Breast cancer (BC) was not initially considered an immunogenic tumor; however, recent data show that immune-related factors are associated with patient prognosis and the response to treatment. Several large adjuvant clinical trials have shown that tumor infiltrating lymphocytes (TIL) are significantly associated with a better prognosis and can also predict responsiveness to pre-operative chemotherapy, particularly in the triple negative (TN) & HER2+ BC subtypes (Carsten Denkert et al. 2010; Loi et al. 2013a). Recently, the presence of ectopic lymph node-like structures characterized by distinct T and B cell zones, called tertiary lymphoid structures (TLS), were identified adjacent to the tumor (Gu-Trantien et al. 2013) in 60% of BC (Buisseret et al. 2017b) and linked with a good prognosis (Gu-Trantien et al. 2013). The mechanisms involved in TLS formation and activities and their impact on tumor immunity is relatively unknown. TIL infiltration and TLS formation are likely regulated, in part, by transcription factors (TF) that control cytokine/chemokine production within the tumor microenvironment (TME) (Pimenta and Barnes, 2014). One such TF, the forkhead box protein 1 (FOXP1) is abnormally expressed in various human tumors and has a known role in regulating immune cell functions. Contradictory data on FOXP1 expression together with a lack of information on its immune regulation led us to explore its role in this tumor type. The first part of this thesis research focused on FOXP1-mediated regulation in BC. Gene/protein analysis was examined in the four BC molecular subtypes, revealing its enriched expression in estrogen receptor positive (ER+) tumors (Luminal A/B). Luminal BC is generally less infiltrated compared with frequently high TIL infiltration in ER negative (ER-) tumors (i.e. HER2+ and TN) [reviewed in (Solinas et al. 2017a) and (Loi et al. 2014)]. We found that high FOXP1 expression in a cohort of untreated primary BC was significantly associated with a lower TIL and fewer TLS compared to FOXP1 low (FOXP1lo) tumors. This observation led us to investigate the effect of FOXP1 on cytokines and chemokines potentially involved in TIL recruitment and/or TLS formation. BC cancer cell lines were used to silence [MCF7; FOXP1hi] or overexpress [MDA-MB-231; FOXP1lo] FOXP1 expression. FOXP1 repression upregulated a number of cytokines and chemokines involved in T and B cell migration and function, while FOXP1 overexpression repressed a majority of the same factors. Expression analysis of the major T and B cell cytokine and chemokine genes was performed for FOXP1lo and FOXP1hi primary BC. These data reveal that FOXP1hi BCs have significant decreases in CXCL9, CXCL10, CXCL11, CXCL13, CX3CL1, CCL20, IL2, IL21, granzyme B and IFNγ and high levels of the immunosuppressive cytokines, IL10 and TGFβ. We next performed a lymphocyte migration assay using primary tumor supernatants prepared from FOXP1lo and FOXP1hi BC finding significantly decreased migration of total CD45+ lymphocytes, B cells, helper (CD4+) and cytotoxic (CD8+) T cells using FOXP1hi compared to FOXP1lo SN. Overall, our data suggest that FOXP1 plays an important role in repressing anti-tumor immune responses by negatively regulating TIL migration directed by specific cytokines and chemokines.The second part of this thesis research focused on the role FOXP1 plays in BC TLS. FOXP1 expression in T and B cell TIL and TLS was evaluated using RT-qPCR, multicolor flow cytometry, immunofluorescence (IF) and immunohistochemistry (IHC) and fresh, fixed and frozen breast tissues. Based on the FOXP1 expression two types of TLS were identified in BC: 1) TLS containing a germinal center (GC-TLS) and 2) TLS lacking a GC (non-GC-TLS). Examination of proteins specifically associated with active humoral immune responses allowed us to identify GC-TLS but not non-GC-TLS as functional. Gene expression analysis of micro-dissected tissues revealed distinct immune profiles that characterize B cell follicles in tonsils and spleen as well as aggregates, non-GC-TLS and GC-TLS in BC. This analysis further demonstrates that ongoing cell-mediated immune responses are associated with GC-TLS. The findings from this thesis research add important information to our understanding of how immune responses are initiated and maintained in BC and provide further insight into the identification and organization of functional immune responses at the tumor site. