Limb-Bud and Heart (LBH) is a novel key transcriptional regulator of vertebrate development. However, the molecular mechanisms upstream of LBH and its role in adult development are unknown. Here we show that in epithelial development, LBH expression is tightly controlled by Wnt signaling. LBH is transcriptionally induced by the canonical Wnt pathway, as evident by the presence of functional TCF/LEF binding sites in the LBH locus and rapid beta-catenin-dependent upregulation of endogenous LBH by Wnt3a. In contrast, LBH induction by Wnt/beta-catenin signaling is inhibited by Wnt7a, which in limb development signals through a non-canonical pathway involving Lmx1b. Furthermore, we show that Lbh is aberrantly overexpressed in mammary tumors of MMTV-Wnt1 transgenic mice and in aggressive basal-subtype human breast cancers that display Wnt/beta-catenin hyperactivation. Deregulation of LBH in human breast cancer appears to be Wnt/beta-catenin dependent as DKK1 and Wnt7a inhibit LBH expression in breast tumor cells. RNAi mediated knockdown of LBH in basal breast cancer cell lines resulted in loss of CD44high/CD24low tumor cells, luminal differentiation, reduced cell growth, reduced colony forming ability, and increased apoptosis, suggesting a novel pro-survival and stem cell maintenance function of LBH in breast cancer. Reciprocal overexpression studies in the basal breast carcinoma line BT549 resulted in increased tumorigenicity in vitro, suggesting that LBH overexpression is indeed oncogenic. Finally, we further characterized LBH protein expression patterns and post-transcriptional regulation. Collectively, this thesis demonstrates that LBH is a direct Wnt target gene in both development and basal breast cancer that promotes the undifferentiated phenotype and survival of basal breast tumor cells.

DOCTOR OF PHILOSOPHY (2019) McMaster University, Hamilton, Ontario (Medical Sciences) TITLE: Serotonergic antagonists affect the activity of breast tumor initiating cells in human and mouse models of breast cancer. AUTHOR: William D. Gwynne, BSc SUPERVISOR: Dr. John A. Hassell NUMBER OF PAGES: XXI; 255 / Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related death amongst women worldwide. The relatively unchanging breast cancer-associated mortality rate is in part due to the existence of rare tumor cells (breast tumor initiating cells; BTIC) that possess stem-like properties permitting them to survive therapy and initiate disease recurrence. Hence, identifying agents capable of eradicating these cells would be a favourable therapeutic strategy to improve the durability of breast cancer remissions. To achieve the latter objective our lab screened over 35,000 small molecules for their capacity to inhibit the viability of BTIC-enriched mouse tumor cells. Unexpectedly, several antagonists of the serotonin (5-hydroxytryptamine; 5-HT) transporter and select receptors were among the hit compounds identified in the screen. This thesis aims to establish a connection between serotonergic activity and BTIC. We accomplished the latter by assessing whether components of the 5-HT signaling system are expressed in mouse and human breast tumor cells and whether inhibition of their activity affects BTIC frequency using multiple orthogonal assays. Our data suggest that breast tumor cells of both mouse and human origin express the components necessary for 5-HT synthesis, activity and metabolism and that inhibition of these proteins with selective antagonists reduces the capacity of these cells to form tumorspheres. We demonstrate that highly selective antagonists of SERT and HTR5A target BTIC as established ex vivo cell transplantation assays. We also discovered that these agents synergize with chemotherapy in vivo to affect the growth of mouse breast tumor allografts and human breast tumor xenografts. To validate the molecular targets of these agents, we attempted to phenocopy their effects in functional assays by knocking out their respective genes using CRISPR-Cas9 technology. Collectively, this thesis contributes to an understanding of how 5-HT signaling affects BTIC and identifies serotonergic antagonists as novel anticancer agents. / Dissertation / Doctor of Philosophy (PhD) / Despite improvements in screening technologies and the development of targeted therapies breast cancer remains the second leading cause of cancer-related death among Canadian women. Whereas the current standard of care is effective at treating the majority of patients diagnosed with breast cancer, there remains a substantial proportion of patients that experience relapse after undergoing therapy. Recurrence is due in part to the existence of rare, stem-like tumor cells, termed breast tumor-initiating cells (BTIC) that are insensitive to existing anticancer agents. Hence, identifying drugs capable of targeting these cells is a desirable goal. To pursue the latter, our lab screened approximately 35,000 compounds for their capacity to affect the growth of BTIC-enriched tumor cell populations. Among the hit compounds were antagonists of the serotonin transporter and serotonin receptors, including FDA-approved psychiatric medications. Here, we explore a connection between serotonin-related proteins and BTIC activity with the aim of identifying novel therapeutic agents.

