Malgré les récentes avancées dans la prise en charge des neuroblastomes de haut risque, le pronostic des patients qui en sont atteints reste péjoratif. Les modalités thérapeutiques sont agressives et de nombreux enfants souffrent des effets secondaires de celles-ci, dégradant de ce fait leur qualité de vie. L’immunothérapie anti-GD2 offre dans ce contexte une alternative thérapeutique, permettant d’améliorer le pronostic de ces tumeurs, mais leur utilisation est néanmoins limitée par la présence d’une toxicité des molécules existantes. Le GD2 O-acétylé n’étant pas exprimé à la surface des fibres nerveuses périphériques, le choisir comme cible thérapeutique peut s’avérer efficace tout en diminuant la toxicité de l’immunothérapie. Nous avons vérifié l’expression du GD2 O-acétylé à la surface des cellules de 4 lignées cellulaires de neuroblastome (LAN1, LAN5, IMR5, NXS2). Cette expression n’est pas modifiée par les traitements par chimiothérapie utilisés dans les traitements des neuroblastomes de haut grade (cisplatine, doxorubicine et topotecan). Nous montrons que l’anticorps murin 8B6 spécifique du GD2 O-acétylé a une relation synergique in vitro avec les molécules de chimiothérapie testées (cisplatine, doxorubicine et topotecan). Nous montrons par la suite que cet anticorps augmente l’efficacité de la doxorubicine et du topotecan in vivo chez la souris sans diminuer la tolérance globale du traitement multimodal. Nos résultats montrent qu’un traitement combinant chimiothérapie et immunothérapie peut augmenter l’efficacité et la tolérance cliniques des chimiothérapies dans le traitement des neuroblastomes de haut grade. / Despite recent advances in high-risk neuroblastoma therapy, the prognosis for patients remains poor. In addition, many patients suffer from complications related to available therapies that are highly detrimental to their quality of life. New treatment modalities are, thus, urgently needed to further improve the efficacy and reduce the toxicity of existing therapies. Since antibodies specific for Oacetyl GD2 ganglioside display pro-apoptotic activity against neuroblastoma cells, we hypothesized that combination of immunotherapy could enhance tumor efficacy of neuroblastoma chemotherapy. We demonstrate here that combination of anti-Oacetyl GD2 monoclonal antibody 8B6 with topotecan synergistically inhibited neuroblastoma cell proliferation, as shown by the combination index values. Mechanistically, we evidence that mAb 8B6 induced plasma cell membrane lesions, consistent with oncosis. Neuroblastoma tumour cells treated with mAb 8B6 indeed showed an increased uptake of topotecan by the tumor cells and a more profound tumor cell death evidenced by increased caspase-3 activation. We also found that the combination with topotecan plus monoclonal antibody 8B6 showed a more potent anti-tumor efficacy in vivo than either agent alone. Importantly, we used low-doses of topotecan with no noticeable side effect. Our data suggest that chemoimmunotherapy combinations may improve the clinical efficacy and safety profile of current chemotherapeutic modalities of neuroblastoma.