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Cells that cover the body’s inner and outer surfaces are called epithelial cells. Endothelial cells are specialised epithelial cells which, among other things, line the inside of blood vessels. The endothelium is anchored to the basement membrane through molecules called laminins. In an acute inflammation laminins can bind to leukocytes so that they can reach the inflamed tissue. Chemokines are molecules that attract leukocytes and can be synthesized by endothelial cells. This report will investigate what impact stimulation with laminin-332 on endothelial cells has on their gene expression for the chemokines CCL7, CCL8, CCL20, CXCL6 and CXCL10. A previously performed analysis for protein expression which had been performed under the same conditions revealed an upregulation of all chemokines except for CCL8, which was downregulated. The analysis for protein expression was executed with Olink’s Proximity Extension Assay and analysis of gene expression was carried out with qRT-PCR. The results revealed that gene expression for CCL8, CXCL6 and CXCL10 was under the detection limit for the chosen method. Gene expression for CCL7 and CCL20 was detectable and revealed an upregulation of gene expression for both genes, which was consistent with the results from the study that analysed protein expression. This led to the conclusion that stimulus with laminin-332 upregulates mRNA expression, protein production and protein secretion in human umbilical vein endothelial cells for chemokines CCL7 and CCL20. Lastly, the involvement of the chemokines CCL7 and CCL20 in inflammation and cancer diseases is explored as well as their potential role as a biomarker for clinical treatment. / Celler som täcker kroppens inre och yttre ytor kallas för epitelceller. Endotelceller är specialiserade epitelceller som bland annat bekläder insidan av blodkärlen. Endotelet är förankrat till basalmembranet via molekyler kallade lamininer. Vid en akut inflammation kan lamininer binda till leukocyter för att de ska kunna ta sig ut till den inflammerade vävnaden. Kemokiner är molekyler som attraherar leukocyter och som kan produceras av endotelcellerna. I denna rapport utforskas vilken påverkan som laminin-332 har på endotelcellers genuttryck för kemokinerna CCL7, CCL8, CCL20, CXCL6 och CXCL10. En tidigare utförd analys för proteinuttryck som gjorts under samma förhållanden visade en uppreglering av samtliga kemokiner, med undantag för CCL8 som blev nedreglerad. Analysen för proteinuttryck var utförd med Olinks Proximity Extension Assay och analys för genuttryck utfördes med qRT-PCR. Resultaten visade att genuttrycket för CCL8, CXCL6 och CXCL10 var för lågt för att detekteras med den valda metoden. Genuttryck för CCL7 och CCL20 var detekterbart och visade båda en uppreglering av genuttryck vilket överensstämde med resultatet från studien som analyserat proteinuttrycket. Detta ledde till slutsatsen att stimulans med laminin-332 uppreglerar uttryck av mRNA, proteinproduktion och proteinsekretion i humana venösa endotelceller från navelsträng för kemokinerna CCL7 och CCL20. Slutligen, utforskas involveringen av kemokinerna CCL7 och CCL20 vid inflammation och cancerassocierade sjukdomar samt vilken roll de kan spela som biomarkörer vid behandling.