Triple negative breast cancers (TNBCs) account for ~15-20% of human breast cancers in Western countries. TNBCs are associated with poor prognosis and a low five year survival rate due, in part, to high rates of tumor recurrence, multi-drug resistance, metastasis, and therapeutic toxicity. Tumor initiating cells (TICs) or cancer stem cells (CSCs) are proposed to be responsible for the origin and maintenance of tumors as well as cancer recurrence, metastasis and drug resistance. Nutritionally-based low- to non-toxic therapeutic nutrients that eliminate both bulk tumor cells (non-TICs) and TICs have potential for prevention and treatment of primary and metastatic cancers, including TNBCs. Omega-3 fatty acid-docosahexaenoic acid (DHA) and certain vitamin E compounds [gamma- and delta- tocopherols [mathematical symbols] and tocotrienols [mathematical symbols]], separately and in combination, were investigated for their ability to eliminate non-TICs and TICs in human TNBCs and the mechanisms of action were studied. DHA induced apoptosis in several human TNBC cell lines via activation of endoplasmic-reticulum stress (ER stress) mediated C/EBP (CCAAT/enhancer binding protein) homologous protein (CHOP)/death receptor-5 (DR5) pro-apoptotic signaling involving caspases-8 and 9. DHA eliminated TICs as measured by elimination of aldehyde dehydrogenase active (ALDH⁺) population and inhibition of mammosphere formation. DHA eliminated TICs via suppression of phosphorylated Signal transducers and activators of transcription 3 (pStat-3) as well as downstream mediators cellular myelocytomatosis oncogene (c-Myc) and cyclin D1. SiRNA to Stat-3 reduced the number of ALDH⁺ TNBCs cells and reduced pStat-3, c-Myc, and cyclin D1 mediators, showing that Stat-3 is necessary for maintaining ALDH⁺ population and that c-Myc and cyclin D1 are downstream mediators of Stat-3. Studies also demonstrated that vitamin E compounds possess distinct anticancer activities. In summary, studies provide novel insights into therapeutic potential of DHA and certain vitamin E compounds for treatment of TNBCs. / text

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19965号 / 医博第4155号 / 新制||医||1017(附属図書館) / 33061 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 伊達 洋至, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The high rate of relapse often seen in breast cancer patients has been suggested to be the result of a small subset of chemotherapy-resistant cancer stem cells (CSCs), believed to be responsible for initiating tumor formation. These CSCs possess the capability to self-renew and give rise to a hierarchy of cells which makes up the bulk of a tumor. Neurotransmitters have been suggested to influence CSC self-renewal and proliferation capabilities, and antagonists of neurotransmission pathways have been implicated as possible treatment methods for chemo-resistant tumors. Using nicotinic acetylcholine receptor (nAChR) antagonists in sphere-forming assays, we have identified a very promising candidate compound: MG624. We found this compound to have a high selectivity for sphere-forming cells over non-sphere-forming cells in vitro, in a dose-dependent relationship, across a panel of cell lines as well as in patient-derived xenograft cells. This was validated in two ex vivo assays, where tumor formation was significantly delayed in mice injected with MG624-treated HCC1954 cells at both the IC50 and IC90 of the compound, indicating that MG624 does indeed target functional BTICs. MG624 was also found to synergize with both taxotere and doxorubicin chemotherapies in vitro, and shrink tumors in NOD/SCID mice when combined with taxotere in vivo. MG624 in combination with taxotere was found to induce apoptosis, and prevent cells from entering into the M-phase of the cell cycle. Interestingly, MG624 was found to eliminate intratumoral fibroblasts in combination with taxotere, despite taxotere being found to recruit fibroblasts to the tumor site when used on its own. Most importantly, the combination of MG624 and taxotere was found to significantly delay tumor progression/relapse in mice, indicating that MG624 may be an excellent candidate compound to one day be combined with chemotherapy to provide durable remission to breast cancer patients. / Thesis / Master of Science (MSc)

