Transplanting pancreatic islets into the liver through the portal vein is currently the most common procedure in clinical islet transplantations for treating patients with brittle type 1 diabetes. However, most islet grafts fail within a 5-year period necessitating retransplantation. The vascular connections are disrupted at islet isolation and implanted islets depend on diffusion of oxygen and nutrients in the immediate posttransplantation period. Rapid and efficient revascularization is of utmost importance for the survival and long-term function of transplanted islets. In this thesis, the influence of the implantation microenvironment for islet engraftment and function was studied. Islets were transplanted into the liver, the renal subcapsular site or the pancreas. Islets implanted into the liver contained fewer glucagon-positive cells than islets implanted to the kidney and endogenous islets. Intraportally transplanted islets responded with insulin and glucagon release to secretagogues, but only when stimulated through the hepatic artery. Thus, the intrahepatic grafts were selectively revascularized from the hepatic artery. The vascular density in human islets transplanted into the liver of athymic mice was markedly lower when compared to human islets grafted to the kidney. Islets implanted into their physiological environment, the pancreas, were markedly better revascularized. Insulin content, glucose-stimulated insulin release, (pro)insulin biosynthesis and glucose oxidation rate were markedly decreased in transplanted islets retrieved from the liver, both when compared to endogenous and transplanted islets retrieved from the pancreas. Only minor changes in metabolic functions were observed in islets implanted into the pancreas when compared to endogenous islets. The present findings demonstrate that the microenvironment has a major impact on the engraftment of transplanted islets. Elucidating the beneficial factors that promote engraftment would improve the survival and long-term function of transplanted islets. Ultimately, islet transplantation may be provided to an increased number of patients with type 1 diabetes.

Οι μέχρι τώρα ενδείξεις από τη βιβλιογραφία εισηγούνται ένα πιθανό προστατευτικό ρόλο των οιστρογόνων στην καρκινογένεση στο παχύ έντερο. Η έκφραση των οιστρογονικών υποδοχέων στο φυσιολογικό βλεννογόνο του παχέος εντέρου, στα αδενώματα και τα καρκινώματα και οι αλληλεπιδράσεις τους με διάφορους συμπαράγοντες θα πρέπει να μελετηθούν υπό το πρίσμα των πολύπλοκων μοριακών δικτύων μεταξύ επιθηλιακών κυττάρων και μυοϊνοβλαστών του στρώματος, αλλά και της θεωρίας των βλαστικών κυττάρων που φαίνεται να ενέχονται στην καρκινογένεση. Η έκφραση των ERα, ERβ1, ΑΙΒ-1, TIF-2, PELP1, NCoR και ALDH1 μελετήθηκε ανοσοϊστοχημικά σε 107 καρκινώματα παχέος εντέρου, σε 77 δείγματα φυσιολογικού βλεννογόνου και σε 29 αδενώματα του ίδιου οργάνου. Εκτιμήθηκαν τόσο τα επιθηλιακά κύτταρα όσο και οι μυοϊνοβλάστες. Για την ακριβέστερη εκτίμηση των μυοϊνοβλαστών, συνεχόμενες ιστολογικές τομές υποβλήθηκαν σε ανοσοϊστοχημικό έλεγχο με χρήση των anti-αSMA και CD34 αντισωμάτων. Τα αποτελέσματά μας έδειξαν ότι η έκφραση των ERβ1, ΑΙΒ-1, ΤΙF-2 και PELP1 ήταν πιο συχνή σε μυοϊνοβλάστες του στρώματος των καρκινωμάτων σε σχέση με τα αδενώματα και το φυσιολογικό βλεννογόνο. Επίσης, στους μυοϊνοβλάστες των καρκινωμάτων, ο NCoR εντοπιζόταν αποκλειστικά στο κυτταρόπλασμα των κυττάρων. Αντίθετα, δεν υπήρχε διαφορά στο ποσοστό έκφρασης των δεικτών αυτών στα επιθηλιακά κύτταρα μεταξύ του φυσιολογικού βλεννογόνου, των αδενωμάτων και των καρκινωμάτων. Ωστόσο, η κυτταροπλασματική εντόπιση του ERβ1 ήταν συχνότερη στα επιθηλιακά κύτταρα των καρκινωμάτων σε σχέση με το φυσιολογικό βλεννογόνο και τα αδενώματα. Επίσης, ο NCoR εκφραζόταν