Étude du rôle des antigènes ABH et Lewis dans deux pathologies : le cancer colique et les gastro-entérites à Novovirus

Alors que la structure et la biosynthèse des antigènes glucidiques de groupes sanguins sont bien élucidées, leurs fonctions biologiques restent mal connues. Au cours de cette thèse, nous nous sommes attachés à mieux définir le rôle de ces antigènes de structures dans deux pathologies humaines : le cancer et les gastro-entérites à Norovirus (virus de type Norwalk). Chez l'homme, l'expression des antigènes ABH et Lewis est modifiée au cours de la cancérogenèse. Afin de mieux comprendre la signification biologique de ces altérations, nous avons utilisé un modèle d'adénocarcinome colique de rat. En modulant l'expression des antigènes H et A à la surface cellulaire par des expériences de transfection des gènes de glycosyltransférases responsables de leur synthèse, nous avons pu observer l'impact de ces antigènes sur la tumorigénicité et la biologie des cellules tumorales. Nous avons ainsi pu mettre en évidence que les antigènes A et H augmentaient la tumorigénicité des cellules cancéreuses et leur permettaient d'échapper au système immunitaire. Cette agressivité accrue corrèle avec une augmentation de la résistance des cellules tumorales à l'apoptose induite par privation de sérum ou par un choc thermique. La présence d'antigène H rend également les cellules plus résistantes à la lyse par les cellules LAK. Bien que le mécanisme moléculaire responsable de ce phénomène de résistance à la mort cellulaire par apoptose ne soit pas élucidé, il pourrait impliquer la molécule CD44 porteuse de ces antigènes sur les cellules de notre modèle. Les antigènes de groupes sanguins interviennent également dans les interactions avec des pathogènes. Dans l'étude réalisée ici, nous nous sommes intéressés aux Norovirus. Ces virus sont responsables d'épidémies de gastro-entérite et affectent des individus de tous ages sur l'ensemble du globe. Par homologie avec un autre membre de cette famille, le RHDV qui utilise l'antigène H type 2 comme ligand sur les cellules épithéliales de lapin, nous avons cherché le récepteur du virus Norwalk parmi les antigènes de groupes sanguins. Il s'avère que ce virus utilise l'antigène H type 1/3 pour se fixer sur ces cellules cibles. Une étude sur des volontaires, en collaboration avec l'équipe de C Moe (USA), nous a permis de conclure que cet antigène est bel et bien le récepteur viral. La collaboration avec l'équipe de J Jiang (USA), nous a également permis de dégager plusieurs profils de fixation des Norovirus sur la salive d'un échantillon de population en fonction des phénotypes ABO, sécréteur et Lewis des individus. Ces virus semblent pouvoir infecter l'ensemble de la population, mais tous les Norovirus ne peuvent pas infecter tous les individus. L'ensemble des résultats expérimentaux est discuté de façon à proposer des hypothèses quant à la signification biologique des antigènes ABH et Lewis.The structure and biosynthesis of blood group antigens are well defined, but their biological roles remain poorly understood. In this thesis, we try to define the function of these antigens in two pathologies: cancer and Norovirus gastro-enteritis (Norwalk-like virus). In man, the ABH and Lewis antigens expression is modified during cancerogenesis. To gain insights into the biological meaning of these alterations, we used a rat colon carcinoma model. By modulating H and A antigens expression through transfection of glycosyltransferases genes needed for their synthesis, we observed that the A and H antigens increased the tumorigenicity of cancerous cells and allowed their immune escape. This increased tumorigenicity correlated with an increased resistance to apoptosis induced by heat shock or serum deprivation. Cells with H antigen were aditionally more resistant to LAK cell lysis than cells devoid of the antigen. The molecular mechanism of this resistance to apoptosis is not defined but it could implicate the CD44 protein which is the major carrier molecule of these antigens in our model. Blood group antigens are involved in interactions with pathogens. In the present work we studied noroviruses. These viruses are the most important cause of non-bacterial epidemics of acute gastroenteritis worldwide. By homology with another member of this virus family, the RHDV which uses the H type 2 antigen for its binding on rabbit epithelial cells, we searched for the Norwalk virus receptor among carbohydrate blood group antigens. We show that Norwalk virus uses H type 1/3 to bind to the intestinal epithelial cells. A work with volunteers, in collaboration with the team of C Moe (USA) allowed us to conclude that H type 1/3 is the Norwalk virus receptor since individuals genetically devoid of this antigen were never infected. A study with J Jiang team (USA) revealed distinct binding patterns of noroviruses to the saliva of volunteers in relation with their ABO, Lewis and secretor phenotypes. Collectively, these viruses may infect almost all (over 98%) humans, but not all blood types are susceptible to all noroviruses. The experimental results are discussed to propose hypotheses as to the ABH and Lewis biological meaning.NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Dexamethasone-induced cell death is restricted to specific molecular subgroups of multiple myeloma