Breast cancer therapy resistance and disease recurrence are driven by an infrequent population of stem-like tumor cells, termed breast cancer stem cells or tumor-initiating cells (BTIC). Whereas drugs that target BTIC could be combined with conventional therapies to provide durable remissions, identifying such agents has been difficult. To achieve the latter, our lab screened more than 35,000 compounds for their capacity to reduce the activity of BTIC-enriched mouse mammary tumorspheres, wherein we identified numerous antagonists of multiple serotonin receptors (HTRs). The serotonergic antagonist that prevented sphere formation with the highest potency is a highly selective antagonist of HTR5A, SB-699551. We subsequently demonstrated that this agent affects BTIC activity in breast tumor cell lines representative of all clinical and molecular subtypes of breast cancer. Whereas the primary target of SB-699551 is known, the downstream signaling pathways responsible for its anti-BTIC effect remains enigmatic. The goal of this thesis work was to elucidate the signaling pathways downstream of HTR5A in human breast tumor cell lines. We used a phospho-proteomic approach to establish that treatment of human SB-699551 affects the phosphorylation of proteins involved in the Gi-coupled and the PI3K/AKT/mTOR signaling axes. Moreover, we demonstrated that selective antagonists of PI3K, AKT, and mTOR phenocopied the effect of SB-699551 in tumorsphere forming assays. Taken together, our data suggests that SB-699551 elicits its effect through the PI3K/AKT/mTOR signaling pathways downstream of HTR5A. / Thesis / Master of Health Sciences (MSc) / Accumulating data suggests that the progression of breast cancer is driven by a rare population of breast tumor-initiating cells (BTIC). BTIC lie dormant during conventional therapy and initiate recurrence after such therapies are withdrawn. Hence, there is an urgent need to develop drugs that target BTIC that can be combined with the current standard of care to improve the durability of remission. With the latter objective in mind, our lab previously determined that antagonists of serotonin signaling target BTIC. One of the agents that we identified in our screen inhibits the activity of serotonin receptor 5A (HTR5A). The exact signaling mechanism whereby inhibition of HTR5A leads to a loss in BTIC activity was enigmatic. Hence, this thesis aims to elucidate the signaling pathways downstream of HTR5A in breast cancer. Knowledge of the latter will help identify a plausible mechanism in addition to identifying biomarkers of therapy efficacy.

Dielectrophoresis (DEP), an electrokinetic force, is the motion of a polarizable particle in a non-uniform electric field. Contactless DEP (cDEP) is a recently developed cell sorting and isolation technique that uses the DEP force by capacitavely coupling the electrodes across the channel. The cDEP platform sorts cells based on intrinsic biophysical properties, is inexpensive, maintains a sterile environment by using disposable chips, is a rapid process with minimal sample preparation, and allows for immediate downstream recovery. This platform is highly competitive compared to other cell sorting techniques and is one of the only platforms to sort cells based on phenotype, allowing for the isolation of unique cell populations not possible in other systems. The original purpose of this work was to determine differences in the bioelectrical fingerprint between several critical cancer types. Results demonstrate a difference between Tumor Initiating Cells, Multiple Drug Resistant Cells, and their bulk populations for experiments conducted on three prostate cancer cell lines and treated and untreated MOSE cells. However, three significant issues confounded these experiments and challenged the use of the cDEP platform. The purpose of this work then became the development of solutions to these barriers and presenting a more commercializable cDEP platform. An improved analysis script was first developed that performs whole cell detection and cell tracking with an accuracy of 93.5%. Second, a loading system for doing smart sample handling, specifically cell focusing, was developed using a new in-house system and validated. Experimental results validated the model and showed that cells were successfully focused into a tight band in the middle of the channel. Finally, a proof of concept for an on-chip micropump is presented and achieved 4.5% in-plane deformation. When bonded over a microchannel, fluid flow was induced and measured. These solutions present a stronger, more versatile cDEP platform and make for a more competitive commercial product. However, these solutions are not just limited to the cDEP platform and may be applicable to multitudes of other microfluidic devices and applications. / Master of Science