πιο συχνά στο κυτταρόπλασμα και σπανιότερα στον πυρήνα των κακοήθων επιθηλιακών κυττάρων σε σχέση με τα φυσιολογικά επιθηλιακά κύτταρα. Η κυτταροπλασματική έκφραση του NCoR στα επιθηλιακά κύτταρα σχετιζόταν με μεγαλύτερη ελεύθερη νόσου και συνολική επιβίωση και αποτελούσε ανεξάρτητο προγνωστικό δείκτη της ελεύθερης νόσου επιβίωσης. Τα αποτελέσματά μας υποδεικνύουν ένα πιθανό ρόλο της ενεργοποίησης του μονοπατιού των οιστρογονικών υποδοχέων στους μυοϊνοβλάστες του στρώματος, στην ανάπτυξη των καρκινωμάτων του παχέος εντέρου. Επίσης, η κυτταροπλασματική μετατόπιση, από τον πυρήνα, του NCoR στα επιθηλιακά κύτταρα, πιθανότατα επηρεάζει διάφορα μοριακά δίκτυα στον πυρήνα των κυττάρων, επιφέροντας ταυτόχρονα ογκοπροαγωγές δράσεις στα επιθηλιακά κύτταρα του βλεννογόνου και ογκοκατασταλτικές επιδράσεις στα αναπτυσσόμενα νεοπλάσματα. Δεδομένου ότι, η πυρηνική έκφραση του NCoR έχει προταθεί ως δείκτης των stem κυττάρων, τα ελάχιστα κύτταρα, στα οποία παρατηρήθηκε πυρηνική έκφραση στον καρκίνο παχέος εντέρου, πιθανότατα, αντιστοιχούν σε καρκινικά stem κύτταρα. Η ALDH1 αποτελεί, επίσης, δείκτη φυσιολογικών και καρκινικών stem κυττάρων σε διάφορα όργανα. Στη μελέτη μας η ALDH1 εκφράστηκε έντονα σε κύτταρα του φυσιολογικού βλεννογόνου, τα οποία βρίσκονταν στη βάση των κρυπτών και πιθανότατα αντιπροσωπεύουν τα stem/προγονικά κύτταρα του εντερικού επιθηλίου. Κατά αντιστοιχία, η έκφραση της ALDH1 στα καρκινικά κύτταρα σχετιζόταν με παρουσία μεταστάσεων και χειρότερη ελεύθερη νόσου επιβίωση. Το εύρημα αυτό, πιθανόν, να αποτελεί ένα δείκτη των καρκινικών stem/προγονικών κυττάρων. Αντίθετα, η έκφραση ALDH1 στους μυοϊνοβλάστες των καρκινωμάτων σχετιζόταν με ευνοϊκούς προγνωστικούς παράγοντες και μεγαλύτερο ελεύθερο νόσου διάστημα. Επίσης, περιστατικά με χαμηλή έκφραση ALDH1 στους μυοϊνοβλάστες και υψηλή έκφραση στα επιθηλιακά κύτταρα σχετιζόταν με μικρότερο διάστημα ελεύθερη νόσου επιβίωσης, αλλά και συνολικής επιβίωσης. Τα ευρήματα αυτά υποδεικνύουν το σημαντικό ρόλο των πολύπλοκων αλληλεπιδράσεων επιθηλίου-στρώματος κατά την καρκινογένεση στο παχύ έντερο και επισημαίνουν τα πολύπλοκα μοριακά δίκτυα που ρυθμίζουν τη λειτουργία των κυττάρων. Η συστημική προσέγγιση των επιθηλιακών κυττάρων και της παθολογίας τους προϋποθέτει τη μελέτη των μορίων τους μέσα σε πολύπλοκα δίκτυα που επηρεάζουν τη δράση τους με μη γραμμικό τρόπο και περιλαμβάνει αμφίδρομες αλληλεπιδράσεις από τα περιβάλλοντα κύτταρα. / Background. The stochastic model of carcinogenesis is recently challenged by the stem cell model. The later suggests that cancer develops from uncontrolled proliferation and aberrant differentiation of adult stem cells or progenitor cells that acquire stem cell-like properties. Microenvironment regulates function and differentiation of normal epithelial cells creating a protective niche for stem cells. Additionally, microenvironment plays a critical role in induction and progression of carcinomas. Both mutations in adult stem cells and changes in signals emanating from the stem cell niche contribute to the initiation of carcinomas. Recent findings suggest a protective role of estrogens in colorectal carcinogenesis. However, estrogens exert various actions on cells depending on the molecular microenvironment and their cross-talk with intracellular cascades and coregulators of transcription. Additionally, estrogens modulate the function of stromal cells and might influence carcinogenesis by indirect actions. Elucidation of the molecular networks implicated in estrogen signaling is very important in view of the potential use of selective estrogen receptor modulators in chemoprevention and targeted anticancer therapy. Materials and methods. An immunohistochemical study was designed to analyze the estrogen receptors α and β and the