Le glioblastome multiforme (GBM) est la tumeur cérébrale maligne la plus fréquente et la plus agressive chez l’adulte. Malgré l’utilisation concomitante de la chirurgie avec la radiothérapie et la chimiothérapie (protocole de Stupp), le pronostic des patients reste sombre avec un taux de survie à 5 ans inférieur à 10 %. La présence de cellules souches cancéreuses (CSC) favorise le maintien de la tumeur puis l’échappement tumoral responsable de la rechute des patients. Il est donc nécessaire d’identifier de nouvelles cibles thérapeutiques pour améliorer la prise en charge de ces patients. Dans ce contexte, l’immunothérapie, stratégie utilisant le système immunitaire pour traiter le cancer, représente une voie prometteuse.Nous identifions ici le OAcGD2 comme un nouvel antigène tumoral du GBM et a partir de biopsie tumorale provenant d’une cohorte de 37 patients. L’utilisation de primoculture de GBM nous permet de plus de démontrer que ce marqueur est également exprimé par les cellules souches de GBM. Nous apportons la preuve de concept de l’immunothérapie du GBM à l’aide d’anticorps monoclonaux anti-OAcGD2 in vitro et in vivo. A côté de la cytotoxicté immunologique, nous identifions un nouveau mécanisme de mort programmé, l’oncose, mis en jeu par les AcM anti-OAcGD2 permettant de sensibiliser ces cellules aux agents de chimiothérapie comme le témolozomide. L’utilisation de l’immunothérapie ciblant le marqueur gangliosidique tumoral OAcGD2 pourrait ainsi permettre d’améliorer l’efficacité de la thérapeutique actuelle (protocole de Stupp) du GBM / Glioblastoma multiforme (GBM) is the most common and agressive primary brain tumors in adults. Despite the concomitant use of surgery with radiotherapy and chemotherapy, the prognosis of patients remains extremely low. The presence of cancer stem cells (CSC) promotes the maintenance of the tumor and then the tumor escape responsible for the relapse of the patients. Therefore it is necessary to identify new therapeutic targets to improve the management of these patients. In the past few decades, immunotherapy represents an important part of treating certain type of cancer. It uses the immune system to treat cancer. Here, we found that O-acetyl GD2 (OAcGD2) is expressed in surgically resected human glioblastoma tissue. In addition, we demonstrated that 8B6 monoclonal antibody specific for OAcGD2 could effectively inhibit glioblastoma cell proliferation in vitro and in vivo. Mostly, we found that OAcGD2 was expressed on the GBM stem cells. We also observed that mAb 8B6 promoted the elimination of GBM via a oncosis-like mechanism. Moreover, this mechanism of programmed cell death induced by anti-OAcGD2 mAbs, sensitizes GBM cells and CSCs to chemotherapy agents such as temolozomide (TMZ). Taken together, these results indicate that O-acetylated GD2 represents a novel antigen for immunotherapeutic-based treatment of high-grade gliomas, and that anti-OAcGD2 mAbs combined with TMZ could enhance therapeutic response in GBM.

Neuroblastoma is the third most common pediatric cancer. Dinutuximab is a recently approved monoclonal antibody targeting GD2, a ganglioside ubiquitously present on neuroblastoma. Recent studies have shown that αGD2 therapy activates PD1-PDL1 signalling, resulting in the inhibition of its full therapeutic potential. The PD1-PDL1 signalling axis is a cellular checkpoint that inhibits immune responses. The blocking of this interaction has been successful in the treatment of numerous cancers, including in combination with anti-GD2 therapy. The Inhibitor of apoptosis (IAP) proteins are commonly upregulated in cancers and prevent cell death through the inhibition of caspases and through the control of NF-κB activity. Smac mimetic compounds (SMCs) have been designed to target IAP activity, thereby promoting cancer cell death. Here, I used the SMC, LCL161, to improve αGD2 antibody treatment against a GD2+ syngeneic neuroblastoma mouse model. I found that murine cell lines NXS2 and N2a were resistant in vitro to LCL161-mediated apoptosis, despite expressing apoptotic components often silenced in neuroblastoma. In vivo, I observed a slight delay in tumour growth induced by LCL161 and I confirmed an in vivo anti-angiogenic effect of LCL161 through ultrasound imaging and necropsy evaluation. I then combined LCL161 and αGD2 antibody (clone ME361-S2a) treatment and reported a delay in NXS2 subcutaneous tumour growth, which was further potentiated with the addition of an αPD-L1 antibody. With optimization, there is potential for SMCs to be used in combination with αGD2 therapy in GD2+ cancers like neuroblastoma.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / Cell isolation method may have effects on the characteristics of the cells isolated from porcine umbilical cords. As stem cells age or approach senescence, it is hypothesized that their properties change. We expect that isolation method and age of cells will have effects on the phenotype of porcine umbilical cord (PUC) cells during in vitro expansion. We investigated the effects of three isolation methods on PUC population doublings, ability to produce colony forming units (CFU), and amount of ganglioside GD2 (GD2) expression over eleven passages. Isolation methods were explant (Exp) in which the Wharton's Jelly was removed from cords, minced and plated, enzyme digest (Dig), and stomacher assisted enzyme digestion (Stom). Cell isolates were analyzed for GD2 expression, CFU, and population doublings at early (3), middle (7), and late (11) passage. The Exp method produced greater (P<0.05) population doublings and more (P<0.05) CFU at passage 7. Explant isolates also were numerically more likely to survive to passage 11 (9/9 isolates vs 5/9 for Dig and 7/9 for Stom). In contrast, the percent cells expressing GD2 was greater (P<0.05) for Stom isolates than Exp isolates at passage 11. There were no trends for increased passage number to decreased population doubling, CFU formation, or percent GD2 positive cells. In summary, our results indicate that the Exp isolation method produced the greatest number of population doublings over 11 passages and there were minimal effects of isolation method on CFU and GD2 expression. Although Exp may be more difficult to scale up to isolate all of the PUCs in a cord, it provided greater in vitro expansion than the enzyme methods in our experiment and may provide the most cells for biotechnological and biomedical applications.