O câncer de mama é a doença maligna que mais acomete as mulheres no mundo. Apesar dos inúmeros tratamentos, o óbito se deve principalmente à doença metastática que pode se desenvolver a partir do tumor primário. Esta progressão tumoral decorre da dificuldade de se estabelecer um prognóstico mais preciso. Atualmente, a teoria de células iniciadoras de tumor vem sendo estudada para tentar explicar a biologia do câncer e descrever novos alvos para prognósticos e terapias. O carcinoma mamário foi o primeiro tumor sólido para o qual foi identificada uma subpopulação celular, definida como CD44+/CD24-, apresentando as características de células iniciadoras tumorais. Embora este fenótipo venha sendo muito utilizado para descrever as células iniciadoras tumorais de mama, muitos trabalhos tem questionado a relevância clínica desses marcadores, enfatizando que outros marcadores devem ser identificados. Assim, o objetivo deste trabalho é analisar e caracterizar marcadores de células-tronco que possam estar relacionados com o grau de malignidade no modelo de câncer de mama. Inicialmente, analisou-se a expressão de 10 marcadores de células-tronco em diferentes linhagens de câncer de mama que apresentam graus crescentes de malignidade. O CD90 foi selecionado devido à alta expressão desse marcador na linhagem mais agressiva Hs578T. Para a caracterização deste marcador, realizou-se ensaios funcionais, através do silenciamento do CD90 na linhagem tumorigênica Hs579T e sua superexpressão na linhagem não-tumorigênica MCF10A. As linhagens celulares geradas foram caracterizadas quanto ao crescimento celular, potencial invasivo e metastático. Foi possível observar que houve uma alteração da morfologia nas linhagens transformadas com o CD90 e, também, um maior tempo de dobramento na linhagem Hs578T-CD90- e um menor na MCF10A-CD90+. Além disso, a linhagem MCF10-CD90+ foi capaz de crescer independentemente de EGF. Através da análise da via EGF, foi possível observar que houve um aumento da expressão da forma fosforilada do receptor e dos fatores Erk, c-Jun, e Jnk na linhagem MCF10A-CD90+ e uma diminuição dos mesmos na linhagem Hs578T-CD90-. A análise da atividade do elemento responsivo do fator de transcrição AP1 comprovou que a via de EGF é funcional na linhagem MCF10-CD90+. Também foram analisados os marcadores de transição epitélio-mesenquimal, verificando-se aumento da expressão dos marcadores mesenquimais na linhagem MCF10A-CD90+ e diminuição na linhagem Hs578T-CD90-. Os ensaios in vitro de invasão mostraram que as células MCF10-CD90+ são capazes de migrar e invadir e as células Hs578T-CD90- apresentam diminuição significativa da habilidade de migração e invasão. Além disso, os ensaios de metástase in vitro e in vivo, mostraram que a superexpressão de CD90 levou à malignização das células MCF10A. Por outro lado, a linhagem Hs578T-CD90- apresentou menor potencial metastático in vitro. Portanto, neste trabalho, pela primeira vez, o CD 90 foi caracterizado funcionalmente como um marcador envolvido na transformação maligna do carcinoma mamário, contribuindo, assim, para melhor entendimento da biologia do câncer de mama e para que se possa desenvolver novas ferramentas de diagnóstico/prognóstico e novos protocolos clínicos e terapêuticos. / Breast cancer is the malignant disease which affects the highest number of women in the world. In spite of the numerous treatments available, death is primarily due to the metastatic disease that may develop from the primary tumor. This tumor progression occurs because of the difficulty in establishing an accurate diagnosis/prognosis. Currently, the tumor initiating cells theory is being applied in an attempt to explain cancer biology and to unveil new diagnostic and therapeutic targets. Mammary carcinoma was the first solid tumor in which a cellular subpopulation, defined as CD44+/CD24-, was associated with tumor initiating cells. Although this phenotype has been widely used to describe breast tumor initiating cells, several studies have questioned the clinical relevance of these markers, emphasizing