Elucidation of Tenascin-X involvement during tumor progression : focus on pancreatic carcinogenesis

La matrice extracellulaire est un réseau de macromolécules ayant un rôle architectural et pouvant influer sur le comportement cellulaire. Parmi les glycoprotéines matricielles, la famille des Ténascines comporte 4 membres (TN-C, -W, -R et -X) partageant une structure modulaire commune, mais présentant des patrons d’expression et des rôles distincts. La Ténascine-X (TNX), objet principal de cette thèse, est le membre le plus largement exprimé, puisque retrouvé dans la grande majorité des tissus conjonctifs sains adultes. Elle possède (1) un rôle architectural, par la régulation de l’espacement entre les fibres de collagène, et (2) des fonctions de signalisation grâce à son interaction avec plusieurs facteurs de croissance. L’implication des Ténascines en cancérologie a été inégalement étudiée et le statut et le rôle de la TNX restaient à élucider. L’adénocarcinome canalaire pancréatique (ADKP), représente 90% des cas de cancer du pancréas et est extrêmement agressif. Les patients atteints de ce cancer présentent un taux de survie à 5 ans inférieur à 10%, du fait d’un diagnostic tardif et d’un arsenal thérapeutique encore inefficace. Ce type de tumeur se développe lentement, à partir de lésions pré-tumorales, dont les principales sont les PanINs (Pancreatic Intraepithelial Neoplasia). L’ADKP est également caractérisé par un important microenvironnement tumoral, aussi appelé stroma tumoral, résultant notamment d’un dépôt abondant de matrice extracellulaire (réaction desmoplasique). Mon objectif au cours de cette thèse était donc de comprendre le rôle de la TNX au cours de la progression tumorale en focalisant essentiellement mon étude sur la carcinogenèse pancréatique. Notre première étude fut d’analyser le statut de la TNX dans les 6 cancers les plus incidents et mortels au monde. Dans cette étude, nous avons observé une diminution drastique de la TNX dans la plupart des cancers, à l’exception des tumeurs cérébrales. En outre, nous avons mis en évidence qu’une expression faible du gène TNXB était associée à un mauvais pronostic dans les tumeurs mammaires et pulmonaires. Par la suite, nous avons montré que la TNX était également perdue dans des échantillons humains d’ADKP, phénomène associé à une survie diminuée des patients. Dans cette deuxième étude, nous avons aussi décrit un dépôt dynamique de TNX au cours de la carcinogenèse pancréatique. En effet, celui-ci est renforcé et forme une gaine autour des lésions pré-tumorales de bas grade, qui s’amoindrit puis semble disparaître dans les stades les plus avancés. Une analyse plus fine de la localisation de la TNX a montré qu’elle était absente du stroma situé à proximité directe des lésions et positif pour αSMA (marqueur des fibroblastes activés). Enfin, nous avons montré in silico, par une approche de Gene Ontology, qu’une faible expression de TNXB dans des échantillons d’ADKP humains était associée à une forte prolifération et une dérégulation du système immunitaire notamment lié à l’inflammation. Dans les étapes précoces de la carcinogenèse pancréatique, nous supposons donc l’existence de 2 stromas, (1) l’un pro-tumoral et localisé en contact direct avec les cellules tumorales et (2) l’autre potentiellement anti-tumoral, ne participant pas à la réaction desmoplasique, mais renfermant la TNX qui régulerait la prolifération tumorale et l’inflammation. Mes travaux de recherche m’ont conduit à utiliser différents modèles dont un modèle murin prédisposé au cancer du pancréas. Cependant, nous avons pu observer, dans 10% des souris, le développement d’une tumeur thymique associée à une accélération de la carcinogenèse pancréatique et à une dérégulation du système immunitaire. L’analyse en profondeur de ce biais expérimental a conduit à la rédaction d’un troisième article. En conclusion, ces travaux de thèse ont permis de mettre en évidence l’implication de la TNX dans le processus tumoral, et souligne les limites de l’utilisation des modèles murins en biologie fondamentale.The extracellular matrix (ECM) is a network of macromolecules having an architectural role and which can influence cellular behavior. Among the matrix glycoproteins, the tenascin (TN) family is composed of 4 members (TNC, TNW, TNR and TNX) sharing a common modular structure, but exhibiting very different expression patterns and roles. Tenascin-X (TNX), the main subject of this thesis, is the most widely expressed member, since it is found in the vast majority of adult healthy connective tissues. It has (1) an architectural role, by regulating the spacing between collagen fibers, and (2) signaling functions through its interaction with several growth factors. The implication of tenascins in oncology has been unequally studied and the status and role of TNX remained to be elucidated. Pancreatic ductal adenocarcinoma (PDAC), which accounts for 90% of pancreatic cancer cases, is extremely aggressive. Indeed, patients suffering from PDAC have a 5-year survival rate less than 10%, due to late diagnosis and a still ineffective therapeutic arsenal. This type of tumor evolves slowly, from pre-tumor lesions, the main ones being PanINs (Pancreatic Intraepithelial Neoplasias). PDAC is also characterized by an important tumor microenvironment, also called tumor stroma, notably resulting from an abundant deposition of extracellular matrix (desmoplastic reaction). My objective during this thesis was therefore to understand the role of TNX during tumor progression by mainly focusing my study on pancreatic carcinogenesis. Our first study was to analyze the status of TNX in the 6 most common and lethal cancers in the world. In this work, we observed a drastic decrease of TNX in most cancers, except brain tumors. In addition, we have shown that low expression of the TNXB gene was associated with a poor prognosis in breast and lung tumors. Subsequently, we showed that TNX was also lost in human PDAC samples, a phenomenon associated with decreased patient survival. In this second study, we also described a dynamic deposition of TNX during pancreatic carcinogenesis. Indeed, it is reinforced and forms a sheath around low-grade pre-tumor lesions, which diminishes and then seems to disappear in the most advanced stages. A more detailed analysis of the location of TNX showed that it was absent from the stroma located in close proximity of the lesions and positive for αSMA (marker of activated fibroblasts). Finally, we have shown, in silico, thanks to a Gene Ontology approach, that a low expression of TNXB in human PDAC samples was associated with a strong proliferation and deregulation of the immune system, particularly linked to inflammation. In the early stages of pancreatic carcinogenesis, we therefore assume the existence of 2 stroma, (1) one pro-tumor and localized in direct contact with tumor cells and (2) the other potentially anti-tumor, not participating to the desmoplastic reaction, but containing TNX, which would regulate tumor proliferation and inflammation. My research has led me to use different models including a mouse model predisposed to pancreatic cancer. However, we observed, in 10% of the mice, the development of thymic tumor associated with an acceleration of pancreatic carcinogenesis and dysregulation of the immune system. The in-depth analysis of this experimental bias led us to the writing of a third article. In conclusion, this thesis work highlighted the involvement of TNX in the tumor process, and underlines the limits of the use of murine models in fundamental biology