various co-regulators of transcription expression of along with that of a proposed functional stem cell marker, ALDH1, in normal colonic mucosa, adenomas and colorectal carcinomas. One hundred seven cases of colorectal carcinoma were retrieved from the Pathology files of the University Hospital of Patras, Greece. None of the patients had received preoperative chemotherapy or radiotherapy. All female patients were at the postmenopausal age. Follow-up was available for all patients. Paired normal mucosa and adenoma specimens were evaluated in 77 and 29 cases, respectively, in an effort to examine the whole spectrum of the multistage progression of colorectal carcinogenesis. Primary antibodies against ERα, ERβ1, AIB-1, TIF-2, PELP1, NCoR and ALDH1 and the Envision polymer-based detection system were employed. Epithelial cells and stromal myofibroblasts were separately assessed. α-SMA and CD34 staining of serial histologic sections was valuable for the recognition of the myofibroblastic nature of the cells. Results. ERα expression was extremely rare and was noted in <1% of the epithelial cells in two cases of colorectal carcinoma. ERβ1, TIF-2, and NCoR were expressed in the nuclei and cytoplasm of epithelial cells and myofibroblasts. AIB-1 and PELP-1 were expressed in the nuclei of epithelial cells and myofibroblasts. PELP-1 displayed a dot-like pattern of staining in the nuclei of cells that is possibly attributed to the presence of focally increased concentration of PELP1 in multiprotein co-regulator complexes within the nuclei of cells. Statistical analysis revealed that nuclear and cytoplasmic expression of ERβ1 and TIF-2 and nuclear expression of AIB-1 and PELP1 in myofibroblasts increased from normal mucosa through adenoma to carcinomas. NCoR was expressed in the cytoplasm of carcinoma-associated fibroblasts but not in myofibroblasts of normal mucosa. Thus, various components of estrogen signaling namely ERβ1 (both genomic and non-genomic actions-associated localization) and co-regulators of transcription, are enhanced in cancer associated myofibroblasts, whereas co-repressor NCoR is expressed in the cytoplasm of the cells implying that ER signaling is enhanced in myofibroblasts of carcinomas. In contrast, nuclear expression of ERβ1, AIB-1, TIF-2, and PELP-1 in epithelial cells was not different among normal mucosa, adenomas and carcinomas. Cytoplasmic expression of ERβ1 was higher in colorectal carcinomas, implying activation of non-genomic actions of ERβ in colorectal carcinogenesis. A translocation of NCoR from the nucleus to the cytoplasm was noted in colorectal carcinomas, since nuclear expression was more common in normal mucosa and cytoplasmic expression was noted in the majority of carcinomas. Cytoplasmic expression of NCoR in epithelial cells was associated with favorable prognosis. These findings might suggest that derepression of NCoR repressed transcription is an important feature of colorectal carcinogenesis and correlates with patients’ prognosis. ALDH1 expression was noted in the nuclei and the cytoplasm of myofibroblasts and epithelial cells. Expression in myofibroblasts was more often noted in carcinomas compared to normal mucosa and was associated with absence of metastasis and favorable prognosis. Epithelial cells of normal mucosa expressed high levels of ALDH1 expression. A distinct pattern of ALDH1 expression along the crypt axis was noted. Nuclear expression was more common and cytoplasmic expression was intensified at the base of the crypts (compartment where epithelial stem cells reside) compared to superficial epithelium. Carcinomas displayed heterogenous expression of ALDH1 in epithelial cells. Increased cytoplasmic expression was associated with the presence of metastasis and