that additional markers should be identified. The objective of the present study is to analyze and characterize stem cell markers that may be related to malignancy stages in the breast cancer model. Initially, the expression of 10 stem cell markers was analyzed in different breast cancer cell lines displaying different malignancy grades. CD90 was selected due to its high expression levels in the most aggressive cell line, namely: Hs578T. In order to further characterize this marker, a functional study was performed in which CD90 was silenced in the Hs578T tumorigenic cell line and overexpressed in the non-tumorigenic MCF10A cell line. The resulting cell lines were characterized relative to growth rate and invasive and metastatic potential. A change in morphology readily was observed in the cell lines overexpressing CD90. In addition, the Hs578T-CD90-cell line presented an increased doubling time (DT), while the MCF10A-CD90+ cell line displayed a lower DT.. Furthermore, MC10-CD90+ cells were able to grow in the absence of EGF. Analysis of components of the EGF pathwayrevealed increased expression levels of the phosphorylated form of Erk, c-Jun and Jnk receptors in the MCF10-CD90+ cell line, while Hs578T-CD90- cells presented decreased expression of the same factors and receptors. Analysis of the activity of the AP1 responsive element allowed confirmation that the EGF pathway is functional in the MCF10-CD90+. . Epithelial-mesenquimal transition markers presented increased expression levels in the MCF10A-CD90+ cell line, accompanied by decreased expression levels in Hs578T-CD90- cells. In vitro invasion assays showed that MCF10A-CD90+ cells are capable of migrating and invading, while Hs578T-CD90- cells presented a significant decrease in their ability to migrate and invade. Additionally, in vitro and in vivo metastasis assays showed that malignization ensued upon overexpression of CD90 in MCF10A cells and a lower tendency to form metastasis in vitro was observed for the Hs578T-CD90- cell line. Therefore, the present study presents, for the first time in the literature, the functional characterization of CD90 as a genetic marker involved in the malignant transformation of mammary carcinoma, leading to a better understanding of the breast cancer biology, which may, in turn, lead to the development of new clinical and therapeutic protocols.

O câncer de mama é a doença maligna que mais acomete as mulheres no mundo. Apesar dos inúmeros tratamentos, o óbito se deve principalmente à doença metastática que pode se desenvolver a partir do tumor primário. Esta progressão tumoral decorre da dificuldade de se estabelecer um prognóstico mais preciso. Atualmente, a teoria de células iniciadoras de tumor vem sendo estudada para tentar explicar a biologia do câncer e descrever novos alvos para prognósticos e terapias. O carcinoma mamário foi o primeiro tumor sólido para o qual foi identificada uma subpopulação celular, definida como CD44+/CD24-, apresentando as características de células iniciadoras tumorais. Embora este fenótipo venha sendo muito utilizado para descrever as células iniciadoras tumorais de mama, muitos trabalhos tem questionado a relevância clínica desses marcadores, enfatizando que outros marcadores devem ser identificados. Assim, o objetivo deste trabalho é analisar e caracterizar marcadores de células-tronco que possam estar relacionados com o grau de malignidade no modelo de câncer de mama. Inicialmente, analisou-se a expressão de 10 marcadores de células-tronco em diferentes linhagens de câncer de mama que apresentam graus crescentes de malignidade. O CD90 foi selecionado devido à alta expressão desse marcador na linhagem mais agressiva Hs578T. Para a caracterização deste marcador, realizou-se ensaios funcionais, através do silenciamento do CD90 na linhagem tumorigênica Hs579T e sua superexpressão na linhagem não-tumorigênica MCF10A. As linhagens celulares geradas foram caracterizadas quanto ao crescimento celular, potencial invasivo e metastático. Foi possível observar que houve uma alteração da morfologia nas linhagens transformadas com o CD90 e, também, um maior tempo de dobramento na linhagem Hs578T-CD90- e um menor na MCF10A-CD90+. Além disso, a linhagem MCF10-CD90+ foi capaz de crescer independentemente de EGF. Através da análise da via EGF, foi possível observar que houve um aumento da expressão da forma fosforilada do receptor e