Das Chemokin SDF-1 spielt eine wichtige Rolle bei der Hämatopoese, bei Immunreaktionen sowie bei der Entwicklung des Herzens, der Extremitätenmuskulatur und des zentralen und peripheren Nervensystems. Lange Zeit galt CXCR4 als der einzige Chemokinrezeptor für SDF-1, bis vor wenigen Jahren CXCR7 als ein alternativer Rezeptor für SDF-1 identifiziert wurde. Da alle Zelltypen des zentralen Nervensystems (ZNS) sensitiv für SDF-1 sind, sollte in dieser Arbeit die Funktion der beiden Rezeptoren in primärer Mikroglia und primären Astrozyten untersucht werden. Bisher konnte CXCR7 nur als Scavenger-Rezeptor für SDF-1 oder als atypischer Chemokinrezeptor nachgewiesen werden. Die Untersuchungen ergaben einen mitogenen und chemotaktischen Effekt von SDF-1 auf primäre Mikroglia, wobei sowohl CXCR4 als auch CXCR7 für das SDF-1-Signalverhalten essentiell sind. Nach Aktivierung von Mikroglia in vitro und in vivo wurden beide Rezeptoren verstärkt expremiert. In primären Astrozyten ergab sich ein ligandenabhängiges Signalverhalten von CXCR7. So führte die Bindung von SDF-1 an CXCR7 zu einer Aktivierung von G-Proteinen, während die Kopplung von interferon-inducible T cell alpha chemoattractant (I-TAC), als zweiten Liganden von CXCR7, eine Signalweiterleitung über ß-Arrestin2 zur Folge hatte. Zudem konnte die G-Protein-gekoppelte Rezeptorkinase (Grk)2 als ein positiver Regulator des SDF-1-CXCR7-Signalverhaltens in Astrozyten identifiziert werden.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第20296号 / 人健博第44号 / 新制||人健||4(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 高桑 徹也, 教授 三谷 章, 教授 浅野 雅秀 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Le glioblastome qui correspond au stade de malignité le plus élevé des gliomes est associé à un très mauvais pronostic car il envahit le parenchyme cérébral de manière diffuse, ce qui rend son exérèse complète généralement impossible, et il résiste aux traitements conventionnels en raison de sa résistance intrinsèque aux stimuli pro-apoptotiques. Le cancer de l’œsophage est également un cancer très agressif car invasif et résistant également aux stimuli pro-apoptotiques. <p>Les chémokines sont des cytokines chémotactiques responsables de la migration des leucocytes et exprimées en réponse à des cytokines inflammatoires, à des facteurs de croissance et à des stimuli pathogènes. De nombreux cancers possèdent un réseau complexe de ces chémokines. Les chémokines de type CXCL et plus particulièrement CXCL8 et CXCL12 sont impliquées dans la biologie des gliomes et du cancer de l’oesophage. Au cours de mon travail de thèse de doctorat, nous avons étudié l’expression des 15 chémokines CXCL et des 9 récepteurs aux chémokines CXCL dans divers modèles de gliomes et de cancers de l’œsophage. Cette étude menée par RT-PCR nous a permis de mettre en évidence la présence d’un patron d’expression complexe de ces chémokines CXCL dans les divers modèles analysés. Nous avons observé une expression plus importante des chémokines CXCL pro-angiogéniques par rapport aux chémokines anti-angiogéniques dans ces deux types de cancers. Nous avons également pu mettre en évidence une implication potentielle des chémokines CXCL2, CXCL3 et CXCL8 dans l’acquisition de la résistance au traitement par témozolomide des gliomes d’origine astrogliale. <p>Les glioblastomes et les cancers de l’œsophage étant deux types de cancers résistants aux stimuli pro-apoptotiques, et le témozolomide étant la seule molécule dotée de bénéfices thérapeutiques réels dans le cas du glioblastome, nous avons également testé le témozolomide dans nos modèles de cancer de l’œsophage in vitro et in vivo. Nous avons pu ainsi montrer un bénéfice thérapeutique réel apporté par cette molécule in vivo sur des animaux immunodéficients greffés avec des cellules humaines de carcinome épidermoïde de l’œsophage. Ce bénéfice thérapeutique peut être expliqué en partie par différents mécanismes d’action tels que l’induction de processus soutenus d’autophagie suivis par de l’apoptose mais également par des effets anti-angiogéniques. Enfin, nous avons pu montrer que la diminution d’expression même transitoire de la chémokine CXCL2 dans nos modèles in vitro de glioblastome et de carcinome épidermoïde de l’œsophage entraîne une diminution de la croissance de ces populations cellulaires cancéreuses, suggérant un rôle important de cette chémokine dans la biologie de ces deux types de cancers. Enfin, nous avons démontré un effet anti-angiogénique in vivo pour le témozolomide dans un modèle de xénogreffes de cancers oesophagiens humains chez la souris immunodéficiente.<p><p>En conclusion, l’ensemble de nos résultats suggèrent que le témozolomide, bien qu’il devienne bientôt un générique sous sa forme d’administration i.v. (la forme orale étant déjà générique), pourrait représenter une molécule d’intérêt pour combattre le cancer de l’œsophage, comme on le sait déjà depuis 2005 en ce qui concerne les glioblastomes. Nos résultats montrent ensuite l’importance du patron d’expression des chémokines CXCL dans la biologie des cellules gliales tumorales et des cellules cancéreuses de l’œsophage. Enfin, nos résultats montrent que le témozolomide détruit en partie ce réseau de chémokines CXCL au sein de ces deux types de cancers.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

No description available.