poor prognosis. Thus, ALDH1 expression had distinct impacts on metastatic potential of carcinomas and patients’ prognosis, accordingly to the cell where it is expressed. Additionally, patients with increased expression in epithelial cells and decreased expression in myofibroblasts had worse prognosis compared to patients displaying all other combinations of ALDH1 expression in epithelial cells and myofibroblasts. Our findings imply a possible role of ALDH1 as a stem/progenitor cell marker in normal mucosa. The association of ALDH1 expression in malignant cells with metastatic potential and worse prognosis implies that it might represent a marker of carcinomas with increased stem/progenitor cell content. The favorable prognostic role of ALDH1 expression in myofibroblasts might be associated with its role in local retinoic acid production. Retinoic acid has various tumor suppressive roles in colorectal carcinomas and can potentially be used in chemopreventive or chemotherapeutic strategies especially in patients with low local production levels. Thus, a comprehensive analysis of molecular networks both in any single cell and among the different cells of colorectal carcinomas and non neoplastic mucosa are mandatory in order to elucidate the role of estrogen signaling in colorectal carcinogenesis in view of development of targeted clinical applications. Conclusions 1.ERβ is the predominant estrogen receptor in colonic tissue. 2.ERβ1 dependent signaling is enhanced in cancer-associated myofibroblasts. 3.PELP1 is associated with genomic actions in both epithelial cells and myofibroblats. 4.In epithelial cells, loss of NCoR nuclear expression correlates with colorectal carcinogenesis possibly through derepression of transcription mediated by various transcription factors. 5.ALDH1 emerges as a marker of normal and cancer stem cells of colorectal carcinomas.

No description available.

Le traitement du cancer à l’aide d’une exposition aux radiations ionisantes peut mener au développement de plusieurs effets secondaires importants, dont un retard de réparation et de régénération du tissu hématopoïétique. Les mécanismes responsables de ces effets demeurent encore inconnus, ce qui limite le développement de nouvelles approches thérapeutiques. À l’aide d’un modèle murin de prise de greffe, nos résultats démontrent que l’endommagement du microenvironnement par l’irradiation a un impact limitant sur le nichage hématopoïétique. Parce que le microenvironnement est composé principalement de cellules dérivées des cellules souches mésenchymateuses (CSM), nous avons évalué le potentiel des CSM à régénérer le tissu hématopoïétique par la reconstitution de la niche osseuse. Cette thérapie a mené à une augmentation remarquable du nichage hématopoïétique chez les souris irradiées. Les causes moléculaires impliquées dans le nichage hématopoïétiques sont encore inconnues, mais nous avons remarqué l’augmentation de la sécrétion de la cytokine « granulocyte-colony stimulating factor » (G-CSF) dans l’espace médullaire suite à l’irradiation. Le G-CSF est impliqué dans la mobilisation cellulaire et est fort possiblement nuisible à une prise de greffe. Nous avons évalué le potentiel d’une thérapie à base de CSM sécrétant le récepteur soluble du G-CSF afin de séquestrer le G-CSF transitoirement et les résultats obtenus démontrent que le blocage du G-CSF favorise le nichage hématopoïétique. Globalement, les données présentées dans ce mémoire démontrent que le microenvironnement osseux et le niveau de G-CSF dans la moelle sont importants dans le processus de nichage hématopoïétique et que la baisse du potentiel de régénération du tissu hématopoïétique suite à l’irradiation peut être renversée à l’aide d’une thérapie cellulaire de CSM