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The Impact of Neutrophils on the Sensitivity of Lymphoma B Cells to Cancer Therapy Taghreed Hirz

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The Impact of Neutrophils on the Sensitivity of Lymphoma B Cells to Cancer Therapy Taghreed Hirz Polymorphonuclear neutrophils and cancer : the impact of neutrophils on the sensitivity of lymphoma B cells to cancer therapy Taghreed Hirz To cite this version: Taghreed Hirz. Polymorphonuclear neutrophils and cancer : the impact of neutrophils on the sensi- tivity of lymphoma B cells to cancer therapy. Cancer. Université Claude Bernard - Lyon I, 2015. English. NNT : 2015LYO10327. tel-01417800 HAL Id: tel-01417800 https://tel.archives-ouvertes.fr/tel-01417800 Submitted on 16 Dec 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. No d’ordre 327-2015 Année 2015 THESE DE L’UNIVERSITE DE LYON Délivrée par L’UNIVERSITE CLAUDE BERNARD LYON I ECOLE DOCTORALE BIOLOGIE MOLECULAIRE, INTEGRATIVE ET CELLULAIRE DIPLOME DE DOCTORAT (arrêté du 7 août 2006) soutenue publiquement le 14 Décembre 2015 par HIRZ Taghreed TITRE Neutrophiles polymorphonucléaires et cancer: L'impact des neutrophiles sur la sensibilité des cellules de lymphome B aux thérapies anti-cancéreuses Directeur de thèse : DUMONTET Charles Jury: Mme. Catherine THIEBLEMONT, Professeure, Paris, France M. Christophe CAUX, Directeur de Recherche, Université Lyon 1, France M. Charles DUMONTET, Professeur, Université Lyon 1, France M. Sebastien JAILLON, Directeur de Recherche, Milan, I UNIVERSITE CLAUDE BERNARD - LYON 1 Président de l’Université M. François-Noël GILLY Vice-président du Conseil d’Administration M. le Professeur Hamda BEN HADID Vice-président du Conseil des Etudes et de la Vie M. le Professeur Philippe LALLE Universitaire M. le Professeur Germain GILLET Vice-président du Conseil Scientifique M. Alain HELLEU Secrétaire Général COMPOSANTES SANTE Faculté de Médecine Lyon Est – Claude Bernard Directeur : M. le Professeur J. ETIENNE Faculté de Médecine et de Maïeutique Lyon Sud – Charles Administrateur provisoire : M. le Professeur G. Mérieux KIRKORIAN UFR d’Odontologie Directeur : M. le Professeur D. BOURGEOIS Institut des Sciences Pharmaceutiques et Biologiques Directeur : Mme la Professeure C. VINCIGUERRA. Institut des Sciences et Techniques de la Réadaptation Directeur : M. le Professeur Y. MATILLON Département de formation et Centre de Recherche en Biologie Humaine Directeur : M. le Professeur P. FARGE COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE Faculté des Sciences et Technologies Directeur : M. le Professeur F. De MARCHI Département Biologie Directeur : M. le Professeur F. FLEURY Département Chimie Biochimie Directeur : Mme le Professeur H. PARROT Département GEP Directeur : M. N. SIAUVE Département Informatique Directeur : M. le Professeur S. AKKOUCHE Département Mathématiques Directeur : M. le Professeur A. GOLDMAN Département Mécanique Directeur : M. le Professeur H. BEN HADID Département Physique Directeur : Mme S. FLECK Département Sciences de la Terre Directeur : Mme la Professeure I. DANIEL UFR Sciences et Techniques des Activités Physiques et Sportives Directeur : M. C. COLLIGNON Observatoire de Lyon Directeur : M. B. GUIDERDONI Polytech Lyon Directeur : M. P. FOURNIER Ecole Supérieure de Chimie Physique Electronique Directeur : M. G. PIGNAULT Institut Universitaire de Technologie de Lyon 1 Directeur : M. C. VITON Institut Universitaire de Formation des Maîtres Directeur : M. R. BERNARD Institut de Science Financière et d'Assurances Directeur : Mme la Professeure V. MAUME- DESCHAMPS 2 Acknowledgements I would like to thank my advisor Pr. Charles Dumontet for his support. Without his leading, I would not have been able to achieve this work. I would like to thank the committee members: Dr. Catherine Thieblemont, Dr. Christophe Caux and Dr. Sebastien Jaillon for reviewing my thesis and for their time and effort. I would extend my appreciation to my colleagues and friends in Antibodies Anticancer team. My gratitude to Eva who assisted me in several technical skills. Ivana, thanks for being the best lab mate and friend I would ever have. To my parents, thanks for your constant encouragement and love I have relied throughout my time in France. It is to them that I dedicate this work. 3 RESUME en français Alors que le rôle des cellules du système immunitaires innées sur la progression tumorale est l'objet d'une investigation croissante, le rôle des neutrophiles sur la sensibilité à la thérapie n'a pas été précédemment décrit. Jusqu’au présent, nous avons effectué des cocultures de neutrophiles et des différentes lignées cellulaires de lymphome non hodgkinien (LNH) en