La Radiothérapie occupe la deuxième place dans la liste de traitement du cancer le plus important après chirurgie. Le progrès technique récent, comme la radiothérapie avec modulation d'intensité (IMRT) ou la radiothérapie guidée par l'image (IGRT), en combinaison avec de nouveaux médicaments à action spécifique tels que les anticorps monoclonaux, sont une garantie d'augmentation de l'index thérapeutique. Cependant, la radiothérapie peut provoquer un’ altération du processus normal de réparation et d'induire le développement d'un cadre de fibrose dans un sous-ensemble de patients sensibles et dans les survivants à long terme au cancer. La principale caractéristique de la fibrose radio-induit est l'accumulation excessive et anormale de collagène composé principalement des éléments fibrillaire et immatures de la matrice extracellulaire (ECM).Les organes qui peuvent être touchés par ce phénomène sont le foie, la peau, les intestins, les reins et les poumons. D'un point de vue clinique, la fibrose peut être considérée comme une condition irréversible, sans solution. Nous et d'autres ont récemment montré que, outre l'activation de la TGF-β/Smad canonique, d'autres voies sont activées dans les tissus fibreux tels que la cascade de signalisation intracellulaire Rho/ROCK. Fait intéressant, la façon dont Rho/ROCK semble spécifiquement activé dans la fibrose intestinale radio-induite, fournis une justification pour un stratégie anti-fibrotique ciblé. L’ inhibition pharmacologique de Rho avec les statines, en fait, est en mesure de prévenir et même inverser les phénomènes de fibrose post-actinique intestinale.Avec ces prémisses, dans nos études, nous avons montré le rôle des statines (Simvastatine et Pravastatine) et d'un inhibiteur spécifique de ROCK (Y-27632) dans un modèle murin de fibrose pulmonaire obtenue avec une approche pharmacologique (Bléomycine - BLM) . Par la suite, nous avons développé un modèle de fibrose pulmonaire induite par l'irradiation complet du thorax et évalué la réponse à l'administration de la Pravastatine. La confirmation de la participation de la voie Rho/ROCK/CTGF dans la fibrose pulmonaire a été montré par immunohistochimie: le traitement à la Pravastatine normalise l'expression de trois marqueurs: RhoB, TGFβ-RII et CTGF.Après, dans deux modèles de fibrose radio-induite (intestinal et pulmonaire), nous avons analysé, grâce à l’immunohistochimie, les mécanismes sous-jacents l'action antifibrotique de la Pravastatine via l’axe MMP2-TIMP2. Très intéressant, quand la pravastatine a été administré à titre préventif ou curatif, nous avons trouvé un impact différent sur la fibrolyse.Enfin, in vitro, nous avons étudié par zymographie l'expression des gélatinases (MMP2 et MMP9) dans des cultures primaires des fibroblastes pulmonaire murins exposées à différentes doses de rayonnement et de Pravastatine. Le métalloprotéases semble être à son tour impliquée dans les mécanismes pro-fibrolytiques induits par les statines.Dans notre modèle animal de fibrose pulmonaire, la Pravastatine est capable d'inverser le processus fibrotique et les métalloprotéases semblent être impliqués à leur tour, in vivo et in vitro, dans les mécanismes pro-fibrolyse induits par le médicament.La multiplicité des acteurs impliqués dans la physiopathologie de lésions fibrotiques explique pourquoi la mise en place d'une stratégie thérapeutique efficace est si complexe. La recherche dans les processus mécaniques de dommages aux tissus normaux ont ouvert la voie à de nouvelles approches thérapeutiques. Ces nouvelles cibles comprennent la réduction de l’inflammation, de l'activation vasculaire et de la thrombose, ainsi que la découverte de nouvelles cibles moléculaires. Il existe une variété de modèles précliniques et des stratégies efficaces, mais de nombreux efforts doivent être déployés pour atteindre l'objectif difficile de protéger les tissus normaux des effets secondaires de la radiothérapie. / Radiotherapy is the second most important treatment modality after surgery in the treatment of cancer. Recent technical advancements, such as intensity-modulated radiation therapy (IMRT) or image-guided radiation therapy (IGRT), combined with new targeted drugs have significant promise for therapeutic outcome. However radiation treatment could result in disabling normal tissue injury and in the development of progressive