génétiquement modifiées ou non. / Cancer treatment using ionizing radiation may lead to significant side effects, including delayed hematopoietic tissue repair and regeneration. The mechanisms mediating these defects remain unknown, thus limiting the development of new therapeutic approaches. Using a mouse engraftment model, our results show that microenvironment damage by irradiation limits hematopoietic homing. Since the microenvironment is mainly composed of mesenchymal stem cells (MSCs)-derived cells, we evaluated the potential of MSCs to improve hematopoietic tissue regeneration by bone marrow niche reconstitution. This therapy led to remarkable enhancement of hematopoietic homing in irradiated mice. The molecular causes involved in hematopoietic homing remain unknown, but we noticed an increased in “granulocyte-colony stimulating factor” (G-CSF) secretion within the medullary space after irradiation. G-CSF is involved in cellular mobilization and may possibly be harmful to engraftment. We evaluated the therapeutical potential of MSC genetically-engineered to secrete a soluble G-CSF decoy receptor that would transiently sequester G-CSF. Results obtained show that G-CSF blocking improved hematopoietic homing. Overall, the findings presented in this thesis indicate that bone marrow microenvironment and G-CSF levels are important in hematopoietic homing process, and that the decline in hematopoietic tissue regeneration potential following irradiation can be reversed by cellular therapy using MSC genetically modified or not.

Wound healing and carcinogenesis are defined as complex, adaptive processes which are controlled by intricate communications between the host and the tissue microenvironment. A number of phenotypic similarities are shared by wounds and cancers in cellular signaling and gene expression. Radiotherapy is the second most effective modality of cancer treatment after surgery and can be used, either alone or in combination with chemotherapy. Recent findings suggest that radiotherapy apart from tumor cell death also rapidly and persistently modifies the tissue microenvironment. These modifications affect cell phenotype, tissue metabolism, bidirectional exchanges and signaling events between cells. The complex interactions between stromal cells and cancer cells are of immense interest and in The First Part of My Thesis, I tried to explore the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts, irrespective of their RhoB status, do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms respectively, TGF-β1 and MMP-mediated. We also found that co-irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response. Secondly, our in vivo experiments, tends to confirm the in vitro data showing that irradiated tumor bed does not stimulate tumor growth and escape. Our results also challenges the view that irradiated stroma would promote migration of carcinoma cells as we show that independently from their genotype co-irradiation of fibroblasts and carcinoma cells repressed carcinoma cell migration and confirmations studies are currently performed in vivo. The Third Part of My Project, was dedicated to investigate the effect on CTC release after radiotherapy. Consistently with the results reported after surgery , the number of CTC increases in the blood stream after radiotherapy probably due to radiation-induced vascular injury induced or/and by EMT induction in tumor cells but these cells seemed to be entrapped into the cardiac cavity. The significance of these CTC to metastatic development is still under investigation but there is evidence for a metastasis-promoting effect of RT from animal studies.Thus the microenvironment can exert antagonist stimulatory or inhibitory effects on malignant cells.