Elucidation du rôle de la Ténascine-X dans la progression tumorale : focus sur la carcinogenèse pancréatique

The extracellular matrix (ECM) is a network of macromolecules having an architectural role and which can influence cellular behavior. Among the matrix glycoproteins, the tenascin (TN) family is composed of 4 members (TNC, TNW, TNR and TNX) sharing a common modular structure, but exhibiting very different expression patterns and roles. Tenascin-X (TNX), the main subject of this thesis, is the most widely expressed member, since it is found in the vast majority of adult healthy connective tissues. It has (1) an architectural role, by regulating the spacing between collagen fibers, and (2) signaling functions through its interaction with several growth factors. The implication of tenascins in oncology has been unequally studied and the status and role of TNX remained to be elucidated. Pancreatic ductal adenocarcinoma (PDAC), which accounts for 90% of pancreatic cancer cases, is extremely aggressive. Indeed, patients suffering from PDAC have a 5-year survival rate less than 10%, due to late diagnosis and a still ineffective therapeutic arsenal. This type of tumor evolves slowly, from pre-tumor lesions, the main ones being PanINs (Pancreatic Intraepithelial Neoplasias). PDAC is also characterized by an important tumor microenvironment, also called tumor stroma, notably resulting from an abundant deposition of extracellular matrix (desmoplastic reaction). My objective during this thesis was therefore to understand the role of TNX during tumor progression by mainly focusing my study on pancreatic carcinogenesis. Our first study was to analyze the status of TNX in the 6 most common and lethal cancers in the world. In this work, we observed a drastic decrease of TNX in most cancers, except brain tumors. In addition, we have shown that low expression of the TNXB gene was associated with a poor prognosis in breast and lung tumors. Subsequently, we showed that TNX was also lost in human PDAC samples, a phenomenon associated with decreased patient survival. In this second study, we also described a dynamic deposition of TNX during pancreatic carcinogenesis. Indeed, it is reinforced and forms a sheath around low-grade pre-tumor lesions, which diminishes and then seems to disappear in the most advanced stages. A more detailed analysis of the location of TNX showed that it was absent from the stroma located in close proximity of the lesions and positive for αSMA (marker of activated fibroblasts). Finally, we have shown, in silico, thanks to a Gene Ontology approach, that a low expression of TNXB in human PDAC samples was associated with a strong proliferation and deregulation of the immune system, particularly linked to inflammation. In the early stages of pancreatic carcinogenesis, we therefore assume the existence of 2 stroma, (1) one pro-tumor and localized in direct contact with tumor cells and (2) the other potentially anti-tumor, not participating to the desmoplastic reaction, but containing TNX, which would regulate tumor proliferation and inflammation. My research has led me to use different models including a mouse model predisposed to pancreatic cancer. However, we observed, in 10% of the mice, the development of thymic tumor associated with an acceleration of pancreatic carcinogenesis and dysregulation of the immune system. The in-depth analysis of this experimental bias led us to the writing of a third article. In conclusion, this thesis work highlighted the involvement of TNX in the tumor process, and underlines the limits of the use of murine models in fundamental biology.La matrice extracellulaire est un réseau de macromolécules ayant un rôle architectural et pouvant influer sur le comportement cellulaire. Parmi les glycoprotéines matricielles, la