly higher in the papillary carcinomas limited to the thyroid gland (without extrathyroidal extension or metastasis). The other markers analyzed did not presented significant or consistent relation with recurrence or other prognostic factors. CONCLUSIONS: In thyroid cancer, the immune microenvironment seems to relate with pathological features of aggressiveness. This study showed that in papillary thyroid carcinomas without significant thyroiditis, the density of tumoral and peritumoral infiltration by CD8+ T lymphocytes is inversely related with lymph node metastasis rate and probably with recurrence, being therefore a marker of better prognosis. These data suggest that these lymphocytes play an anti-tumoral role in papillary thyroid carcinoma, supporting the implication of immune response in the progression of this neoplasm

O osteossarcoma é um tumor ósseo maligno, de maior ocorrência em cães, possui rápido crescimento e alto potencial metastático. Assim, o cão é um modelo útil para o estudo da doença em humanos, tendo em vista as semelhanças clínicas e histopatológicas que ocorrem em ambas às espécies. Atualmente, os estudos a respeito de células-tronco são promissores considerando seu alto potencial terapêutico. Entretanto, ainda prevalecem muitas dúvidas referentes ao tratamento de tumores utilizando a terapia celular. Este tema é pouco conhecido e estudado, por isso, o objetivo deste trabalho foi avaliar a interação entre células-tronco obtidas da polpa dentária canina com as células derivadas osteossarcoma canino. Foram realizados cocultivos celulares das células derivadas de polpa dentária canina, osteossarcoma canino e derivadas de osso normal canino. Analisou-se os aspectos morfológicos das células cocultivadas e controle, assim como a atividade proliferativa, a morte celular, o potencial elétrico mitocondrial e a expressão gênica. A partir dos resultados obtidos, concluiu-se que a interação entre a célula-tronco da polpa dentária canina imatura e as células de osteosarcoma canino não apresentam alterações morfológicas. Entretanto, as células-tronco derivadas da polpa dentária canina e de osso fetal canino sadio parecem servir de suporte para o crescimento tumoral. Além disso, a cocultura celular, em todos os grupos testados, promove alterações na expressão gênica e proteica. / Osteosarcoma is a malignant bone tumor most frequent in dogs. It has fast growth and high metastatic potential. Thus, the dog is an useful model for the study of human disease, due to the clinical and histological similarities found in both species. Currently, studies about stem cells are promising considering its high therapeutic potential. However, many doubts still exist regarding the treatment of tumors using cell therapy. This theme is little known and studied. Therefore, the aim of this study was to evaluate the interaction between stem cells obtained from canine immature dental pulpstem cells with osteosarcoma cells derived from dogs. Cellular coculture were performed using cells derived from canine dental pulp, canine osteosarcoma and canine normal bone. The morphological aspects of cocultured cells and control were analyzed, as well as proliferative activity, cell death, the mitochondrial membrane electric potential and gene expression. In summary, it was concluded that the interaction between stem cells from canine immature dental pulp and canine osteosarcoma cells did not show morphological changes. However, stem cells derived from canine dental pulp and healthy canine fetal bone serve to support tumor growth. Furthermore, the cell coculture in all groups tested, causes changes in gene and protein expression.