Submitted by Mayara Caroline Rosolem null (mayara_rosolem@yahoo.com.br) on 2017-12-13T13:13:58Z No. of bitstreams: 1 Tese_Mayara_Caroline_Rosolem.pdf: 2522128 bytes, checksum: af877102b33947d3515c6cd2c29b01af (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2017-12-13T13:40:57Z (GMT) No. of bitstreams: 1 rosolem_mc_dr_jabo.pdf: 2522128 bytes, checksum: af877102b33947d3515c6cd2c29b01af (MD5) / Made available in DSpace on 2017-12-13T13:40:57Z (GMT). No. of bitstreams: 1 rosolem_mc_dr_jabo.pdf: 2522128 bytes, checksum: af877102b33947d3515c6cd2c29b01af (MD5) Previous issue date: 2017-11-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os tumores mamários malignos possuem várias formas de evadir o sistema imune e, dentre elas está a modulação das células dendríticas (DCs), por interferência na sua maturação, resultando em apresentação de antígenos ineficiente aos linfócitos T e consequente indução da tolerância imunológica. As DCs moduladas pelo tumor recrutam muitos linfócitos T regulatórios (Tregs) para o microambiente tumoral, o que amplia os efeitos imunossupressores locais. A forte relação entre as DCs e os linfócitos Tregs no microambiente tumoral ainda foi pouco estudada, principalmente em cães. Por isso, este estudo teve o objetivo de avaliar a relação existente entre as DCs e os linfócitos Tregs em carcinomas mamários caninos do tipo simples de cadelas, por meio da técnica de imunohistoquímica. Foram utilizadas 10 amostras de glândula mamária sem tumor (G1) e 40 amostras de neoplasias mamárias (G2: adenomas; G3: carcinomas papilares; G4: carcinomas tubulares e G5: carcinomas sólidos), que foram submetidas a imunodetecção de linfócitos Treg (FOXP3+), linfócitos T CD4 e T CD8, MHC-II, DCs mieloides (imaturas e maduras), as citocinas TGF-β, IL-10, a enzima Indoleamine 2,3-dioxigenase (IDO) e dos receptores de quimiocina (CCR6 e CCR7). A maioria das células, citocinas e receptores imunológicos mostraram correlação positiva dentro da população tumoral e controles avaliados, principalmente sobre o efeito fixo “idade”, que teve alta correlação positiva com o tamanho tumoral e com CCR6. Quanto às correlações negativas, o anticorpo CD83 foi o único que não teve correlação significativa positiva com nenhuma outra variável. Observou-se que houve ocorreu relação entre as DCs mieloides imaturas/maduras e linfócitos Tregs, bem como TGF-β e IL-10, e a enzima IDO apresentaram marcante presença nas amostras malignas. A imunodetecção de CCR6 e CCR7 ocorreu principalmente nos tumores mais agressivos, onde CCR6 teve alta relação com as pacientes mais idosas e com tumores maiores, o que, no final de tudo pode refletir que o envelhecimento do sistema imunológico possa ser mais um fator pró-tumoral. / The malignant mammary tumors have several ways of evading the immune system, including the modulation of dendritic cells (DCs), by interfering with their maturation, resulting in inefficient presentation of antigens to T cells and consequent induction of immunological tolerance. Tumor-modulated DCs recruit many regulatory T cells (Tregs) into the tumor microenvironment, which amplifies local immunosuppressive effects. The strong relationship between DCs and Tregs cells into the tumor microenvironment was poorly studied, especially in dogs. Therefore, this study aimed to evaluate the relationship between DCs and Tregs cells in the simple type canine mammary carcinomas, using the immunohistochemical technique. Ten samples of mammary gland without tumor (G1) and 40 samples of mammary neoplasms (G2: adenomas, G3: papillary carcinomas, G4: tubular carcinomas and G5: solid carcinomas) were submitted to immunodetection of Treg cells (FOXP3 +), CD4 and CD8 T cells, MHC-II, myeloid DCs (immature and mature), cytokines TGF-β, IL10, Indoleamine 2,3-dioxygenase (IDO) and chemokine receptors (CCR6 and CCR7). Most of the cells, cytokines and immunological receptors showed positive correlation within the tumor population and controls evaluated, mainly on the fixed effect "age” that had high positive correlation with the tumor size and with CCR6. As for the negative correlations, the CD83 antibody was the only one that had no significant positive correlation with any other variable. It was observed that there was a relationship between immature / mature myeloid DCs and Tregs cells, as well as TGFβ and IL-10, and the IDO enzyme showed a marked presence in the malignant samples. Immunodetection of CCR6 and CCR7 occurred mainly in the most aggressive tumors, where CCR6 had a high relation with older patients and with larger tumors, which, in the end, may reflect that the aging of the immune system may be more of a pro- tumor. / FAPESP: 2012/09385-0