famille des Ténascines comporte 4 membres (TN-C, -W, -R et -X) partageant une structure modulaire commune, mais présentant des patrons d’expression et des rôles distincts. La Ténascine-X (TNX), objet principal de cette thèse, est le membre le plus largement exprimé, puisque retrouvé dans la grande majorité des tissus conjonctifs sains adultes. Elle possède (1) un rôle architectural, par la régulation de l’espacement entre les fibres de collagène, et (2) des fonctions de signalisation grâce à son interaction avec plusieurs facteurs de croissance. L’implication des Ténascines en cancérologie a été inégalement étudiée et le statut et le rôle de la TNX restaient à élucider. L’adénocarcinome canalaire pancréatique (ADKP), représente 90% des cas de cancer du pancréas et est extrêmement agressif. Les patients atteints de ce cancer présentent un taux de survie à 5 ans inférieur à 10%, du fait d’un diagnostic tardif et d’un arsenal thérapeutique encore inefficace. Ce type de tumeur se développe lentement, à partir de lésions pré-tumorales, dont les principales sont les PanINs (Pancreatic Intraepithelial Neoplasia). L’ADKP est également caractérisé par un important microenvironnement tumoral, aussi appelé stroma tumoral, résultant notamment d’un dépôt abondant de matrice extracellulaire (réaction desmoplasique). Mon objectif au cours de cette thèse était donc de comprendre le rôle de la TNX au cours de la progression tumorale en focalisant essentiellement mon étude sur la carcinogenèse pancréatique. Notre première étude fut d’analyser le statut de la TNX dans les 6 cancers les plus incidents et mortels au monde. Dans cette étude, nous avons observé une diminution drastique de la TNX dans la plupart des cancers, à l’exception des tumeurs cérébrales. En outre, nous avons mis en évidence qu’une expression faible du gène TNXB était associée à un mauvais pronostic dans les tumeurs mammaires et pulmonaires. Par la suite, nous avons montré que la TNX était également perdue dans des échantillons humains d’ADKP, phénomène associé à une survie diminuée des patients. Dans cette deuxième étude, nous avons aussi décrit un dépôt dynamique de TNX au cours de la carcinogenèse pancréatique. En effet, celui-ci est renforcé et forme une gaine autour des lésions pré-tumorales de bas grade, qui s’amoindrit puis semble disparaître dans les stades les plus avancés. Une analyse plus fine de la localisation de la TNX a montré qu’elle était absente du stroma situé à proximité directe des lésions et positif pour αSMA (marqueur des fibroblastes activés). Enfin, nous avons montré in silico, par une approche de Gene Ontology, qu’une faible expression de TNXB dans des échantillons d’ADKP humains était associée à une forte prolifération et une dérégulation du système immunitaire notamment lié à l’inflammation. Dans les étapes précoces de la carcinogenèse pancréatique, nous supposons donc l’existence de 2 stromas, (1) l’un pro-tumoral et localisé en contact direct avec les cellules tumorales et (2) l’autre potentiellement anti-tumoral, ne participant pas à la réaction desmoplasique, mais renfermant la TNX qui régulerait la prolifération tumorale et l’inflammation. Mes travaux de recherche m’ont conduit à utiliser différents modèles dont un modèle murin prédisposé au cancer du pancréas. Cependant, nous avons pu observer, dans 10% des souris, le développement d’une tumeur thymique associée à une accélération de la carcinogenèse pancréatique et à une dérégulation du système immunitaire. L’analyse en profondeur de ce biais expérimental a conduit à la rédaction d’un troisième article. En conclusion, ces travaux de thèse ont permis de mettre en évidence l’implication de la TNX dans le processus tumoral, et souligne les limites de l’utilisation des modèles murins en biologie fondamentale

