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<p>The majority (75%) of renal cell carcinoma (RCC) is comprised of clear cell renal cell carcinoma (ccRCC). Despite ccRCC being the most aggressive form of RCC, our knowledge regarding the pathogenesis of the disease remains limited. We have identified upregulation of the extracellular protein periostin (POSTN) in ccRCC. Western blot analysis of ccRCC tumours from 27 patients demonstrated high POSTN protein expression in 26 tumours compared to their respective adjacent normal kidney tissue (ANK). Immunohistochemistry (IHC) analysis revealed high levels of stromal POSTN protein in the ccRCC primary tumours and 16 metastasized ccRCCs. Intriguingly, abundant stromal POSTN in the tumour and non tumour boundary were observed in local and metastaized ccRCC, and in A498 ccRCC cell-derived xenograft tumours. Collectively, these results suggest that the ccRCC-associated POSTN was derived from the stroma. This notion was supported by the co-existence of POSTN with α-smooth muscle actin (αSMA) in both local and metastasized ccRCC tumours. αSMA is a marker of activated stromal fibroblasts (myofibroblasts). Furthermore, co-culture of NIH3T3 murine fibroblasts with human A498 or 786-0 ccRCC cells dramatically enhanced POSTN transcription and secretion from NIH3T3 cells. Extracellular POSTN significantly enhanced A498 cell attachment. Upregulation of POSTN in NIH3T3 cells enhanced their proliferation. Taken together, my research demonstrates that 1) ccRCC induces fibroblast-mediated accumulation of extracellular POSTN, 2) stromal POSTN enhances ccRCC attachment, and 3) high levels of POSTN promotes fibroblasts' proliferation. These observations suggest a critical role for POSTN in mediating the co-evolving process between ccRCC and its stroma during ccRCC pathogenesis.</p> / Master of Science (MSc)

Head and neck squamous cell carcinomas are still challenging despite progress in the oncological treatment. Study of the molecular biology allows to deeply characterize tumor properties and to predict the prognosis for affected patients. Nowadays there are many drugs clinically tested in the group of targeted therapy medicine Experimental work comprised both in vitro and in situ assays, being performed thanks to the collaboration between a number of departments of the 1st Faculty of Medicine of the Charles University in Prague, Academy of Sciences of the Czech Republic, Institute of Hematology and Blood Transfusion and Faculty of Veterinary Medicine of the Ludwig-Maxmillian University Munich. Galectin-1 is important inductor of the myofibroblasts/cancer associated fibroblasts. These fibroblasts are regarded as negative prognostic markers thanks to their capability of invasive cancer cells induction. On the other hand, Galectin-9 is not present in the carcinoma and in the case of dysplasia, its expression indicate aberrant features together with aberrant expression of keratin 14 and 19. Except from galectins using as prognostic markers, we focused on the galectins as a therapeutics instruments as well. Presented work with mutant variants of galectin-2 proved their effect on both pharmacodynamics and...