International audienceDue to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic heterogeneity of MM because sensitive cell lines were restricted to MAF and MMSET signature subgroups, whereas all CCND1 cell lines (n = 10) were resistant to dexamethasone. We demonstrated that the glucocorticoid receptor expression was an important limiting factor for dexamethasone-induced cell death and we found a correlation between glucocorticoid receptor levels and the induction of glucocorticoid-induced leucine zipper (GILZ) under dexamethasone treatment. By silencing GILZ, we next demonstrated that GILZ is necessary for Dex induced apoptosis while triggering an imbalance between anti- and pro-apoptotic Bcl-2 proteins. Finally, the heterogeneity of the dexamethasone response was further confirmed in vivo using myeloma xenograft models. Our findings suggested that the effect of dexamethasone should be re-evaluated within molecular subgroups of myeloma patients to improve its efficacy and reduce its adverse effects

Targeted alpha-therapy with 211At-anti-mCD138 mAb in a syngeneic murine Multiple Myeloma model : can repeated doses or fractionation protocol overpass single dose efficiency?

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Improved Functionality of the Vasculature during Conventionally Fractionated Radiation Therapy of Prostate Cancer

<div><p>Although endothelial cell apoptosis participates in the tumor shrinkage after single high-dose radiotherapy, little is known regarding the vascular response after conventionally fractionated radiation therapy. Therefore, we evaluated hypoxia, perfusion and vascular microenvironment changes in an orthotopic prostate cancer model of conventionally fractionated radiation therapy at clinically relevant doses (2 Gy fractions, 5 fractions/week). First, conventionally fractionated radiation therapy decreased tumor cell proliferation and increased cell death with kinetics comparable to human prostate cancer radiotherapy. Secondly, the injection of Hoechst 33342 or fluorescent-dextrans showed an increased tumor perfusion within 14 days in irradiated tumors, which was correlated with a clear reduction of hypoxia. Improved perfusion and decreased hypoxia were not explained by increased blood vessel density, size or network morphology. However, a tumor vascular maturation defined by perivascular desmin+/SMA+ cells coverage was clearly observed along with an increase in endothelial, zonula occludens (ZO)-1 positive, intercellular junctions. Our results show that, in addition to tumor cell killing, vascular maturation plays an uncovered role in tumor reoxygenation during fractionated radiation therapy.</p></div

Development and in vitro evaluation of a copper-64 radiolabeled antibody for targeting the CXCR4 receptor in human multiple myeloma

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PRIMA-1Met induces myeloma cell death independent of p53 by impairing the GSH/ROS balance

International audienceThe aim of this study was to assess the efficiency of p53 reactivation and induction of massive apoptosis (PRIMA-1(Met)) in inducing myeloma cell death, using 27 human myeloma cell lines (HMCLs) and 23 primary samples. Measuring the lethal dose (LD50) of HMCLs revealed that HMCLs displayed heterogeneous sensitivity, with an LD50 ranging from 4 μM to more than 200 μM. The sensitivity of HMCLs did not correlate with myeloma genomic heterogeneity or TP53 status, and PRIMA-1(Met) did not induce or increase expression of the p53 target genes CDKN1A or TNFRSF10B/DR5. However, PRIMA-1(Met) increased expression of NOXA in a p53-independent manner, and NOXA silencing decreased PRIMA1(Met)-induced cell death. PRIMA-1(Met) depleted glutathione (GSH) content and induced reactive oxygen species production. The expression of GSH synthetase correlated with PRIMA-1(Met) LD50 values, and we showed that a GSH decrease mediated by GSH synthetase silencing or by and L-buthionine sulphoximine, an irreversible inhibitor of γ-glutamylcysteine synthetase, increased PRIMA-1(Met)-induced cell death and overcame PRIMA-1(Met) resistance. PRIMA-1(Met) (10 μM) induced cell death in 65% of primary cells independent of the presence of del17p; did not increase DR5 expression, arguing against an activation of p53 pathway; and synergized with L-buthionine sulphoximine in all samples. Finally, we showed in mouse TP53(neg) JJN3-xenograft model that PRIMA-1(Met) inhibited myeloma growth and synergized with L-buthionine sulphoximine in vivo

Fractionated irradiation induces vascular remodeling.