As células tronco mesenquimais de tecido adiposo (MSCs-TA) são células progenitoras que residem entre adipócitos e contribuem para o turnover do tecido adiposo. Gangliosídios são glicoensfigolipídios localizados na membrana das células, envolvidos na regulação do crescimento celular, interação de superfície, sinalização transmembrana e diferenciação celular. O gangliosídio neural GD2 foi relatado como um marcador de superfície de células tronco mesenquimais de medula óssea e cordão umbilical, mas existem poucos dados sobre a expressão do GD2 em MSCs-TA indiferenciadas e nas diferenciadas para adipócito ou osteoblasto. Nosso principal objetivo foi estudar a expressão de gangliosídios nas MSCs-TA, em especial o GD2, durante a diferenciação adipogênica e osteogênica. Para isso, as MSCs-TA foram isoladas de lipoaspirado humano, cultivadas e induzidas para diferenciação adipogênica e osteogênica. As análises foram feitas por HPTLC, microscopia confocal, citometria de fluxo e PCR em tempo real. Por HPTLC, as MSCs-TA indiferenciadas e MSCs-TA diferenciadas para adipócitos e osteoblasto mostraram aumento do perfil de gangliosídios complexos. A microscopia confocal evidenciou os gangliosídios GM3, GM1 e GD2 na superfície das células e, por citometria de fluxo, identificamos uma subpopulação de células GD2 positivas nas MSCs-TA e MSCs-TA diferenciadas para adipócito ou osteoblasto. Entretanto, o percentual de células GD2 positivas decresceu com a diferenciação. A expressão do mRNA da GD2 sintase aumentou na diferenciação adipogênica e diminui na diferenciação osteogênica. O GD2 é um substrato para a biosíntese de gangliosídios complexos e o aumento da expressão da GD2 sintase pode estar relacionado com o aumento de gangliosídios complexos que ocorre durante a diferenciação adipogênica. / Mesenchymal Stem Cells from Adipose Tissue (MSCs-TA) are progenitor cells that reside between adipocytes, and contribute to the turnover of adipose tissue. Gangliosides are glycosphingolipids localized in cell membrane, involved in cell growth regulation, surface interaction, transmembrane signaling and differentiation. The neural ganglioside GD2 has been reported as surface marker for MSCs from bone marrow and umbilical cord, but sparse data exist about the expression of GD2 in MSCs-TA and during the differentiation to adipocytes and osteoblast. Our aim was to study the expression of glangliosides, in special of GD2 in MSCs-TA and during the adipogenic and osteogenic differentiation. Thus MSCs-TA were isolated from lipoaspirate, cultured and induced to adipogenic and osteogenic differentiation. Then, we examined the gangliosides expression by HPTLC, confocal microscopy, flow citometry and real-time PCR. By HPTLC, the MSCs-TA and MSCs-TA differentiated into adipocytes and osteoblast demonstrate an increased complex gangliosides profile. The confocal microscopy showed the presence of GM3, GM1, and GD2 on the cell surface. By the flow cytometry, we identified a GD2 positive subpopulation in MSCs-TA and in MSCs-TA differentiated to adipocytes and osteoblast. However, the percentage of GD2 positive cells decreased with the differentiation. The expression of GD2 synthase mRNA increased during the adipogenic differentiation and decreased in osteogenic differentiation. GD2 is a substrate for the complex gangliosides biosynthesis, and the increase in GD2 synthase expression could be related with the increase in complex gangliosides that occurs during the adipogenic differentiation.