Loss of Tenascin-X expression during tumor progression: A new pan-cancer marker

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Conversion of adult human fibroblasts into neural precursor cells using chemically modified mRNA

Direct reprogramming offers a unique approach by which to generate neural lineages for the study and treatment of neurological disorders. Our objective is to develop a clinically viable reprogramming strategy to generate neural precursor cells for the treatment of neurological disorders through cell replacement therapy. We initially developed a method for directly generating neural precursor cells (iNPs) from adult human fibroblasts by transient expression of the neural transcription factors, SOX2 and PAX6 using plasmid DNA. This study advances these findings by examining the use of chemically modified mRNA (cmRNA) for direct-to-iNP reprogramming. Chemically modified mRNA has the benefit of being extremely stable and non-immunogenic, offering a clinically suitable gene delivery system. The use of SOX2 and PAX6 cmRNA resulted in high co-transfection efficiency and cell viability compared with plasmid transfection. Neural positioning and fate determinant genes were observed throughout reprogramming with ion channel and synaptic marker genes detected during differentiation. Differentiation of cmRNA-derived iNPs generated immature GABAergic or glutamatergic neuronal phenotypes in conjunction with astrocytes. This represents the first time a cmRNA approach has been used to directly reprogram adult human fibroblasts to iNPs, potentially providing an efficient system by which to generate human neurons for both research and clinical application

Open-CSAM, a new tool for semi-automated analysis of myofiber cross-sectional area in regenerating adult skeletal muscle

Abstract Adult skeletal muscle is capable of complete regeneration after an acute injury. The main parameter studied to assess muscle regeneration efficacy is the cross-sectional area (CSA) of the myofibers as myofiber size correlates with muscle force. CSA analysis can be time-consuming and may trigger variability in the results when performed manually. This is why programs were developed to completely automate the analysis of the CSA, such as SMASH, MyoVision, or MuscleJ softwares. Although these softwares are efficient to measure CSA on normal or hypertrophic/atrophic muscle, they fail to efficiently measure CSA on regenerating muscles. We developed Open-CSAM, an ImageJ macro, to perform a high throughput semi-automated analysis of CSA on skeletal muscle from various experimental conditions. The macro allows the experimenter to adjust the analysis and correct the mistakes done by the automation, which is not possible with fully automated programs. We showed that Open-CSAM was more accurate to measure CSA in regenerating and dystrophic muscles as compared with SMASH, MyoVision, and MuscleJ softwares and that the inter-experimenter variability was negligible. We also showed that, to obtain a representative CSA measurement, it was necessary to analyze the whole muscle section and not randomly selected pictures, a process that was easily and accurately be performed using Open-CSAM. To conclude, we show here an easy and experimenter-controlled tool to measure CSA in muscles from any experimental condition, including regenerating muscle