La contribution des Cellules Souches/Stromales Mésenchymateuses (CSM) dans le développement des Leucémies Aiguës Myéloblastiques (LAM) n’est pas encore clairement établie. L'objectif de ce travail a été de rechercher de potentielles modifications phénotypiques et fonctionnelles au sein des CSM médullaires de patients atteints de LAM de novo au diagnostic. Nous montrons que ces cellules présentent un défaut prolifératif accompagné d’une augmentation de l’apoptose et d’un déficit d’expression de certains facteurs de la niche (Ang-1, SCF, TPO et VCAM-1). De façon intéressante, ce défaut prolifératif est indépendamment associé à une évolution péjorative de la maladie. Néanmoins, ces anomalies des CSM de LAM ne semblent pas affecter leur capacité de soutien de l’hématopoïèse physiologique ou leucémique in vitro. En effet, comme les CSM normales, elles protègent les cellules leucémiques de l’apoptose, induisent leur quiescence (principalement par contact direct) et ainsi diminuent la proportion des cassures double-brin d’ADN. Ces données suggèrent que les modifications des CSM de LAM, probablement une des conséquences délétères de la prolifération tumorale, n'auraient pas un rôle spécifique dans le développement du processus leucémique. / The contribution of Mesenchymal Stem/Stromal Cells (MSCs) to the development of Acute Myeloid Leukemias (AMLs) remains poorly understood. In the present study, we investigated potential functional and phenotypic modifications of Bone Marrow (BM)-derived MSCs from patients with AML de novo at diagnosis. We showed that BM-derived MSCs from most of AML patients display proliferative defect, had increased apoptosis levels and demonstrated defective expression of several niche-related factors (Ang-1, SCF, TPO and VCAM-1). Interestingly, this proliferative defect was independently associated with disease progression. Nevertheless, these abnormalities in AML MSCs did not affect their in vitro capacity to support physiological but also leukemic hematopoiesis. Indeed, as normal MSCs do, they protect blast cells from apoptosis, induce their quiescence (mainly by direct contact), and decreased yields of DNA double-strand breaks. Consequently, in AML de novo these stromal cell alterations, probably a consequence of the deleterious effect of the tumor cell growth on BM MSCs, do not appear to have a specific role in the development of the leukemic process.

L’hématopoïèse se déroule dans un microenvironnement spécialisé appelé niche où les cellules souches hématopoïétiques (CSH) sont en contact étroit avec les cellules stromales mésenchymateuses. Cette interaction cellulaire associée à d’autres facteurs environnementaux, comme la présence des espèces réactives à l’oxygène, est cruciale pour la régulation des CSH normales, mais aussi leucémiques. Pour étudier ce microenvironnement, il est donc important de développer un modèle in vitro de niche humaine qui mime la physiologie in vivo. Nous avons choisi comme modèle deux lignées mésenchymateuses stromales humaines HS-27a et HS-5, très peu décrites dans la littérature. Le premier objectif a été de déterminer la qualité de cette niche tant du point de vue cellulaire, moléculaire que fonctionnel. Nos résultats montrent clairement que les cellules HS-27a participent à la formation d’une niche « quiescente » alors que les cellules HS-5 représentent une niche « proliférative ». Le deuxième objectif a été de créer une niche contrôlée pour le métabolisme oxydatif en régulant l’expression d’une protéine antioxydante, la glutathion peroxydase 3 ou GPx3. L’originalité de ce travail repose sur l’utilisation d’une méthode non virale de transfert de gène par le transposon piggyBac. Le plasmide porteur du gène d'intérêt a été apporté sous forme d’ADN et une source de transposase, enzyme catalysant la réaction d'intégration sous forme d’ARNm. Notre travail montre que GPx3 est un régulateur clé de l’homéostasie hématopoïétique favorisant le maintien des progéniteurs immatures. Pour la première fois, nous créons par ingénierie in vitro une niche hématopoïétique « calibrée » capable de mimer