Une forte production de la chimiokine CCL2 par les cellules malignes et les cellules stromales a été démontrée dans la plupart des cancers humains. Ainsi, l’axe chimiokinique CCR2/CCL2 est un important marqueur du développement des cancers ; ce même axe est associé à la récurrence de tumeurs après thérapie anticancéreuses. Les macrophages associés aux tumeurs (TAM) et les lymphocytes T régulateurs (Treg) ont des capacités immunosuppressives robustes et contribue à la croissance tumorale. Durant cette thèse, je me suis intéressé à la fonction de l’expression du récepteur de chimiokine CCR2 par ces cellules dans le contexte de thérapies anticancéreuses. Nous avons montré que le récepteur de chimiokines CCR2 contrôle la migration des Treg en contexte tumoral, chez l’homme et la souris, et que son expression par les Treg peut servir de biomarqueur de la réponse à la chimiothérapie. Notre étude indique une nouvelle fonction de CCR2 et définie un nouveau sous-type de Treg impliqué dans la régulation de l’immunité antitumorale. En parallèle, nous avons pu mettre en évidence que les métastases pulmonaire sont composées à la fois de macrophages résident du tissu et de macrophages recrutés via l’axe CCR2. La présence de macrophages résidents au sein des tumeurs pourrait contribuer à l’hétérogénéité des microenvironnements de diffèrent type de tumeurs. Le récepteur CCR2 est important pour le la phase de rechute après chimiothérapie, indiquant un rôle limité des macrophages résidents dans ce phénomène. De plus, nous avons montré que le VEGF joue un rôle direct dans la survie des TAM. Ainsi, la combinaison de la chimiothérapie avec un anticorps anti-VEGF cible simultanément les TAM résidents et recrutés et permet d’augmenter l’efficacité de la chimiothérapie. / Malignant and stromal cells are strong producer of the chemokine CCL2 in most human cancers. The chemokine axis CCR2/CCL2 is thus a key marker of cancer development, but is also associated with relapse following therapy. Tumour associated macrophages (TAM) and regulatory T cells (Treg) display robust immunosuppressive capacities and contribute to tumour growth. My thesis work focused on the function of the expression of the chemokine receptor CCR2 by these cell types in the context of anticancer therapies. We have shown that CCR2 controls the migration of Treg in tumoral context, in both human and mice, and that the expression of this receptor by Treg could serve as a biomarker of the response to chemotherapy. Our study indicate a novel function of CCR2, defining at the same time a new Treg subset implicated in the regulation of antitumor immunity.We have also demonstrated that pulmonary metastases are composed of both tissue resident and recruited macrophages. The presence of resident macrophages within tumours could contribute to the heterogeneity of the microenvironment of different tumour types. CCR2 is largely implicated in the relapse phase following chemotherapy, indicating a limited role for resident macrophages in this phenomenon. Meanwhile, we have demonstrated that VEGF plays a direct role in TAM survival. The combination of chemotherapy with an anti-VEGF antibody targets both resident and recruited TAM, thereby enhancing the efficacy of chemotherapy. Finally, we have shown that the CCR2/CCL2 axis is implicated in the response to radiotherapy by enhancing the recruitment of both Treg and TAM. This work provides evidences for a central role of the CCR2/CCL2 axis in mediating Treg and TAM co-localization in response to anticancer therapy, this axis could also contribute to establishment of immunosuppressive networks in tumours. Our results provide a better understanding of the immune mechanism implicated in resistance to anticancer therapies.

Parmi les cellules stromales présentes dans le microenvironnement du cancer du sein, les adipocytes représentent des acteurs émergents dans la progression tumorale. Etudier le dialogue croisé entre adipocytes et cellules tumorales est d'importance en médecine, car l'obésité est un facteur reconnu de mauvais pronostic dans de nombreux cancers, notamment le cancer du sein. Nos résultats montrent que lorsque les adipocytes sont maintenus en présence des cellules tumorales pendant un temps prolongé, ils se réorientent progressivement en cellules fibroblastiques, présentant des caractéristiques de fibroblastes-Associés au Cancer. Nous avons nommé ces cellules, ADFs pour " Adipocytes Derived Fibroblasts " et montré que ces derniers, présents dans des tumeurs mammaires humaines, favorisent de manière importante l'invasion des cellules tumorales. D'autre part, nous avons démontré que les adipocytes favorisent une résistance pléiotropique aux cellules tumorales via un mécanisme d'efflux original et que cette chimiorésistance médiée par les adipocytes est amplifiée en conditions d'obésité. Ces travaux pourraient expliquer, en moins en partie, le mauvais pronostic des cancers du sein chez les patientes obèses. / Among stromal cells present in breast cancer, adipocytes represent emerging players in tumor progression. Studying the role of adipocytes in cancer is a major interest since epidemiological studies have convincingly established that obesity is associated with a poor outcome for several cancers, especially breast cancer. We demonstrated that some CAFs (Cancer Associated Fibroblasts) present in the breast tumor stroma arise from the "dedifferenciation" of adipocytes upon prolonged stimulation by tumor cells. This population was named ADFs (Adipocytes-Derived Fibroblasts) and was found in clinical samples of breast cancer. We further demonstrated that ADFs stimulate the invasive capacities of tumor cells. Moreover, our results suggest that adipocytes promote multidrug resistance in breast cancer cell lines mediated by an original efflux mechanism. Finally, we demonstrated that adipocyte-induced chemoresistance is amplified by obesity. This work may explain, at least in part, the poor prognosis of breast cancer in obese patients.