fibrosis in a subset of sensitive patients and in long-term cancer survivors. The main feature of tissue fibrosis is excessive accumulation of abnormal and cross-linked collagen mainly composed of fibrillar and immature extracellular matrix (ECM) components.The organs that can be affected by this phenomenon are liver, skin, intestine, kidneys and lungs. From a clinical point of view, fibrosis can be seen as an irreversible condition, without solution. We and others recently showed that beside the activation of the canonical TGF-β/Smad pathway, other intracellular signaling cascades including the Rho/ROCK pathway are switched on in fibrotic tissues. Interestingly, the Rho/ROCK pathway seems differentially activated in radiation-induced intestinal fibrosis, thereby providing a rationale for a specific, targeted anti-fibrotic strategy. Pharmacological inhibition of Rho using statins indeed prevent and even reverse intestinal radiation fibrosis.In our studies, we showed the role of Statin (Pravastatin e Simvastatin) and a specific inhibitor ROCK inhibitors (Y-27632) in a mice model of pulmonary induced-fibrosis obtained by a pharmacological approach (Bleomycin – BLM). Indeed, we developed a model of lung fibrosis by complete irradiation of chest and tested Pravastatin action. Confirmation of the involvement of Rho/ROCK/CTGF pathway in lung fibrosis are shown by immunohistochemistry: Pravastatin-treament normalized the expression of three markers: RhoB, TGF-RII and CTGF.Then, in models of radiation induced gut and lung fibrosis, we analysed, from a immunohistological point of view, the underlying mechanisms of the antifibrotic action of Pravastatin via MMP2-TIMP2 axis. Interestingly we found a different impact on fibrolysis when Pravastatin was administered preventively or curatively.Finally, in vitro, we investigate by zymography the expression of Gelatinases (MMP2 and MMP9) in primary lung fibroblasts cultures exposure at the different radiation and Pravastatin doses. Metalloproteases would appear to be in turn involved in pro-fibrolytic mechanisms induced by statin.The multiplicity of actors involved in the pathogenesis of fibrotic lesions explains why the definition of an effective therapeutic strategy is so complex.Researches in mechanistic processes of normal tissue damage paved the way for new therapeutic approaches. These new targets include reduction of vascular activation, inflammation and thrombosis and new molecular targets definition. Effective strategies are multiple on preclinical models, but numerous efforts have to be made to achieve the complicated goal of protection of normal tissues from the side effects of radiation therapy. / La radioterapia è la seconda modalità di trattamento più importante dopo chirurgia neltrattamento delle neoplasie. I recenti progressi tecnici, come la terapia ad intensità modulata(IMRT) o l’image-guided radioterapia (IGRT), in combinazione con nuovi farmaci ad azionemirata come gli anticorpi monoclonali, costituiscono ulteriore garanzia di incrementodell’indice terapeutico. Tuttavia il trattamento radiante può causare un’alterazione delnormale processo di riparazione e indurre lo sviluppo di un quadro di fibrosi in unsottogruppo di pazienti sensibili e nei lungo-sopravviventi al cancro. La caratteristicacardinale della fibrosi radioindotta è l’eccessivo ed anomalo accumulo di collagene compostoprincipalmente di componenti fibrillari e immature della matrice extracellulare (ECM).Gli organi che possono essere interessati da questo fenomeno sono fegato, pelle,intestino, reni e polmoni. Da un punto di vista clinico, la fibrosi può essere vista come unacondizione irreversibile, senza soluzione. Noi ed altri recentemente abbiamo mostrato cheaccanto alla attivazione della via canonica TGF-β/Smad, altre vie vengono attivate nei tessutifibrotici come la cascata di segnalazione intracellulare della via Rho/ROCK. Interessantenotare che la via Rho/ROCK sembra specificatamente attivata nella radiazione indotta fibrosiintestinale, fornendo così una spiegazione razionale per una specifica, mirata strategia antifibrotica.L'inibizione farmacologica di Rho con le statine infatti è in grado di prevenire eaddirittura invertire i fenomeni di fibrosi intestinale post-attinica.Grazie a queste premesse, nei nostri studi, abbiamo mostrato il ruolo delle statine(Pravastatina