L’adénocarcinome pancréatique (PDA) présente une résistance accrue aux chimiothérapies. Un concept propose que sa composition cellulaire participe à ce processus en limitant l’accès aux drogues tout en modulant les capacités des cellules tumorales. En effet, les cellules non tumorales, principalement mésenchymateuses (CAFs) et immunitaires, représentent 70% de la masse tumorale et forment le microenvironnement intra-tumoral ou stroma. L’impact du stroma dans le développement et la progression des PDA se trouve être au centre d’un large champ d’investigations cliniques. Notre première étude a porté sur un facteur neurotrophique, Slit2, impliqué dans la guidance axonale est sécrété par les CAFs. Ce dernier induit une augmentation de la migration des cellules de Schwann et des changements morphologiques et quantitatifs des cellules neuronales. Ainsi, les nerfs se retrouvent plus nombreux et de taille plus importante dans la tumeur comparée à un pancréas sain, c’est ce qu’on appelle le remodelage neural. Notre second travail a permis d’identifier un complexe multi-protéique (ANXA6/LRP1/TSP1), associé au trafic vésiculaire, présent uniquement dans le compartiment stromal et plus particulièrement dans les CAFs. Ce complexe est porté par des vésicules extracellulaires et procure un avantage prolifératif et pro-migratoire aux cellules tumorales. Les données obtenues au cours de mon travail de thèse constituent un rationnel fort pour étudier le potentiel thérapeutique des éléments permettant le dialogue entre les différents compartiments de la tumeur dans le but de sensibiliser les cellules tumorales aux chimiothérapies et ainsi d’améliorer la survie des patients. / Pancreatic adenocarcinoma (PDA) is particularly resistant to current therapies. A concept suggests that its cellular composition participates in this process, limiting drugs access and affecting tumor cells behavior. Indeed, non-tumor cells, mainly mesenchymal (including Cancer Associated Fibroblasts, CAFs) and immune cells display over 70% of the tumor mass and form the intra-tumoral microenvironment or stroma. The impact of stroma in PDA development and progression is at the center of many clinical investigations. Firstly, we studied a neurogenic factor, Slit2, implicated in axon guidance pathway and secreted by CAFs. Slit2 increases Schawnn cells migration and morphologic changes of neural cells. Indeed, nerve size and density are increased in a tumor compared to a healthy pancreas, that is called, neural remodeling. Secondly, we worked on a multi-proteic complex (ANXA6/LRP1/TSP1), associated to vesicular trafficking, only expressed in stromal compartment, and mainly in CAFs. This complex is present in extracellular vesicles and confers proliferative and pro-migratory capacities to tumor cells. Data obtained during my thesis constitute an important rationale to target the crosstalk between tumor and stromal compartment, in order to sensitize tumor cells to chemotherapy and improve patient survival.

Head and neck squamous cell carcinomas are still challenging despite progress in the oncological treatment. Study of the molecular biology allows to deeply characterize tumor properties and to predict the prognosis for affected patients. Nowadays there are many drugs clinically tested in the group of targeted therapy medicine Experimental work comprised both in vitro and in situ assays, being performed thanks to the collaboration between a number of departments of the 1st Faculty of Medicine of the Charles University in Prague, Academy of Sciences of the Czech Republic, Institute of Hematology and Blood Transfusion and Faculty of Veterinary Medicine of the Ludwig-Maxmillian University Munich. Galectin-1 is important inductor of the myofibroblasts/cancer associated fibroblasts. These fibroblasts are regarded as negative prognostic markers thanks to their capability of invasive cancer cells induction. On the other hand, Galectin-9 is not present in the carcinoma and in the case of dysplasia, its expression indicate aberrant features together with aberrant expression of keratin 14 and 19. Except from galectins using as prognostic markers, we focused on the galectins as a therapeutics instruments as well. Presented work with mutant variants of galectin-2 proved their effect on both pharmacodynamics and...