dos fatores Erk, c-Jun, e Jnk na linhagem MCF10A-CD90+ e uma diminuição dos mesmos na linhagem Hs578T-CD90-. A análise da atividade do elemento responsivo do fator de transcrição AP1 comprovou que a via de EGF é funcional na linhagem MCF10-CD90+. Também foram analisados os marcadores de transição epitélio-mesenquimal, verificando-se aumento da expressão dos marcadores mesenquimais na linhagem MCF10A-CD90+ e diminuição na linhagem Hs578T-CD90-. Os ensaios in vitro de invasão mostraram que as células MCF10-CD90+ são capazes de migrar e invadir e as células Hs578T-CD90- apresentam diminuição significativa da habilidade de migração e invasão. Além disso, os ensaios de metástase in vitro e in vivo, mostraram que a superexpressão de CD90 levou à malignização das células MCF10A. Por outro lado, a linhagem Hs578T-CD90- apresentou menor potencial metastático in vitro. Portanto, neste trabalho, pela primeira vez, o CD 90 foi caracterizado funcionalmente como um marcador envolvido na transformação maligna do carcinoma mamário, contribuindo, assim, para melhor entendimento da biologia do câncer de mama e para que se possa desenvolver novas ferramentas de diagnóstico/prognóstico e novos protocolos clínicos e terapêuticos. / Breast cancer is the malignant disease which affects the highest number of women in the world. In spite of the numerous treatments available, death is primarily due to the metastatic disease that may develop from the primary tumor. This tumor progression occurs because of the difficulty in establishing an accurate diagnosis/prognosis. Currently, the tumor initiating cells theory is being applied in an attempt to explain cancer biology and to unveil new diagnostic and therapeutic targets. Mammary carcinoma was the first solid tumor in which a cellular subpopulation, defined as CD44+/CD24-, was associated with tumor initiating cells. Although this phenotype has been widely used to describe breast tumor initiating cells, several studies have questioned the clinical relevance of these markers, emphasizing that additional markers should be identified. The objective of the present study is to analyze and characterize stem cell markers that may be related to malignancy stages in the breast cancer model. Initially, the expression of 10 stem cell markers was analyzed in different breast cancer cell lines displaying different malignancy grades. CD90 was selected due to its high expression levels in the most aggressive cell line, namely: Hs578T. In order to further characterize this marker, a functional study was performed in which CD90 was silenced in the Hs578T tumorigenic cell line and overexpressed in the non-tumorigenic MCF10A cell line. The resulting cell lines were characterized relative to growth rate and invasive and metastatic potential. A change in morphology readily was observed in the cell lines overexpressing CD90. In addition, the Hs578T-CD90-cell line presented an increased doubling time (DT), while the MCF10A-CD90+ cell line displayed a lower DT.. Furthermore, MC10-CD90+ cells were able to grow in the absence of EGF. Analysis of components of the EGF pathwayrevealed increased expression levels of the phosphorylated form of Erk, c-Jun and Jnk receptors in the MCF10-CD90+ cell line, while Hs578T-CD90- cells presented decreased expression of the same factors and receptors. Analysis of the activity of the AP1 responsive element allowed confirmation that the EGF pathway is functional in the MCF10-CD90+. . Epithelial-mesenquimal transition markers presented increased expression levels in the MCF10A-CD90+ cell line, accompanied by decreased expression levels in Hs578T-CD90- cells. In vitro invasion assays showed that MCF10A-CD90+ cells are capable of migrating and invading, while Hs578T-CD90- cells presented a significant decrease in their ability to migrate and invade. Additionally, in vitro and in vivo metastasis assays showed that malignization ensued upon overexpression of CD90 in MCF10A cells and a lower tendency to form metastasis in vitro was observed for the Hs578T-CD90- cell line. Therefore, the present study presents, for the first time in the literature, the functional characterization of CD90 as a genetic marker involved in the malignant transformation of mammary carcinoma, leading to a better understanding of the breast cancer biology, which may, in turn, lead to the development of new clinical and therapeutic protocols.