Chemokines are chemotactic cytokines, which mediate cell trafficking and play a key role in mobilisation of leukocytes. More recently, chemokines and their cognate receptors have been described as key players in different aspects of cancer biology contributing to proliferation, angiogenesis and metastasis. In particular, chemokines CCL19 and CCL21 acting on their associated receptor CCR7 are postulated to be key drivers of lymph node metastasis in a number of malignancies including breast, colon, gastric, & thyroid cancers. It has been reported that the cleavage of the pre-cysteine bridge N-terminal residues of CCL21 (SDGGAQD) and of CCL19 (GTNDAED) renders both peptides incapable of fully activating CCR7. However, little is known about the nature of the interactions that occur between the N-terminal residues of CCL19 or CCL21 and the CCR7 receptor, or the role they have in activation of CCR7. The aim of this study is to investigate the role of the residues in the N-terminus of CCL19 and in particular CCL21 in the context of CCR7 activation and to use this information in the discovery of novel CCR7 antagonists or agonists. To achieve this, we synthesised a number of short (three to seven amino acids) peptides and peptidomimetics inspired by the seven N-terminal amino acid residues of CCL19 and CCL21 and pharmacologically characterised their ability to activate CCR7 or block the activation of CCR7 using a number of in vitro assays such as calcium flux, trans-well (Boyden chamber), and Western blotting. We also carried out computational studies to better understand and predict the activity of these peptides. Our results demonstrate that some of these peptides are indeed capable of acting as agonists or antagonists of CCR7. / Kuwait Health Ministry and Kuwait Civil Services Commission

Metastasis is a major cause of death in cancer patients but currently there are no drugs available for its treatment. Hence there is an urgent clinical need for identifying and developing anti-metastatic drugs. The activation of CC chemokine receptor 7 (CCR7) and C-X-C chemokine receptor type 4 (CXCR4) plays an important role in lymph node metastasis in a variety of cancers. Indeed, in patients with tumours which are positive for CCR7 and/or CXCR4 expression, prognosis and survival are poorer than those whose tumours are negative for these receptors. CCR7 and/or CXCR4 activation, in addition to being involved in inducing invasive phenotypes in cancer cells, promotes tumour cell growth and survival. Our group has previously identified a series of sulfonamides as CCR7 antagonists. This project aims to extend on those studies and to develop a dual CCR7 and CXCR4 antagonist to reduce metastasis in cancer. Novel potent biaryl sulfonamide CCR7 antagonists were synthesised and assessed by calcium flux assay. Several potential dual CCR7 and CXCR4 biaryl sulfonamide antagonists have been synthesised, these are hybrid compounds incorporating features from CCR7 antagonists of this project, and from known sulfonamide CXCR4 antagonists. The most potent of such compound was able to inhibit CCR7 activation in calcium flux assay (95% inhibition at 1 µM), however, the relative potency of these compounds as CXCR4 antagonists was low. Molecular docking was used to investigate the binding mode of the synthesised compounds in CCR7 and CXCR4. The generated docking poses were able to rationalise some of the calcium flux assay results.

No description available.

No description available.