Les cancers ovariens, se développant de façon silencieuse, diagnostiqués à des stades tardifs et de mauvais pronostiques, requièrent urgemment la mise au point de nouvelles options thérapeutiques. Ma thèse s'est attachée à caractériser les propriétés physiques et biologiques des cancers ovariens Séreux de Haut Grade (HGSOC), représentant 75% des tumeurs ovariennes.En premier lieu, nous avons démontré la valeur pronostique de la protéine MAP3K8 s'accumulant dans les HGSOC. Nous avons montré que MAP3K8 contrôle la prolifération et la migration des cellules cancéreuses via la transition G 1/S et les mécanismes d'adhésion dynamique. Aussi, nous avons mis en évidence que MAP3K8 active majoritairement la voie MEK, présentant ainsi un potentiel prédictif des inhibiteurs de MEK, les positionnant comme une stratégie thérapeutique prometteuse, en combinaison des thérapies conventionnelles, chez les HGSOC.Dans un second axe de ma thèse, nous avons montré que la rigidité augmente avec la taille tumorale, chez les HGSOC présentant une signature moléculaire « Fibrose ». Cette rigidification tumorale s'associe à une accumulation de stroma et un remodelage du réseau de collagène, mais aussi à une activation spécifique de la voie MEK. De façon intéressante, la rigidification tumorale accompagne un « switch » métabolique glycolytique, restreint au centre de la tumeur, la périphérie demeurant plus molle, et différant par une production élevée de collagène et un métabolisme OXPHOS. La rigidité pourrait donc être au carrefour de 3 processus majeurs, tels un remodelage de la matrice, l'activation de MEK et un switch métabolique stromal, expliquant, au moins en partie, la progression des HGSOC. / Ovarian cancers, which develop in a silent manner in the peritoneal cavity, resulting in a late diagnosis and a poor prognosis, urgently require new therapeutic strategies. In this context, my thesis aimed at better characterize the physical and biological properties of the High Grade Serous ovarian cancers (HGSOCs), accounting for 75% of the tumours.First, we found that the protein MAP3K8 accumulates in HGSOC and is a potential prognostic marker for these tumours. We demonstrated that MAP3K8 controls cancer cell proliferation and migration by regulating key players in Gl/S transition and adhesion dynamics. Importantly, we highlighted that MAP3K8 function is mainly mediated by the MEK pathway, and exhibits a predictive potential for MEK inhibitors, defining them as a promising therapeutic option, in combination with conventional therapy, for HGSOC patients.In a second part of my thesis, we showed that tumor stiffness is increased during tumor growth in HGSOC presenting a "Fibrosis" molecular signature. Moreover, tumor stiffening is associated with high stromal content and remodeling of the collagen network. Interestingly, the MEK kinase was specifically activated upon tumor stiffening. Furthermore, tumor stiffness accompanies a glycolytic metabolic switch, restricted to the central part of stiff tumors. Indeed, the periphery of stiff tumors remains softer than the central part with stromal cells secreting high levels of collagens and showing an OXPHOS metabolism. Thus, tumor stiffness could be at the crossroad of three major processes, i.e. matrix remodeling, MEK activation and stromal metabolic switch, that might explain, at least in part, the progression of HGSOC.

The majority of cancer research projects mainly focus on the epithelial cancer cell, while the role of the tumour stroma has been largely neglected. Conventional 2D techniques, such as well plates and other kinds of tissue culture plastic, and animal models are mainly used to broaden our understanding of how tumours arise, develop, and induce metastasis. However, there is accumulating evidence suggesting a tremendous impact of the non‐cancerous tumour stroma on carcinogenesis, while other publications illustrate the great importance of advanced 3D in vitro models for cancer research. The overall goal of this work was to investigate how cancer associated fibroblasts (CAFs; the most abundant component in the tumour stroma) and normal prostate fibroblasts (NPFs), isolated from patients diagnosed with aggressive forms of prostate cancer, contribute to angiogenesis, an important hallmark of cancer progression. For this purpose, a 3D in vitro angiogenesis co‐culture model was established. At first, two (semi‐) synthetic hydrogel platforms, gelatine methacrylate (GelMA) and star‐shaped (star)PEG‐heparin hydrogels were characterised and their physicochemical properties were compared with each other. Interestingly, GelMA gels shrank while starPEG‐heparin gels swelled in cell culture medium over the course of 24 hours. The cell concentration, in addition to the stiffness, was critical for the formation of endothelial networks, and the knowledge of swelling behaviour enabled the adjustment of initial cell density to ensure the density between both gel types was comparable. Moreover, preliminary tests with mesenchymal stem cells demonstrated that the hydrogel can be actively remodelled, as evaluated by stiffness parameters at day one and seven of incubation. Growth factors (GFs) affect cellular fate and behaviour, and storage, presentation and administration of such chemokines can be critical for certain cellular applications. Due