As células tronco mesenquimais de tecido adiposo (MSCs-TA) são células progenitoras que residem entre adipócitos e contribuem para o turnover do tecido adiposo. Gangliosídios são glicoensfigolipídios localizados na membrana das células, envolvidos na regulação do crescimento celular, interação de superfície, sinalização transmembrana e diferenciação celular. O gangliosídio neural GD2 foi relatado como um marcador de superfície de células tronco mesenquimais de medula óssea e cordão umbilical, mas existem poucos dados sobre a expressão do GD2 em MSCs-TA indiferenciadas e nas diferenciadas para adipócito ou osteoblasto. Nosso principal objetivo foi estudar a expressão de gangliosídios nas MSCs-TA, em especial o GD2, durante a diferenciação adipogênica e osteogênica. Para isso, as MSCs-TA foram isoladas de lipoaspirado humano, cultivadas e induzidas para diferenciação adipogênica e osteogênica. As análises foram feitas por HPTLC, microscopia confocal, citometria de fluxo e PCR em tempo real. Por HPTLC, as MSCs-TA indiferenciadas e MSCs-TA diferenciadas para adipócitos e osteoblasto mostraram aumento do perfil de gangliosídios complexos. A microscopia confocal evidenciou os gangliosídios GM3, GM1 e GD2 na superfície das células e, por citometria de fluxo, identificamos uma subpopulação de células GD2 positivas nas MSCs-TA e MSCs-TA diferenciadas para adipócito ou osteoblasto. Entretanto, o percentual de células GD2 positivas decresceu com a diferenciação. A expressão do mRNA da GD2 sintase aumentou na diferenciação adipogênica e diminui na diferenciação osteogênica. O GD2 é um substrato para a biosíntese de gangliosídios complexos e o aumento da expressão da GD2 sintase pode estar relacionado com o aumento de gangliosídios complexos que ocorre durante a diferenciação adipogênica. / Mesenchymal Stem Cells from Adipose Tissue (MSCs-TA) are progenitor cells that reside between adipocytes, and contribute to the turnover of adipose tissue. Gangliosides are glycosphingolipids localized in cell membrane, involved in cell growth regulation, surface interaction, transmembrane signaling and differentiation. The neural ganglioside GD2 has been reported as surface marker for MSCs from bone marrow and umbilical cord, but sparse data exist about the expression of GD2 in MSCs-TA and during the differentiation to adipocytes and osteoblast. Our aim was to study the expression of glangliosides, in special of GD2 in MSCs-TA and during the adipogenic and osteogenic differentiation. Thus MSCs-TA were isolated from lipoaspirate, cultured and induced to adipogenic and osteogenic differentiation. Then, we examined the gangliosides expression by HPTLC, confocal microscopy, flow citometry and real-time PCR. By HPTLC, the MSCs-TA and MSCs-TA differentiated into adipocytes and osteoblast demonstrate an increased complex gangliosides profile. The confocal microscopy showed the presence of GM3, GM1, and GD2 on the cell surface. By the flow cytometry, we identified a GD2 positive subpopulation in MSCs-TA and in MSCs-TA differentiated to adipocytes and osteoblast. However, the percentage of GD2 positive cells decreased with the differentiation. The expression of GD2 synthase mRNA increased during the adipogenic differentiation and decreased in osteogenic differentiation. GD2 is a substrate for the complex gangliosides biosynthesis, and the increase in GD2 synthase expression could be related with the increase in complex gangliosides that occurs during the adipogenic differentiation.