Bien que rares, les tumeurs endocrines développées chez l'Homme demeurent problèmatiques. Une meilleure compréhension des mécanismes qui régulent leur croissance constitue un objectif essentiel pour identifier des cibles thérapeutiques nouvelles.Dans la première partie de cette thèse, nous avons étudié l'impact du microenvironnement tumoral (MeT), définit par l'ensemble des facteurs qui encerclent la niche tumorale primitive, sur la croissance des tumeurs endocrines digestives. In vitro, nous observons un effet prolifératif réciproque entre des fibroblastes, l'une des cellules pivots du MeT, et des lignées cellulaires humaines de tumeurs endocrines pancréatiques, tel qu'il est susceptible d'exister in situ. Dans une seconde partie, nous avons montré que le pegvisomant, un antagoniste du récepteur de l'hormone de croissance utilisé chez des patients atteint d'adénome hypophysaire somatotrope, n'a pas d'effet prolifératif in vitro sur les cellules somatotropes adénomateuses. / Although rare, endocrine tumors developed in Humans remain problematic, such as a better understanding of their regulatory mechanisms of growth represent a step forward to identify new therapeutical targets.In the first part of this thesis, we investigated the impact of the tumor microenvironment (TME), as defined by the factors surrounding the tumor primitive niche, on the growth of human digestive endocrine tumors. We, here, showed the occurrence of a reciprocal proliferation between human fibroblasts, a key cell within the TME, and human pancreatic neuroendocrine tumor cell lines, suggesting that human fibroblasts may constitue a new therapeutical target of interest in the TME of digestive endocrine tumors. In a second part, we showed that pegvisomant (PEG), a growth hormone receptor antagonist currently used in patients with GH-secreting pituitary adenoma, did not impact in vitro the proliferation rate of GH-secreting adenoma cells and therefore is suitable in patients with a persisting GH-secreting pituitary adenoma residue after surgery.

Solid tumors are complex structures comprising besides the cancer cells vasculature, extracellular matrix (ECM), soluble molecules and a plethora of various other cell types. These components form a so-called tumour microenvironment. From the numerous cell types that are part of tumor microenvironment, tumor infiltrating lymphocytes (TILs) play a major role in patient prognosis. Their presence is also of major importance with regard to new biological therapies based on immune checkpoint inhibitors. Crucial role of TILs is also reflected by the new approaches in cancer diagnostics namely by Immunoscore method (currently used in clinical settings). Immunoscore is based on localization and quantification of CD3+ and CD8+ TILs in thin histological sections of tumor tissue. The question remains to which extent the information obtained from 2D slices reflects the situation in tumor microenvironment considering its spatial heterogeneity. The development of new methodological approaches allowing evaluation of histological information in 3D is the key to answer this question. The theoretical part of this work first describes the heterogeneity of the tumor microenvironment and the role of immune cells within it. Then, the role of spatial heterogeneity and its possible influence on the histopathological...

Head and neck cancers are etiologically associated with smoking and alcohol consumption. Part of these tumors is induced by HPV and their incidence is increasing in the last decade. Patients with virally induced tumors have better prognosis even though they are usually diagnosed with tumors in advanced stage. One of the possible explanations may be better stimulation of the immune system by viral antigens. Macrophages are cells of the innate immune system which belong to professional phagocytes. They are called TAM upon infiltration to the tumor where they represent heterogeneous group of cells. Two main phenotypes are antitumor M1 and protumor M2 macrophages. TAMs are a major component of tumor microenvironment of many types of tumors, one of them are also head and neck cancers. In my thesis I focused on the immunohistochemical detection of M1 and M2 macrophages in the head and neck tumors of viral and non-viral etiology and at the same time RT-qPCR analyses of gene expression of macrophage-associated and/or immunosuppressive genes IDO1, ARG1, CD163, NOS2 a PTGS2 was performed. My data showed that HPV- negative tumors had higher number of M2 macrophages with typical markers CD163, ARG1 and PTGS2. It is known that patients with these tumors have worse prognosis of the disease. Due to high...