e Simvastatina) e di uno specifico inibitore di ROCK (Y-27632) in un modellomurino di fibrosi polmonare indotta ottenuto con un approccio farmacologico (bleomicina -BLM). In seguito, abbiamo sviluppato un modello di fibrosi polmonare indottadall’irradiazione completa del torace e valutata la risposta alla somministrazione dellaPravastatina. In questo modello ed in un modello di fibrosi intestinale indotto da radiazioni,abbiamo analizzato, da un punto di vista immunoistologico, i meccanismi sottostanti l'azione9antifibrotica della pravastatina e il ruolo delle metalloproteasi (MMP2 e TIMP2). Infine, invitro, abbiamo indagato, mediante zimografia, l'espressione delle gelatinasi (MMP2 e MMP9)in culture primarie di fibroblasti polmonari murini esposti a differenti dosi di radiazione epravastatina.Nel nostro modello animale di fibrosi polmonare, la Pravastatina è in grado di renderereversibile il processo fibrotico e le metalloproteasi parrebbero essere a loro volta coinvolte,in vivo and in vitro, nei meccanismi pro-fibrolitici indotti dal farmaco.La molteplicità di attori coinvolti nella patogenesi delle lesioni fibrotiche spiegaperché la definizione di una strategia terapeutica efficace è così complessa. Ricerche neiprocessi meccanicistici di danno ai tessuti normali hanno aperto la strada a nuovi approcciterapeutici. Questi nuovi obiettivi comprendono la riduzione dell’ attivazione vascolare,dell'infiammazione e della trombosi, oltre alla definizione di nuovi target molecolari. Esistonomolteplici ed efficaci strategie su modelli preclinici, ma numerosi sforzi devono essere fattiper raggiungere il complicato obiettivo di proteggere i tessuti normali dagli effetti collateralidella radioterapia.

La fibrose radio-induite est une complication sévère et tardive de la radiothérapie. Plusieurs études cliniques ont montré que la combinaison pentoxifylline-vitamine E est un traitement sûr et efficace contre la fibrose. Cependant, les mécanismes moléculaires de son efficacité restent inexplorés. Nous avons montré l’efficacité de la combinaison pentoxifylline-vitamine E dans l’entéropathie radique dans une faisabilité clinique. En parallèle, en utilisant un modèle unique, in vitro, de cellules musculaires lisses intestinales primaires isolées des personnes atteintes de l’entéropathie radique, nous avons montré une synergie entre la pentoxifylline et l’analogue hydrophile de vitamine E (trolox) qui permet à l’association d’inhiber l’expression de TGF-β1 au niveau de l’ARN messager et de la protéine. Cette action inhibitrice intervient au niveau transcriptionnel et conduit à une inhibition conséquente des cibles de la voie de signalisation TGF-β1/Smad (Col Iα1, FN1, PAI-1, CTGF), alors qu’elle semble sans effet sur la voie de signalisation Rho/ROCK. Pour la première fois, dans ces cellules issues de l’entéropathie radique, nous avons montré une surexpression de miR-210 ; microRNA induit par l’hypoxie. L’association pentoxifylline-trolox inverse la surexpression de miR-210 aussi bien dans les conditions normoxique que dans les conditions hypoxiques. L’implication de miR-210 dans l’entéropathie radique n’a pas été préalablement étudiée, néanmoins nous avons montré qu’un inhibiteur de miR-210 diminue l’expression de Col Iα1 dans ce modèle. L’effet anti-fibrosant exercé par l’association pentoxifylline-vitamine E est partiellement induit par l’inhibition de la cascade TGF-β1. L’inhibition de miR-210 est un deuxième mécanisme potentiel nécessitant d’autres investigations. Cette étude renforce les essais clinique antérieurs en montrant in vitro une synergie entre pentoxifylline et vitamine E et permettant de proposer cette association en première ligne thérapeutique dans la fibrose radio-induite. De plus, miR-210 est proposé comme une possible cible thérapeutique pour traiter la fibrose radio-induite. / Radiation-induced fibrosis is a serious late complication of radiotherapy. Pentoxifylline-vitamin E has proven effective and safe in clinical trials as treatment of fibrosis, while the molecular mechanism of its activity is yet unexplored. We showed efficacy of Pentoxifylline-vitamin E combination in radiation-induced enteropathy in a small clinical study. In parallel, using a unique in vitro model of primary smooth muscle cells isolated from intestinal samples isolated from humans with radiation enteropathy