Latent TGF-β Activation Is a Hallmark of the Tenascin Family

International audienceTransforming growth factor-β (TGF-β) isoforms are secreted as inactive complexes formed through non-covalent interactions between bioactive TGF-β entities and their N-terminal pro-domains called latency-associated peptides (LAP). Extracellular activation of latent TGF-β within this complex is a crucial step in the regulation of TGF-β activity for tissue homeostasis and immune cell function. We previously showed that the matrix glycoprotein Tenascin-X (TN-X) interacted with the small latent TGF-β complex and triggered the activation of the latent cytokine into a bioactive TGF-β. This activation most likely occurs through a conformational change within the latent TGF-β complex and requires the C-terminal fibrinogen-like (FBG) domain of the glycoprotein. As the FBG-like domain is highly conserved among the Tenascin family members, we hypothesized that Tenascin-C (TN-C), Tenascin-R (TN-R) and Tenascin-W (TN-W) might share with TN-X the ability to regulate TGF-β bioavailability through their C-terminal domain. Here, we demonstrate that purified recombinant full-length Tenascins associate with the small latent TGF-β complex through their FBG-like domains. This association promotes activation of the latent cytokine and subsequent TGF-β cell responses in mammary epithelial cells, such as cytostasis and epithelial-to-mesenchymal transition (EMT). Considering the pleiotropic role of TGF-β in numerous physiological and pathological contexts, our data indicate a novel common function for the Tenascin family in the regulation of tissue homeostasis under healthy and pathological conditions

Matching Gene Expression with Hypometabolism after Cerebral Ischemia in the Nonhuman Primate

International audienceTo correlate brain metabolic status with the molecular events during cerebral ischemia, a cDNA array was performed after positron emission tomography scanning in a model of focal cerebral ischemia in baboons. Cluster analysis for the expression of 74 genes allowed the identification of 4 groups of genes. In each of the distinct groups, the authors observed a marked inflection in the pattern of gene expression when the CMRo was reduced by 48% to 66%. These patterns of coordinated modifications in gene expression could define molecular checkpoints for the development of an ischemic infarct and a molecular definition of the penumbra

Coupling between Myogenesis and Angiogenesis during Skeletal Muscle Regeneration Is Stimulated by Restorative Macrophages

Summary: In skeletal muscle, new functions for vessels have recently emerged beyond oxygen and nutrient supply, through the interactions that vascular cells establish with muscle stem cells. Here, we demonstrate in human and mouse that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted together to couple myogenesis and angiogenesis in vitro and in vivo during skeletal muscle regeneration. Kinetics of gene expression of ECs and MPCs sorted at different time points of regeneration identified three effectors secreted by both ECs and MPCs. Apelin, Oncostatin M, and Periostin were shown to control myogenesis/angiogenesis coupling in vitro and to be required for myogenesis and vessel formation during muscle regeneration in vivo. Furthermore, restorative macrophages, which have been previously shown to support myogenesis in vivo, were shown in a 3D triculture model to stimulate myogenesis/angiogenesis coupling, notably through Oncostatin M production. Our data demonstrate that restorative macrophages orchestrate muscle regeneration by controlling myogenesis/angiogenesis coupling. : In this study, Chazaud et al. demonstrate that endothelial cells (ECs) and myogenic progenitor cells (MPCs) interacted to couple myogenesis and angiogenesis during skeletal muscle regeneration. EC- and MPC-derived Apelin, Oncostatin M, and Periostin controlled myogenesis/angiogenesis coupling and were required for myogenesis and vessel formation. They show that, via the production of Oncostatin M, restorative macrophages promoted myogenesis/angiogenesis coupling. Keywords: muscle stem cells, myogenesis, angiogenesis, skeletal muscle regeneration, macrophage

Aging Disrupts Muscle Stem Cell Function by Impairing Matricellular WISP1 Secretion from Fibro-Adipogenic Progenitors

Research on age-related regenerative failure of skeletal muscle has extensively focused on the phenotypes of muscle stem cells (MuSCs). In contrast, the impact of aging on regulatory cells in the MuSC niche remains largely unexplored. Here, we demonstrate that aging impairs the function of mouse fibro- adipogenic progenitors (FAPs) and thereby indirectly affects the myogenic potential of MuSCs. Using transcriptomic profiling, we identify WNT1 Inducible Signaling Pathway Protein 1 (WISP1) as a FAP-derived matricellular signal that is lost during aging. WISP1 is required for efficient muscle regeneration and controls the expansion and asymmetric commitment of MuSCs through Akt signaling. Transplantation of young FAPs or systemic treatment with WISP1 restores the myogenic capacity of MuSCs in aged mice and rescues skeletal muscle regeneration. Our work establishes that loss of WISP1 from FAPs contributes to MuSC dysfunction in aged skeletal muscles and demonstrates that this mechanism can be targeted to rejuvenate myogenesis