L’objectif de ce travail a consisté à évaluer le rôle d’un microenvironnement avec une haplo-insuffisance hétérozygote du gène BRCA1 dans les événements précoces de la transformation tumorale du cancer du sein. Dans ce but, nous avons modélisé un stroma BRCA1-muté en utilisant des cellules souches / stromales mésenchymateuses (MSCs) obtenues par différenciation de cellules souches pluripotentes induites (iPSCs) issues d’une patiente porteuse de la mutation (MSCs BRCA1+/-). Ces cellules mutées pour BRCA1 ont été comparées à des MSCs sans la mutation (MSCs BRCA1+/+) générées à partir d’iPSCs BRCA1+/+. Ce travail de thèse a porté sur l’influence du stroma BRCA1-muté à travers deux axes : le caractère pro-angiogénique des MSCs BRCA1+/- et l’induction d’une transition épithélio-mésenchymateuse (TEM) sur des cellules mammaires normales (HME1).Nous montrons que les MSCs BRCA1-muté présentent des propriétés pro-angiogéniques significativement augmentées en surexprimant le facteur hypoxique HIF-1α et des facteurs de la famille du VEGF, PDGF et Angpt se traduisant par des capacités augmentées à former des structures vasculaires in vitro et in vivo. Les MSCs BRCA1-muté présentent également des capacités migratoires supérieures en produisant et sécrétant la périostine (POSTN), une protéine de la matrice extracellulaire impliquée dans l’adhésion, la motilité et la migration cellulaires. Ces capacités ont été validées par une approche de siRNA spécifique pour la POSTN. In vivo, nous montrons que la co-injection de MSCs BRCA1-muté et de cellules malignes mammaires murines (4T1-Luc-GFP) a permis d’augmenter significativement la croissance tumorale et la formation de métastases pulmonaires. Ces résultats sont corrélés avec la détection de la POSTN in situ et avec la formation d’un réseau vasculaire tumoral développé, quantifié par marquage du CD34. Par ailleurs nous avons démontré qu’un surnageant de MSCs BRCA1+/- peut induire une TEM des cellules HME1 en favorisant l’acquisition d’un phénotype souche cancéreux (CD24Low/CD44High) et en accélérant leur migration. Enfin nous avons initié la production in vitro d’organoïdes mammaires en utilisant des MSCs et des HME1 afin d’étudier plus précisément les mécanismes moléculaires de cette TEM après contact et des possibles événements précoces de la transformation maligne. Nos résultats indiquent que les MSCs peuvent participer à l’initiation tumorale et à la progression métastatique dans un contexte d’une mutation hétérozygote du gène BRCA1. La POSTN pourrait représenter à la fois un marqueur pronostique mais également une cible thérapeutique pour ces cancers du sein héréditaires. / The aim of this study was to evaluate the role of a BRCA1 heterozygous haplo-deficient microenvironment in the early events of tumour transformation of breast cancer. For this purpose we modeled a BRCA1-mutated stroma using mesenchymal stem / stromal cells (MSCs) obtained by differentiation of induced pluripotent stem cells (iPSCs) from a patient carrying the mutation (MSCs BRCA1+/-). These BRCA1-mutated cells were compared to MSCs without the mutation (MSCs BRCA1+/+) generated from iPSCs BRCA1+/+. This study focuses on two aspects of BRCA1-mutated stroma, namely the pro-angiogenic properties of BRCA1+/- MSCs and the induction of an epithelial-mesenchymal transition (EMT) on normal breast cells (HME1).We have shown that BRCA1-mutated MSCs exhibit enhanced pro-angiogenic properties by overexpressing the hypoxic factor HIF-1α and factors from VEGF, PDGF and Angpt families resulting in increased capacities to form vascular structures in vitro and in vivo. BRCA1-mutated MSCs exhibit also higher migratory capabilities by production and secretion of periostin (POSTN), an extracellular matrix protein, which is involved in cell adhesion, motility and migration. These capacities have been validated by a specific siRNA approach for POSTN. In vivo, the coinjection of BRCA1-mutated MSCs with murine breast cancer cell line (4T1-Luc-GFP) promotes tumour growth and the formation of lung metastases. These results are correlated with in situ POSTN detection and with the formation of a developed tumour vascular network, quantified by CD34 staining. We also demonstrated that supernatant of BRCA1+/- MSCs can induce an EMT on HME1 cells by promoting the acquisition of stemness properties (CD24Low/CD44High) and accelerating their migration. Finally we initiated the in vitro production of mammary organoids using MSCs and HME1 in order to study more precisely the molecular mechanisms of this EMT after contact and possible early events of the malignant transformation. These results indicate that MSCs can participate to tumour initiation and metastatic progression in heterozygous BRCA1-mutated background. POSTN could represent a prognostic marker and a therapeutic target for these hereditary breast cancers.