<p>(<b>A</b>) Pseudo-confocal images of tumor blood vessels during CFRT and stained for ZO-1/CD31 (top) or SMA/CD31 (bottom). (<b>B</b>,<b>C</b>). Image quantification of ZO-1+/CD31+ (<b>B</b>) and peri-CD31+ SMA surfaces (<b>C</b>). Values represent the average of n≥13 per point ± sem. (<b>D</b>) Image quantification of peri-CD31+ desmin surface and frequency of desmin+/SMA+ vessels. (<b>B</b>,<b>E</b>,<b>D</b>) Statistical comparisons vs. t0. (<b>E</b>) Representative confocal images of a blood vessel from a 14-day treated tumor stained for CD31/desmin/SMA. (<b>F</b>) histogram analysis of CD31/desmin/SMA pseudocolor profile of confocal image cross-section.</p

Maintenance of vascular density and distribution during fractionated irradiation.

<p>(<b>A</b>) Microvessel density in tumors during CFRT. Values represent the average of n≥13 per point ± sem. (<b>B</b>) Distance profile between cells and the closest blood vessel, from tumors during CFRT. Profiles are based on n≥13. Statistical comparisons vs. t0. (<b>C</b>) Pseudo-confocal images of tumor-associated blood vessels (CD31+) stained for TUNEL during CFRT. Arrows: TUNEL+/CD31+ cells. (<b>D</b>) Image quantification of CD31+/TUNEL+ surface. Values represent the average of n≥13 per point ± sem. (<b>E</b>) Representative Z-stack images of 100 µm-thick tumor sections before (t0) or after 2 weeks of CFRT (t14) and stained for blood vessels (CD31+/Fli-1+). (<b>F</b>) Image analysis of blood vessel network from 100 µm-thick tumor sections. Values represent the average of n  =  9 per point ± sem.</p

Fractionated irradiation reduces hypoxia and increases tumor perfusion.

<p>(<b>A</b>) Pseudo-confocal images of tumors during CFRT, stained for hypoxia (EF5) and endothelial cells (CD31). (<b>B</b>) Image quantification of EF5+ surface in tumors during CFRT. Values represent the average of n≥13 per point ± sem. (<b>C</b>) Pseudo-confocal images of tumors perfused with Hoechst 33342 and 10 kDa/2 MDa dextrans before (t0) or after 2 weeks of CFRT (t14). SYBR green was used as a counterstain of total cell nuclei. (<b>D,E,F</b>) Image quantification of Hoechst+ (<b>D</b>), and medium (<b>E</b>) and large (<b>F</b>) dextran+ surfaces in tumors during CFRT (n  =  6). (<b>B</b>,<b>D</b>,<b>E</b>,<b>F</b>). Statistical comparisons vs. t0.</p

ImmunoPET in Multiple Myeloma—What? So What? Now What?

International audienceDespite constant progress over the past three decades, multiple myeloma (MM) is still an incurable disease, and the identification of new biomarkers to better select patients and adapt therapy is more relevant than ever. Recently, the introduction of therapeutic monoclonal antibodies (mAbs) (including direct-targeting mAbs and immune checkpoint inhibitors) appears to have changed the paradigm of MM management, emphasizing the opportunity to cure MM patients through an immunotherapeutic approach. In this context, immuno-positron emission tomography (immunoPET), combining the high sensitivity and resolution of a PET camera with the specificity of a radiolabelled mAb, holds the capability to cement this new treatment paradigm for MM patients. It has the potential to non-invasively monitor the distribution of therapeutic antibodies or directly monitor biomarkers on MM cells, and to allow direct observation of potential changes over time and in response to various therapeutic interventions. Tumor response could, in the future, be anticipated more effectively to provide individualized treatment plans tailored to patients according to their unique imaging signatures. This work explores the important role played by immunotherapeutics in the management of MM, and focuses on some of the challenges for this drug class and the significant interest of companion imaging agents such as immunoPET