to the high anionic charge density of heparin, starPEG‐heparin hydrogels are known to reversibly immobilise several GFs and thereby might mimic the GF reservoir of the extra cellular matrix. Thus, transport processes of GFs with low and high heparin affinity inside these hydrogels were analysed by fluorescence correlation spectroscopy and a bulk diffusion approach. Results indicated that diffusion constants were synergistically decreased with increasing size and heparin affinity of the diffusant. Next, the capability of endothelial cells (ECs) to self‐assemble and organise into 3D capillary networks was tested in GelMA, starPEG‐heparin and Matrigel hydrogels. Only starPEG‐heparin hydrogels allowed the formation of interconnected capillaries in macroscopic hydrogel samples. However, as it is widely used to test for pro‐ and anti‐angiogenic agents, the 2D Matrigel angiogenesis assay was included for subsequent co‐culture experiments of ECs and fibroblasts in order to investigate how the stromal cells influence the formation of endothelial networks. For a detailed characterisation of 3D structures, a conventionally applied 2D method (Maximum Intensity Projection for 3D reconstructed images, MIP) was compared to an optimised 3D analysing tool. As a result, it was discovered that MIP analysis did not allow for an accurate determination of 3D endothelial network parameters, and can result in misleading interpretations of the data set. Indirect co‐cultures of hydrogel‐embedded ECs with a 2D layer of fibroblasts showed that fibroblast‐derived soluble factors, including stromal cell‐derived factor 1 and interleukin 8, affected endothelial network properties. However, only co‐encapsulation of ECs and fibroblasts in starPEG‐heparin hydrogel discs revealed remarkable changes in endothelial network parameters between CAF and NPF samples. In detail, the total length and branching of the capillaries was increased. For two donor pairs, the diameter of capillaries was decreased in CAF samples compared to NPF samples, underlining the high physiological relevance of this model. In contrast, significant differences in 2D Matrigel assays were not detected between, CAF, NPF and control (ECs only) samples. In summary, a 3D angiogenesis co‐culture system was successfully developed and used to characterise stromal‐endothelial interactions in detail. The combination of advanced biomaterials (starPEG‐heparin) and 3D analysing techniques goes beyond conventional 2D in vitro cancer research, and opens new avenues for the development of more complex models to further improve the acquisition of more biologically relevant data.

La progression tumorale est un processus multi-étapes dépendant notamment des interactions entre les cellules cancéreuses et le stroma environnant. Le développement du cancer du sein implique une communication étroite entre les cellules épithéliales mammaires, les cellules inflammatoires, les myofibroblastes et les cellules endothéliales. Ainsi, le microenvironnement tumoral apparaît comme une cible de choix dans le traitement anti-tumoral. L’utilisation de modèles précliniques est une étape clé dans le développement et la validation de nouvelles thérapies. Néanmoins, peu d’études sont disponibles sur le rôle du stroma péri-tumoral dans ces modèles.Dans le but d’étudier le stroma péri-tumoral des modèles précliniques de cancers du sein, nous avons combiné une analyse par cytométrie en flux à une analyse par immunohistochimie afin d’identifier, puis de quantifier, les différentes populations stromales hématopoïétiques (lymphocytes, monocytes/macrophages, polynucléaires) et non hématopoïétiques (myofibroblastes, cellules endothéliales). Vingt et un modèles de xénogreffe de tumeurs humaines de cancers du sein ainsi que 2 modèles transgéniques (MMTV-PyMT et MMTV-ErbB2), ainsi que leurs allogreffes respectives, furent utilisés lors de ce travail.Les analyses des tumeurs humaines et murines ont montré un infiltrat stromal très hétérogène d’une tumeur à l’autre, avec pour composante majoritaire les macrophages. Un infiltrat important en polynucléaires a également été détecté dans les modèles de PDX, caractéristique d’une inflammation locale importante dans ces modèles. L’analyse phénotypique de macrophages a montré une expression variable de marqueurs M1 et M2 dans les modèles de PDX. Les macrophages issus de tumeurs murines transgéniques, spontanées ou allogreffées, présentaient quant à eux un profil majoritairement M1. L’étude transcriptomique de macrophages triés, a permis à la fois de valider les résultats obtenus au niveau protéique mais a également mis en évidence des différences majeures dans l’expression de nombreux gènes, impliqués dans des voies de signalisation variées telles que la croissance tumorale, l’invasion et la métastase.Cette étude nous a permis