As células tronco mesenquimais de tecido adiposo (MSCs-TA) são células progenitoras que residem entre adipócitos e contribuem para o turnover do tecido adiposo. Gangliosídios são glicoensfigolipídios localizados na membrana das células, envolvidos na regulação do crescimento celular, interação de superfície, sinalização transmembrana e diferenciação celular. O gangliosídio neural GD2 foi relatado como um marcador de superfície de células tronco mesenquimais de medula óssea e cordão umbilical, mas existem poucos dados sobre a expressão do GD2 em MSCs-TA indiferenciadas e nas diferenciadas para adipócito ou osteoblasto. Nosso principal objetivo foi estudar a expressão de gangliosídios nas MSCs-TA, em especial o GD2, durante a diferenciação adipogênica e osteogênica. Para isso, as MSCs-TA foram isoladas de lipoaspirado humano, cultivadas e induzidas para diferenciação adipogênica e osteogênica. As análises foram feitas por HPTLC, microscopia confocal, citometria de fluxo e PCR em tempo real. Por HPTLC, as MSCs-TA indiferenciadas e MSCs-TA diferenciadas para adipócitos e osteoblasto mostraram aumento do perfil de gangliosídios complexos. A microscopia confocal evidenciou os gangliosídios GM3, GM1 e GD2 na superfície das células e, por citometria de fluxo, identificamos uma subpopulação de células GD2 positivas nas MSCs-TA e MSCs-TA diferenciadas para adipócito ou osteoblasto. Entretanto, o percentual de células GD2 positivas decresceu com a diferenciação. A expressão do mRNA da GD2 sintase aumentou na diferenciação adipogênica e diminui na diferenciação osteogênica. O GD2 é um substrato para a biosíntese de gangliosídios complexos e o aumento da expressão da GD2 sintase pode estar relacionado com o aumento de gangliosídios complexos que ocorre durante a diferenciação adipogênica. / Mesenchymal Stem Cells from Adipose Tissue (MSCs-TA) are progenitor cells that reside between adipocytes, and contribute to the turnover of adipose tissue. Gangliosides are glycosphingolipids localized in cell membrane, involved in cell growth regulation, surface interaction, transmembrane signaling and differentiation. The neural ganglioside GD2 has been reported as surface marker for MSCs from bone marrow and umbilical cord, but sparse data exist about the expression of GD2 in MSCs-TA and during the differentiation to adipocytes and osteoblast. Our aim was to study the expression of glangliosides, in special of GD2 in MSCs-TA and during the adipogenic and osteogenic differentiation. Thus MSCs-TA were isolated from lipoaspirate, cultured and induced to adipogenic and osteogenic differentiation. Then, we examined the gangliosides expression by HPTLC, confocal microscopy, flow citometry and real-time PCR. By HPTLC, the MSCs-TA and MSCs-TA differentiated into adipocytes and osteoblast demonstrate an increased complex gangliosides profile. The confocal microscopy showed the presence of GM3, GM1, and GD2 on the cell surface. By the flow cytometry, we identified a GD2 positive subpopulation in MSCs-TA and in MSCs-TA differentiated to adipocytes and osteoblast. However, the percentage of GD2 positive cells decreased with the differentiation. The expression of GD2 synthase mRNA increased during the adipogenic differentiation and decreased in osteogenic differentiation. GD2 is a substrate for the complex gangliosides biosynthesis, and the increase in GD2 synthase expression could be related with the increase in complex gangliosides that occurs during the adipogenic differentiation.