Le neuroblastome (NB) est la tumeur solide extracranienne la plus fréquente et mortelle chez les jeunes enfants. Il se caractérise par une résistance à la chimiothérapie possiblement en partie dû à la présence de cellules initiatrices de tumeurs (TICs). Des études ont mis en évidence le rôle de CD133 comme un marqueur des TICs dans divers types de cancers. Les buts de notre travail étaient d’abord de démontrer les vertus de TICs des cellules exprimant CD133 et ensuite, en utilisant une analyse globale du génome avec des polymorphismes nucléotidiques simples (SNPs), d’effectuer une analyse différentielle entre les TICs et les autres cellules du NB afin d’en identifier les anomalies génétiques spécifiques. Des lignées cellulaires de NB ont été triées par cytométrie de flux afin d’obtenir deux populations: une enrichie en CD133 (CD133high), l’autre faible en CD133 (CD133low). Afin de déterminer si ces populations cellulaires présentent des propriétés de TICs, des essais sur les neurosphères, les colonies en agar mou et les injections orthotopiques de 500 cellules sélectionnées dans 11 souris ont été réalisées. Après une isolation de l’ADN des populations sélectionnées, nous avons effectué une analyse génotypique par SNP utilisant les puces « Affymetrix Genome-Wide Human SNP Array 6.0 ». Pour vérifier l’expression des gènes identifiés, des Western Blots ont été réalisés. Nos résultats ont démontré que la population CD133 avait des propriétés de TICs in vitro et in vivo. L’analyse génotypique différentielle a permis d’identifier deux régions communes (16p13.3 and 19p13.3) dans la population CD133high ayant des gains et deux autres régions (16q12.1 and 21q21.3) dans la population CD133low possédant des pertes d’hétérozygoties (LOH). Aucune perte n’a été observée. Parmi les gènes étudiés, l’expression protéique d’éphrine-A2 était corrélée à celle de CD133 dans 6 tumeurs et 2 lignées cellulaires de NB. De plus, l’augmentation de la concentration d’anticorps anti-éphrine-A2 dans le milieu diminue la taille des neurosphères. Ainsi, la population CD133high, qui a des vertus de TICs, possède des caractéristiques génotypiques différentes par rapport à celle CD133low. La présence d’éphrine-A2 dans les cellules exprimant CD133 souligne son importance dans le développement des TICs. Ces résultats suggèrent la présence de potentielle cible pour de nouvelles thérapeutiques ciblant les TICs mise en évidence par l’étude génomique. / Neuroblastoma (NB) is the most common and deadly extracranial solid tumor of childhood characterized by a resistance to chemotherapy possibly due to the presence of tumor initiating cells (TICs). Studies showed the role of CD133 as a marker of TICs in various types of cancers. Our goals were first to demonstrate the stemness of TICs expressing CD133 and then, using a global genomic analysis with single nucleotide polymorphism (SNPs), to perform a differential analysis between TICs and other cells of NB to identify the specific genetic abnormalities. NB cell lines were sorted by flow cytometry to obtain two populations: one enriched in CD133 (CD133high), the other low in CD133 (CD133low). To determine whether these cell populations have TICs properties, we test the ability of cells to form either neurosphères or, colonies in soft agar and we also test their carcinogenic properties by orthotopic injections of 500 selected cells in 11 mice. After a DNA extraction on selected populations, a differential genotyping analysis has been made with Affymetrix Genome-Wide Human SNP Array 6.0. To verify the expression of the genes identified, Western blots had been made. Our results have demonstrated that CD133high population presented TICs properties in vitro and in vivo. The differential genotyping analysis allowed identifying two gains common regions (16p13.3 and 19p13.3) in CD133high population and two others loss of heterozygosity (LOH) (16q12.1 and 21q21.3) in CD133low population . No losses were observed. Among the genes studied, ephrin-A2 protein expression was correlated to CD133 expression in 6 NB tumors and 2 NB cell lines. Also, ephrin-A2’s increased concentration influenced the neurospheres by decreasing their size. Thereby, CD133high population, which had TICs properties, possess different genotyping characteristics compared to CD133low population. The presence of ephrine-A2 in cells expressing CD133 emphasizes its importance in the development of TICs. These results suggest the presence of potential target for new therapies targeting the TICs demonstrated by the genomic study.