we showed that pentoxifylline and the hydrophilic analogous of vitamin E (trolox) synergize to inhibit TGF-β1 protein and mRNA expression. This inhibitory action is mediated at the transcriptional level and leads to subsequent inhibition of TGF-β1/Smad targets (Col Iα1, FN1, PAI-1, CTGF), while it has no effect on the Rho/Rock pathway. We have also demonstrated, for the first time, an overexpression of the hypoxia-induced microRNA miR-210 in the fibrotic cells. Pentoxifylline-trolox combination could reverse this miR-210 overexpression in normoxic and hypoxic conditions. While miR-210 has not been previously shown to be involved in radiation-induced enteropathy, we showed that miR-210 inhibitor could reduce mRNA expression of Col Iα1. The anti-fibrotic effect of combined pentoxifylline-vitamin E is at least in part mediated by inhibition of the TGF-β1 cascade. MiR-210 inhibition is another mechanism which needs further investigations. This study strengthens previous clinical data showing pentoxifylline-vitamin E synergy and supports its use as a first-line treatment of radiation-induced fibrosis. Also, it suggests miR-210 as a new potential therapeutic target for the treatment of this complication.

Les éponges (Porifera) sont l'une des premières lignées d'animaux à avoir émergé. De ce fait, elles sont considérées comme des espèces clés pour retracer l’origine et l'évolution des gènes et des voies de signalisation qui ont sous-tendu l'apparition de la pluricellularité chez les métazoaires. Entre autres, les voies Wnt ont été décrites comme des cascades génétiques essentielles du control de nombreux mécanismes cellulaires (prolifération, communication, adhésion, motilité, etc.) au cours du développement précoce des bilatériens et des cnidaires. C’est pourquoi, l'étude de ces voies, chez les lignées d’émergences plus anciennes sont essentielles afin de comprendre l'origine des plans d’organisation des animaux.J’ai alors entrepris de nombreuses analyses bioinformatiques sur différentes bases de données d’éponges. Il apparait alors que l’ancêtre commun des éponges possédait déjà certainement tous les composants des voies Wnt. Néanmoins, à ce jour, puisque l’intégralité de ces composants n’a été identifiée que dans le genre Oscarella (lignée des Homoscleromorpha), différentes pertes secondaires sembleraient s’être produites chez les démosponges, les éponges calcaires et les hexactinellides. Afin de tester si ces gènes orthologues sont impliqués dans la mise en place du plan d’organisation des éponges, des études fonctionnelles ont été mises en œuvre. Ces approches fonctionnelles réalisées sur deux lignées d’éponges différentes tendent alors à confirmer la conservation des voies de signalisation Wnt dans les processus de mise en place des plans d’organisation des animaux, à la fois au cours de l'embryogenèse mais aussi lors du renouvellement cellulaire chez l'adulte. / Sponges (Porifera) are one of the earliest emerged animal lineages. They are thus considered as key species to retrace early evolution of genes and pathways underlying the emergence of multicellularity in metazoans. Among others, the Wnt pathways have been described as crucial modules controlling cell proliferation, cell communication, cell adhesion and cell motility during the early development of Bilaterians and Cnidarians. Therefore the study of these signaling pathways in more basally branching lineages is essential for unraveling the origin of animal body plans. I performed numerous bioinformatic analyses on different poriferan databases. One of my main results is that the last common ancestor of Porifera probably already possessed all the components of the Wnt pathways. Nevertheless, because, to date, all these components were only retrieved in the Oscarella genus (Homoscleromorpha lineage), several secondary gene losses would have occurred in other sponge lineages, namely Demospongia, Calcarea and Hexactinellida.In order to test whether or not these retrieved orthologous genes, are involved in patterning sponge body plan (as they do in Bilateria and Cnidaria), functional studies were implemented. These functional studies performed on two different lineages tend to confirm that Wnt signaling pathways were conserved from sponges to vertebrates to pattern animal body plan during both embryogenesis and cell renewal in adult.