Das Induzieren und Aufrechterhalten einer tumor-protektiven Immunität sind wesentliche Ziele in der Immuntherapie des Neuroblastoms. Eine Erhöhung der Anzahl von tumor-infiltrierenden Leukozyten könnte ein Weg sein, um dieses Ziel zu erreichen. Fractalkine ist ein besonderes TH1 CX3C Chemokin, welches sowohl Adhäsion und Migration von Leukozyten vermittelt. Gerichtetes IL-2 (ch14.18-IL-2) wurde durch eine genetische Fusion von anti-GD2 Antikörper mit IL-2 hergestellt, damit IL-2 spezifisch in das Mikromilieu von Neuroblastomen gebracht werden kann. In dieser Arbeit habe ich die Hypothese getestet, dass Gentherapie mit dem Chemokin Fractalkine (FKN) eine wirksame Antineuroblastom-Immunantwort induziert, welche durch gerichtetes IL-2 amplifiziert wird. Zu diesem Zweck wurden NXS2-Zellen genetisch verändert, damit sie murines FKN produzieren (NXS2-FKN). Transkription und Expression des mFKN Gens konnte in NXS2-FKN Zellen und Tumorgewebe gezeigt werden. Die chemotaktische Eigenschaft von FKN wurde sowohl in vitro als auch in vivo gezeigt. FKN zeigte eine Reduktion des Primärtumorwachstums, welches durch gerichtetes IL-2 mit nicht-kurativen Dosen von ch14.18-IL-2 deutlich verbessert wurde. Ferner wurden experimentelle Lebermetastasen nur in den Mäusen komplett eradiziert, welche die Kombinationstherapie erhalten haben. Die Mechanismen, welche an dieser Antitumorantwort beteiligt sind, schließen eine wirksame T-Zell-Aktivierung (Hochregulation von CD69, CD25, und von TNF-alpha und INF-gamma), sowie eine Erhöhung der tumorspezifischen CTL-Aktivität mitein. Die Depletion von CD4+ und CD8+ T-Zellen in vivo hat diesen therapeutischen Effekt aufgehoben, was die essentielle Rolle von T-Zellen in diesem immuntherapeutischen Ansatz unterstreicht. Zusammenfassend konnte ich zum ersten Mal zeigen, dass Chemokin-Gentherapie mit FKN durch gerichtetes IL-2 amplifiziert wird, was eine Kombination dieser beiden Strategien zur adjuvanten Therapie beim Neuroblastom nahe legt. / Induction and maintenance of tumor-protective immunity are the major goals of neuroblastoma immunotherapy. Enhancing the amount of tumor infiltrating leukocytes might be a way to achieve these goals since they may be associated with residual evidence of the ineffective immune response. Fractalkine is a unique TH1 CX3C chemokine known to induce both adhesion and migration of leukocytes mediated by a membrane-bound and a soluble form, respectively. Targeted IL-2 (ch14.18-IL-2) was constructed by anti-GD2 antibody fused with IL-2 so that IL-2 can be directed into the microenvironment of neuroblastoma tumor. Here, I tested the hypothesis that chemokine gene therapy with fractalkine (FKN) induces an effective anti-neuroblastoma immune response amplified by targeted IL-2. NXS2 cells were engineered to stably produce murine FKN (NXS2-FKN). Transcrip- tion and expression of the mFKN gene in NXS2-FKN cells and tumor tissue were demonstrated. The chemotactic activity of FKN expressed by NXS2 cells was determined both in vitro and in vivo. Importantly, NXS2-FKN exhibited a reduction in primary tumor growth, which was boosted by targeted IL-2 using non-curative doses of ch14.18-IL-2. Furthermore, experimental liver metastases were completely eradicated in mice receiving the combination therapy, demonstrating the induction of a long-lived tumor protective response. The mechanisms involved in antitumor response included effective T cell activation as indicated by the up-regulation of T-cell activation markers (CD69, CD25) and proinflammatory cytokines (TNF-alpha, INF-gamma) as well as the enhancement of tumor specific CTL activity. The depletion of CD4+ and CD8+ T cells in vivo abrogated the therapeutic effect supporting the crucial role of T cells in this immunotherapeutic approach. In summary, I demonstrated for the first time that chemokine gene therapy with FKN is amplified by targeted IL-2 suggesting a combination of both strategies as an adjuvant therapy for neuroblastoma.