présence de divers agents cytotoxiques ou des thérapies ciblées. Afin d’évaluer l'effet du traitement sur la prolifération cellulaire et la mort des cellules, des marquages CFSE et DAPI ont été effectués respectivement, en utilisant la cytométrie en flux. Les neutrophiles ainsi que les cellules HL60 différenciées avec des propriétés de neutrophiles, ont atténué la sensibilité de cellules de lymphome à des agents anticancéreux in vitro, à la fois dans les modèles 2D et 3D. L'effet protecteur des neutrophiles a été testée in vivo en injectant des cellules de LNH et des neutrophiles chez des souris SCID/CB17 traités avec vincristine. La coinjection de neutrophiles réduit la sensibilité des cellules LNH à la chimiothérapie. Cet effet protecteur a été validé en utilisant des cellules primaires, purifiée à partir de patients atteints de leucémie lymphoïde chronique, exposés à des agents cytotoxiques ou des agents ciblés en présence de neutrophiles autologues. La protection par les neutrophiles est contact dépendante. Elle est médiée par l'interaction de CD11b et ICAM1, exprimé par les neutrophiles et les lymphocytes B, respectivement, et par la molécule d'adhésion CD44. Elle est également dépendante de Mcl1 et est partiellement abrogée par un composé anti-Mcl1. 4 TITRE en anglais Polymorphonuclear neutrophils and cancer: The impact of neutrophils on the sensitivity of lymphoma B cells to cancer therapy RESUME en anglais While the role of innate immune cells on tumor progression is the object of increasing scrutiny, the role of neutrophils on sensitivity to therapy has not been previously described. To this end, we performed cocultures of freshly purified human neutrophils and different non- Hodgkin lymphoma (NHL) cell lines in the presence of various cytotoxic and targeted agents. CFSE and DAPI assays were performed to assess the therapeutic effect on cell proliferation and cell death, respectively, using flow cytometry. Neutrophils and differentiated HL60 cells with neutrophil-like properties attenuated the sensitivity of lymphoma cells to anti-cancer agents both in 2D and 3D models in vitro. The protective effect of neutrophils was tested in vivo using SCID/CB17 mice inoculated with NHL cells together with neutrophils, and treated with vincristine. The co-inoculation of neutrophils reduced the sensitivity of NHL cells to chemotherapy. Similar findings were made on primary cells, purified from patients diagnosed with chronic lymphocytic leukemia, exposed to cytotoxic agents or recently approved targeted agents (ibrutinib and idelalisib) in the presence of autologous neutrophils. Neutrophil-induced protection was dependent on cell-cell contact mediated by the interaction of CD11b and ICAM-1, expressed by neutrophils and B cells respectively, and by the adhesion molecule CD44. This protective effect was Mcl-1-dependent and was partially abrogated by an anti- Mcl-1 compound. DISCIPLINE : Cancérologie KEYWORDS/MOTS-CLES Neutrophils, lymphoma, chemotherapy, targeted therapy, cell-cell interactions Neutrophiles, lymphome, chimiothérapie, thérapie ciblée, interactions cellulaires INSTITUTE ET ADRESSE DU LABORATOIRE Anticorps Anticancer Centre de recherche en cancérologie UMR INSERM 1052/CNRS 5286 8 Avenue Rockefeller, 69373 LYON Cedex 08 5 Un résumé substantiel en français: Chapitre I 1. Introduction Le système immunitaire est un système complexe, mais bien organisé, qui est composé d'un réseau de cellules, des tissus et des organes qui permet d'identifier et d'éliminer les envahisseurs potentiellement nocifs tels que les bactéries, les virus, les champignons ou des parasites, ainsi que des objets endogènes tels que des complexes immuns ou des cellules transformées. Cette défense est divisée en deux types généraux de réactions: réactions de l'immunité innée et de l'immunité adaptative. En général, l'immunité innée est considérée comme une réponse non spécifique, alors que le système immunitaire adaptatif est considéré comme étant très spécifique. En outre, les récepteurs d'antigène de la réponse immunitaire adaptative sont généralement considérés comme «maître» dont leur engagement dicte la fonction des lymphocytes. Les cellules du système immunitaire sont généralement régulé par des hormones de croissance et la promotion ou la suppression de signaux, mais ils sont tous vulnérables aux erreurs, qui pourrais conduire à la transformation maligne. Le cancer est une classe de maladies caractérisées par une division incontrôlée des cellules et la capacité des cellules à envahir les tissus normaux et des dommages que ce soit localement ou au site distant du corps. Un lymphome malin survient quand un seul lymphocyte
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