Embryonic stem (ES) cells can be maintained indefinitely in culture while retaining the ability to give rise to cellular derivatives from the three germ layers. These unique characteristics hold great promise for regenerative medicine and underscore the importance of understanding the molecular mechanisms behind ES cell maintenance. The embryonic stem cell state is supported by a delicate equilibrium of mechanisms that maintain pluripotency, prevent differentiation, and promote proliferation and survival. We sought to find genes that could contribute to one or more of these processes in human ES cells by using a gain-of-function screen of over 8000 human open reading frames (ORFs). We identify Vestigial-like 4 (Vgll4), a co-transcriptional regulator with no previously known function in ES cells, as a positive regulator for survival of human ES cells. Specifically, Vgll4 protects human ES cells from dissociation stress, and enhances colony formation from single cells. These effects may be attributable in part to the ability of Vgll4 to decrease the activity of initiator and effector caspases. Based on global transcriptional analysis, we hypothesize that Vgll4 enhances survival of hES cells at clonal densities by regulating changes in the cytoskeleton, which may in turn regulate pathways known to result in hES cell death. This dissertation introduces a novel approach for studying hES cell survival in the context of cell dissociation and presents Vgll4 as a novel regulator of this process. We also propose that Vgll4 could have multiple functions in hES cells including possible roles in pluripotency, cell cycle dynamics, Hippo pathway regulation, and \(TGF\beta\) signaling. A direct regulator of survival in human embryonic stem cells could have important implications for facilitating the generation of transgenic cell lines and reporters, thus harnessing the therapeutic application of these cells.

Tumour cell invasion is one of the most critical steps in malignant progression. It includes a broad spectrum of mechanisms, including both individual and collective cell migration, which enables them to spread towards adjacent tissue, and form new metastases. Understanding the mechanisms of cell spreading, and invasion, is crucial for effective anticancer therapy. Two modes of individual migration of tumour cells have been established in a three-dimensional environment. Mesenchymally migrating cells use proteases to cleave collagen bundles, and thus overcome the ECM barriers. Recently described protease-independent amoeboid mode of invasion has been discovered in studies of cancer cells with protease inhibitors. During my PhD study, I have focused on determining the molecular mechanisms involved in amoeboid invasion of tumour cells. We have examined invasive abilities in non-metastatic K2 and highly metastatic A3 rat sarcoma cell lines. We have shown that even though highly metastatic A3 rat sarcoma cells are of mesenchymal origin, they have upregulated Rho/ROCK signalling pathway. Moreover, A3 cells generate actomyosin-based mechanical forces at their leading edges to physically squeeze through the collagen fibrils by adopting an amoeboid phenotype. Amoeboid invasiveness is also less dependent on...