Le neuroblastome (NB) est la tumeur extra-crânienne la plus fréquente du jeune enfant. Malgré une thérapie multimodale très agressive, 40% des patients atteints de NB à haut risque rechutent. Le traitement de ces patients consiste à éliminer la tumeur par chirurgie, radiothérapie et chimiothérapie, à reconstituer la moelle osseuse par une greffe de cellules souches autologues et enfin à éliminer la maladie résiduelle (MRD) par une immunothérapie visant l’antigène GD2 exprimé par les neuroblastes. Notre étude préclinique a examiné l’efficacité de deux stratégies de traitements qui visent à potentialiser les thérapies actuelles et réduire leur toxicité. La première consiste à réduire la masse tumorale par la radiothérapie ciblée combinée à des radiosensibilisants. La deuxième approche est basée sur l’activation des cellules natural killer (NK) pour potentialiser l’effet de l’immunothérapie anti-GD2 et éliminer la MRD. L’autophagie est un processus catabolique qui élimine les protéines et organelles endommagées par différents stress incluant les irradiations. Par conséquent, inhiber l’autophagie pourrait sensibiliser les neuroblastes aux irradiations. Or, nous avons montré qu’étant très radiosensibles, les neuroblastes ne sont pas davantage éliminés par les irradiations quand ils sont traités avec un inhibiteur de l’autophagie. De plus, l’absence d’un inhibiteur efficace de l’autophagie à usage thérapeutique ne permet pas actuellement d’adopter cette approche. Notre étude a également permis de révéler une nouvelle approche de stimulation des cellules NK par les cellules dendritiques plasmacytoïdes (pDC) activées par un ligand du récepteur Toll-like, capable d’éradiquer la MRD et prévenir les rechutes de NB. Nos résultats ont permis, d’une part, d’élucider les mécanismes impliqués dans la lyse des cellules NK activées par les pDC contre les neuroblastes et, d’une autre part, de démontrer que l’axe pDC-NK chez le patient est fonctionnel, augmente l’efficacité de l’anti-GD2 et élimine efficacement les neuroblastes. Ainsi, l’immunothérapie par les cellules NK est une stratégie très prometteuse pour traiter le NB. Cette étude préclinique servira de base à l’élaboration d’un essai clinique pour traiter les enfants atteints de NB au CHU Sainte Justine. / Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Despite aggressive multimodal therapy, 40% of patients with high-risk NB relapse. The current therapy comprises an induction treatment with chemotherapy and surgery, a consolidation treatment including radiotherapy and high-dose chemotherapy followed by bone marrow rescue with autologous hematopoietic stem cell transplantation and finally anti-GD2 immunotherapy targeting the disialoganglioside (GD2) antigen expressed by neuroblasts to treat minimal residual disease (MRD). Our preclinical study proposes two treatment strategies to potentiate current therapies and reduced toxicities. The first aim to reduce tumor mass by targeted radiotherapy combined with radiosensitizers. The second approach is based on the activation of natural killer (NK) cells to potentiate the effect of anti-GD2 therapy and eliminate MRD. Autophagy is a catabolic process that recycle damaged proteins and organelles, induced under various conditions of cellular stress including irradiation. Therefore, inhibiting autophagy could sensitize neuroblasts to irradiation. However, our study showed that neuroblasts were highly sensitive to irradiation and autophagy inhibitor failed to increase neuroblasts sensitization to irradiation. In addition, the absence of a potent autophagy inhibitor for therapeutic use does not allow this approach to be adopted. Our preclinical study demonstrated a novel approach based on NK cell stimulation with Toll-like activated plasmacytoid dendritic cells (pDC) that enhances the efficacy of anti-GD2 immunotherapy and prevent NB relapse. We elucidated the mechanisms involved in pDC-activated NK cells killing of neuroblasts. We further demonstrated that neuroblasts were efficiently killed by patient’s NK cells after stimulation by activated pDC. This is further increased by the addition of anti-GD2 antibody. Altogether, our study demonstrates that NK cell-based immunotherapy has a real potential to enhance anti-GD2 immunotherapy effect and prevent NB relapse. This preclinical study will serve as a basis for the development of a clinical trial to treat children with NB at CHU Sainte Justine.

Systemet som förenklar hantering och information av tidrapportering, projekt, kunder, lager och anställda. Syftet är att företaget skall få en mera överblick över deras verksamhet, då all information samlas på samma ställe. De anställda kan via webben snabbt och enkelt rapportera in deras arbetstider under den gångna veckan. Systemet innefattar: SMS-tjänst, automatiskt utskick, hanteringen för lager, projekt, kunder och semester, kontinuerlig statistik med grafiska diagram samt utskriftsfunktioner. Allt är utvecklat i PHP och MySQL. / The system simplifies the management and information, about time reports, projects, customers, storage and employees. The main purpose for this system is that the company shall have a more structured overview about their company, and where all the information is gathered, at the same spot. The employees are able to report in their working progress during the week, in a simple easy way trough the internet. The system contains a SMS service, automatically circular messages, management of storage, projects, clients and employees vacations. The system also contains continuous dynamic statistics, with graphical layout and also print functionality. Everything is developed in PHP with MySQL as database. / Detta är en reflektionsdel till en digital medieproduktion. mange@mdw.se www.mdw.se