de mettre en évidence le rôle de la tumeur sur son microenvironnement. En effet, celle-ci est à la fois capable d’attirer un panel de cellules stromales qui lui et propre et ensuite de l’activer de façon spécifique. / Tumor development is a multi-step process influencing by interactions between tumor cells and surrounding stroma. Breast cancer development involves a high level of communication between mammary epithelial cells, inflammatory cells, myofibroblasts and endothelial cells. So, the tumoral microenvironment appears as a prime target for anti-tumoral treatment. The use of preclinical models is a critical step in development and validation processes of new therapies. Nevertheless, the role of stroma in these models is poorly understood.In order to evaluate stromal cell populations in breast cancer preclinical models, we combined flow cytometry analysis and immunohistochemistry to identify, and then quantify, various stromal populations as hematopoietic cells (lymphocytes, monocytes/macrophages, polymorphonuclear leukocytes) and non-hematopoietic cells (myofibroblasts, endothelial cells). Twenty-one breast cancer patient-derived xenografts as well as 2 transgenic mouse models (MMTV-PyMT and MMTV-ErbB2), and their respective allografts, were studied.Analysis of human and murine tumors showed a strong heterogeneity between tumors regarding infiltrating stroma-cells, with a high proportion of macrophages. A significant amount of polymorphonuclear leukocytes was also detected in PDXs, indicating a local inflammation in these models. The phenotypic analysis of macrophages showed a variable expression of M1 and M2 markers in PDXs. Macrophages infiltrating transgenic mouse tumors, spontaneous or allografted, were mainly M1. Transcriptomic analyses of sorted macrophages, allowed us to validate previous results but also highlighted major differences in the expression of numerous genes implicated in various pathways as tumor growth, invasion and metastasis.Finally, this study highlighted the impact of tumor cells on their surrounding stroma. Indeed, we demonstrate that cancer cells are able to attract a specific panel of stromal cells and activate them in a specific way.

Head and neck squamous cell carcinoma is the fifth most common cancer worldwide. They are associated with high morbidity and mortality. Despite considerable advances in surgical and oncological treatment over the past two decades, overall treatment outcome has only slightly improved. In my thesis I focused on serum gene expression analysis of head and neck cancer patients, which followed the tissue gene expression analysis in same patients. Further we investigated gene expression analysis in tumour stroma, which is now considered as significant factor in cancer initiation and progression. We revealed several candidate genes, which are involved in signalling pathways connected with cell differentiation and proliferation and are involved in apoptotic pathway (BCl-2, BCl-XL a MAX). As well we detected down-regulation of the main tumour suppressor p 53 protein. In peritumoural tissue we detected overexpression of cytokines typical for embryonal development and ectoderm differentiation - IGF-2 and BMP-4, which significantly influence the phenotype of normal keratinocytes. Further we identified several candidate genes relating with overexpression of Gal-1 in stromal myofibroblasts rich tissue (SPIN1, FUSIP1, TRIM23, SLC25A40, PTPLAD1, MP3K2). HNSCC is a heterogeneous disease despite the presence of...

Head and neck squamous cell carcinoma is the fifth most common cancer worldwide. They are associated with high morbidity and mortality. Despite considerable advances in surgical and oncological treatment over the past two decades, overall treatment outcome has only slightly improved. In my thesis I focused on serum gene expression analysis of head and neck cancer patients, which followed the tissue gene expression analysis in same patients. Further we investigated gene expression analysis in tumour stroma, which is now considered as significant factor in cancer initiation and progression. We revealed several candidate genes, which are involved in signalling pathways connected with cell differentiation and proliferation and are involved in apoptotic pathway (BCl-2, BCl-XL a MAX). As well we detected down-regulation of the main tumour suppressor p 53 protein. In peritumoural tissue we detected overexpression of cytokines typical for embryonal development and ectoderm differentiation - IGF-2 and BMP-4, which significantly influence the phenotype of normal keratinocytes. Further we identified several candidate genes relating with overexpression of Gal-1 in stromal myofibroblasts rich tissue (SPIN1, FUSIP1, TRIM23, SLC25A40, PTPLAD1, MP3K2). HNSCC is a heterogeneous disease despite the presence of...