Radiotherapy is the second most important treatment modality after surgery in the treatment of cancer. Recent technical advancements, such as intensity-modulated radiation therapy (IMRT) or image-guided radiation therapy (IGRT), combined with new targeted drugs have significant promise for therapeutic outcome. However radiation treatment could result in disabling normal tissue injury and in the development of progressive fibrosis in a subset of sensitive patients and in long-term cancer survivors. The main feature of tissue fibrosis is excessive accumulation of abnormal and cross-linked collagen mainly composed of fibrillar and immature extracellular matrix (ECM) components.The organs that can be affected by this phenomenon are liver, skin, intestine, kidneys and lungs. From a clinical point of view, fibrosis can be seen as an irreversible condition, without solution. We and others recently showed that beside the activation of the canonical TGF-β/Smad pathway, other intracellular signaling cascades including the Rho/ROCK pathway are switched on in fibrotic tissues. Interestingly, the Rho/ROCK pathway seems differentially activated in radiation-induced intestinal fibrosis, thereby providing a rationale for a specific, targeted anti-fibrotic strategy. Pharmacological inhibition of Rho using statins indeed prevent and even reverse intestinal radiation fibrosis.In our studies, we showed the role of Statin (Pravastatin e Simvastatin) and a specific inhibitor ROCK inhibitors (Y-27632) in a mice model of pulmonary induced-fibrosis obtained by a pharmacological approach (Bleomycin - BLM). Indeed, we developed a model of lung fibrosis by complete irradiation of chest and tested Pravastatin action. Confirmation of the involvement of Rho/ROCK/CTGF pathway in lung fibrosis are shown by immunohistochemistry: Pravastatin-treament normalized the expression of three markers: RhoB, TGF-RII and CTGF.Then, in models of radiation induced gut and lung fibrosis, we analysed, from a immunohistological point of view, the underlying mechanisms of the antifibrotic action of Pravastatin via MMP2-TIMP2 axis. Interestingly we found a different impact on fibrolysis when Pravastatin was administered preventively or curatively.Finally, in vitro, we investigate by zymography the expression of Gelatinases (MMP2 and MMP9) in primary lung fibroblasts cultures exposure at the different radiation and Pravastatin doses. Metalloproteases would appear to be in turn involved in pro-fibrolytic mechanisms induced by statin.The multiplicity of actors involved in the pathogenesis of fibrotic lesions explains why the definition of an effective therapeutic strategy is so complex.Researches in mechanistic processes of normal tissue damage paved the way for new therapeutic approaches. These new targets include reduction of vascular activation, inflammation and thrombosis and new molecular targets definition. Effective strategies are multiple on preclinical models, but numerous efforts have to be made to achieve the complicated goal of protection of normal tissues from the side effects of radiation therapy.

Les cellules iNKT et T CDS innées sont présumées contribuer à l'irnmunosurveillance (IS) des cancers et sont fonctionnellement déficientes dans la leucémie myéloïde chronique (LMC). Notre hypothèse était que ces défauts résultent de l'incapacité des cellules dendritiques myéloïdes (mDC) à les activer. Des analyses par cytométrie en flux et microscopie confocale ont révélé une baisse de l'expression membranaire de CD 1 d, qui présente les antigènes aux cellules iNKT, à la surface des mDC des patients LMC, par comparaison aux sujets sains. Ce défaut n'est associé ni à un défaut de maturation des mDC, comme le montre l'expression normale de HLA-DR et de CDS6, ni à une baisse d'expression intracellulaire de CDld ou de son transcrit. Ces résultats sont conciliables avec une rétention intracellulaire. Le traitement in vitro des mDC des patients LMC avec un inhibiteur de la protéine ROCK restaure partiellement l'expression de surface de CD 1 d et la présentation antigénique par CD Id, alors qu'il n'a eu aucun effet sur les mDC des sujets sains. Nous proposons que la protéine ROCK, qui est activée par le domaine DH-PH de