le microenvironnement normal et leucémique. Ce modèle permet non seulement d’identifier les acteurs clés de la régulation des cellules médullaires, mais aussi de développer des stratégies thérapeutiques ciblées. / Hematopoiesis occurs in a hypoxic microenvironment or niche in which hematopoietic stem cells (HSCs) are in close contact with mesenchymal stromal cells. Cellular interactions as well as microenvironmental factors such as reactive oxygen species are crucial for the maintenance of normal and leukemic HSCs. Developing an in vitro human culture system that closely mimcs marrow physiology is therefore essential to study the niche. Here, we present a model using two human stromal cell lines, HS-27a and HS-5. Previously poorly described in the literature, we have further characterized both of these cell lines. The first objective was to assess the quality of HS-27a and HS-5 niches by investigating their cellular, molecular and functional characteristics. Our results clearly show that HS-27a cells display features of a “quiescent” niche whereas HS-5 cells rather represent a “proliferative” niche. The second objective was to engineer a hematopoietic niche where the oxidative metabolism is optimized for the expression of an antioxidant protein, glutathione peroxidase 3 (GPx3). The originality of this work is the use of a non-viral gene transfer system by using the transposon piggyBac. This strategy was achieved by delivering a DNA plasmid carrying the gene of interest, and an mRNA source of transposase, the enzyme which catalyzes the transgene integration. Functionally, GPx3 was shown to be a key regulator for sustaining hematopoietic homeostasis by maintaining immature progenitor cells. For the first time, an original non-viral gene transfer has been used to create an in vitro hematopoietic niche that recapitulates the complexity of normal and leukemic microenvironment. This niche not only provides a platform to identify regulatory factors controlling medullary cells, but may also help in the development of targeted therapeutic strategies.

No description available.

Malgré le rôle des variants constitutivement actifs du récepteur des androgènes (RA) et du stroma tumoral dans le cancer de la prostate résistant à la castration (CRPC), leurs relations restent inconnues. Nous rapportons l’impact de l’interleukine-6 (IL-6) sécrétée par les cellules stromales prostatiques (PrSC) sur les cellules épithéliales tumorales prostatiques exprimant les variants autonomes du RA. Le milieu de culture conditionné par les PrSC (CMPrSC) contenait des taux élevés d’IL-6 et induisait une augmentation de l’activité transcriptionnelle de STAT3 dans les LNCaP et C4-2b exprimant le variant RAQ640X, via une activation de pY705-STAT3. Cette activité de STAT3 était inhibée par la neutralisation de l’IL-6. L’analyse par mRNA array et RT-qPCR a mis en évidence un profil transcriptomique spécifique lié à l’expression du RAQ640X et à l’exposition au CMPrSC, impliquant les fonctions de motilité, d’invasion et de migration cellulaires, et l’expression de gènes favorisant la dissémination métastatique. Ainsi, nos résultats illustrent une coopération épithélio-stromale «privilégiée» en présence de variants autonomes du RA, impliquée dans la progression tumorale. / Constitutively active androgen receptor (AR) variants and stromal microenvironment are involved in castration resistant prostate cancer (CRPC), but their relationship remains unknown. We describe the effects of interleukin-6 (IL6) secreted from prostate stromal fibroblast cells (PrSC) towards prostate epithelial cancer cells expressing constitutively active AR variants. Conditioned culture medium from PrSC (CMPrSC) contained high levels of IL-6 and led to an increased STAT3 transcriptional activity in LNCaP and C4-2b cells expressing the ARQ640X variant, through pY705-STAT3 activation. This STAT3 activity was significantly diminished with neutralizing antibody anti-IL6. Gene expression analysis using mRNA array and RT-qPCR highlighted a specific transcriptional profile related to ARQ640X expression and PrSC exposure, resulting in cellular motility, invasion and cellular migration, and IL-6 genes expression promoting metastatic dissemination. Overall, our data emphasize a “preferred” epithelio-stromal cooperation when expressing constitutive active RA variants, which contributes to tumor progression.