BCR-ABL, interfere avec la réponse immunitaire dépendant des lymphocytes iNKT au cours de la LMC par régulation négative de l'expression membranaire de CDld des mDC. Le fait que les cellules iNKT et T CDS innées retrouvent des fonctions normales après rémission complète de la LMC est en faveur d'une génération de cellules T CD8 innées dépendante des cellules iNKT, comme décrit chez la souris. Notre travail suggère une implication des cellules iNKT et T CD8 innées dans l'IS de la LMC et révèle l'axe ROCK/mDC comme une nouvelle cible thérapeutique dans la maladie. / CDld-restricted iNKT cells and innate CD8 T cells are believed to play a key role in cancer immune surveillance and are functionally deficient in chronic myeloid leukemia (CML). Herein, we have hypothesized that this defect might originate from BCR-ABL-dependent dysfunctions in myeloid dendritic cells (mDC). Indeed, flow cytometry and confocal microscopy revealed that cell-surface expression of CDld was downregulated in CML mDC, relative to healthy donor (HD) controls. The decreased cell-surface display of CDld could not be ascribed to defective mDC differentiation, as attested by normal expression of HLA-DR and the CD86 maturation marker. On the other hand, reduced membrane expression was not associated with decreased intracytoplasmic levels of CDld or its mRNA transcripts, consistent with intracellular retention. ln vitro treatrnent of CML mDC with the Rho-associated protein Kinase (ROCK) inhibitor Y-27632 partially restored both cell-surface CDld expression and CDld-mediated antigen presentation, while it had no effect on HD mDC. We propose that ROCK, which is most likely activated by the DH-PH domain of BCR-ABL, mediates iNKT-cell immune subversion in CML patients by downregulating CDld expression on CML mDC. Remarkably, both iNKT cells and innate CD8 T cells retumed to nonnal after complete CML remission, a finding consistent with a iN KT cell-dependent generation of innate CD8 T cells, similarly to the observations in mice. Ali in ali, our study supports the possible contribution of iNKT/innate CD8 T cells to tumor surveillance in CML, and reveals the ROCK/mDC axis as a new potential target to restore immune surveillance in CML.

Neuroblastoma is a childhood cancer of the peripheral sympathetic nervous system, emerging from cells of the neural crest. In Sweden, neuroblastoma accounts for 20 cases out of all, 300-350, pediatric cancer cases each year (Barncancerfonden 2019, Turup on behalf of Cancer Centrum 2019). This cancer often appears in the sympathetic ganglia and/or the adrenal gland and has a high rate of metastasis that often results in morbidity (Matthay et al. 2016). Recent findings implicating a mutation in the Rho/Rac signaling pathway, a pathway involved in neural crest differentiation and migration, were found in every fourth neuroblastoma patient (Dyberg et al. 2017) These mutations tend to shift Rho to a more active state which is believed to lead to more downstream Rho-associated Kinase (ROCK) activation. While inhibition of ROCK has been seen to promote MYCN protein degradation, induce neuroblastoma cell differentiation and repress neuroblastoma growth in vitro and in vivo (Dyberg et al. 2017). Rho/ROCK signaling pathway effects on cytoskeletal arrangement and cell shape have also been suggested to be involved in tumor promoted changes of the TME (Johan and Samuel, 2018). In this master’s thesis project, we explore the effects of the Rho/ROCK pathway on the tumor microenvironment (TME) and immune response (IR) in neuroblastoma. More specifically we are focusing on populations of T cells, macrophages and fibroblasts in tumors, and looking into tumor vascular structure (such as blood vessel) and extracellular matrix (ECM) formation after ROCK inhibitor treatment within neuroblastoma tumors from transgenic mice model TH-MYCN and multi-cellular tumor spheroids (MCTS), a three-dimensional (3D) in vitro model simulating TME in neuroblastoma cell lines. Through our studies we hope to find insights into the Rho/ROCK signaling pathway and involvement of the tumor microenvironment in cancer therapy, while elucidating potential new drugs and drug targets for improving outcomes in neuroblastoma treatment.