<p>Bmi1 is a member of the Polycomb Group proteins and has been demonstrated as being vital in stem cell regulation. Bmi1 is overexpressed in many cancers, including glioblastoma, and has been shown to regulate cancer cell self-renewal and proliferation both <em>in vitro</em> and <em>in vivo</em>. This study aimed to determine if Bmi1 modulates brain tumour initiating cell properties using a spontaneous primary glioblastoma cell line and a commercial glioblastoma cell line. To determine the role of Bmi1 in glioblastoma cells, stem cell assays and <em>in vivo</em> analysis of tumour formation was performed on both control cells and Bmi1 knockdown cells. In both cell lines, Bmi1 was found to play a positive regulatory role in stem cell properties. When Bmi1 was knocked down in brain tumour initiating cells, properties such as self-renewal, proliferation and tumour formation were impaired compared to control cells. This study supports recent literature which shows that Bmi1 regulates stem cell properties in glioblastoma cells and supports the potential use of Bmi1 as a therapeutic target in glioblastoma brain tumours.<strong><br /> </strong></p> / Master of Science (MSc)

<p>Brain metastases are most common in adults suffering from lung cancer, predicting uniformly poor patient outcome and short survival time. Despite their frequency and severity, very little is known about the tumorigenesis of brain metastases. Previously developed primary brain tumour-initiating cell (BTIC) models were used to determine the presence of a stem-like population in brain metastases from the lung. Use of clinical samples and the NCI-H1915 cell line allowed for the development of useful strategies for study of brain metastasis.</p> <p>The sphere formation capacity and expression of known BTIC markers in brain metastases was suggestive of a self-renewing population. Differentiation studies demonstrated that neither clinical samples nor NCI-H1915 cells had neural lineage potential. Intracranial xenotransplant of clinical samples and NCI-H1915 cells into NOD-SCID mice led to formation of multiple focal masses throughout the ventricles; the tumours were also serially transplantable, further implicating a TIC population. Of known BTIC markers, only CD15 expression levels and patterns were similar enough in clinical samples and NCI-H1915 cells to warrant prospective sorting experiments in the cell line. Use of CD15 failed to identify a CSC or TIC population in NCI-H1915 cells.</p> <p>These findings suggest that a TIC population is present in brain metastases; however, this remains to be identified. It is recommended that due to the limitations of cell surface markers, the study of brain metastasis should use a selective gene expression approach, in order to target genes and pathways essential to metastasis. It was shown that NCI-H1915 cells could be useful for such an approach, studying the effects on proliferation, sphere formation, and tumour formation capacity of brain metastases from the lung. Further study using this model could ultimately lead to the disruption of pathways essential to the metastatic process, transforming a uniformly fatal disease into a more localized and treatable one.</p> / Master of Science (MSc)

Much interest has recently accumulated of the role of adult stem cells in both normal tissue homeostasis and carcinogenesis. Whereas normal and cancerous mammary epithelial stem cells have been identified and isolated from bulk primary tissue, little remains known about their regulation in vivo. Here we describe the molecular profile of mammary epithelial stem cells cultured in vitro and that of their tumourigenic counterparts, breast cancer stem cells. Our studies of gene transcription reveal potential mechanisms that may cooperate in the regulation of normal and cancer stem cells in vitro, and may also reflect their in vivo behaviour. These data bear consequences for the design of novel breast cancer therapeutics, as cancer stem cells are thought to resist conventional treatments and persist thereafter, causing disease relapse and seeding metastases. To address this issue we have devised a functional genomics approach to screen for novel biomarkers and therapeutic targets of breast cancer modeled in vitro; this culture system is centered on bona fide stem cells and may therefore offer improved relevance to human disease when compared with breast cancer cell lines. / Thesis / Master of Science (MS)

Clinical management of esophageal adenocarcinoma (EAC) relies on radiation therapy, yet radioresistance is a pervasive challenge in this disease. The mechanisms of EAC radioresistance remain largely unknown due to a paucity of validated preclinical models. The present studies report on the development of seven primary xenograft models established from patient tumours. These models are used to interrogate the range of radiosensitivities and mechanisms of radioresistance in EAC tumours. We found that radiation enriches the tumour-initiating cell population in two xenograft lines tested. Furthermore, three tested xenograft lines respond to irradiation by upregulating Hedgehog transcripts, a pathway involved in stem cell maintenance and proliferation. Upregulation occurs in autocrine and paracrine patterns simultaneously, suggesting that Hedgehog signalling may have a complex role in the radioresponse of EAC tumours. These findings suggest that inhibiting stem cell pathways in combination with radiotherapy may have an important role in the clinical management of EAC.

Clinical management of esophageal adenocarcinoma (EAC) relies on radiation therapy, yet radioresistance is a pervasive challenge in this disease. The mechanisms of EAC radioresistance remain largely unknown due to a paucity of validated preclinical models. The present studies report on the development of seven primary xenograft models established from patient tumours. These models are used to interrogate the range of radiosensitivities and mechanisms of radioresistance in EAC tumours. We found that radiation enriches the tumour-initiating cell population in two xenograft lines tested. Furthermore, three tested xenograft lines respond to irradiation by upregulating Hedgehog transcripts, a pathway involved in stem cell maintenance and proliferation. Upregulation occurs in autocrine and paracrine patterns simultaneously, suggesting that Hedgehog signalling may have a complex role in the radioresponse of EAC tumours. These findings suggest that inhibiting stem cell pathways in combination with radiotherapy may have an important role in the clinical management of EAC.

Parmi les cancers cutanés, le mélanome métastatique est le moins fréquent (5 à 7%) mais le plus meurtrier par sa forte résistante aux thérapies conventionnelles. Bien qu'immunogène, aucun traitement efficace n'existe actuellement pour traiter ce cancer, ce qui fait qu'il est urgent de trouver de nouvelles cibles thérapeutiques. Dans ce contexte, nous avons évalué si l'Insulin-Like Growth Factor-1 (IGF-1) pouvait représenter une cible d'intérêt thérapeutique dans le mélanome en inhibant l'expression du facteur IGF-1, à l'aide d'un épisome antisens, dans deux modèles cellulaires : des cellules de mélanome primaire B16-F0 et métastatique B16-F10 (cellules appelées B16-F0mod et B16-F10mod lorsque l'expression d’IGF-1 est inhibée).Dans des modèles expérimentaux in vivo, nos résultats montrent que la réduction d'expression d'IGF-1 induit une diminution de la tumorigénicité des cellules de mélanome, en générant des tumeurs sous-cutanées plus petites (B16-F0 et B16-F10 dans les souris C57BL/6) et en inhibant totalement (souris C57BL/6) ou fortement (souris NSG) la capacité des B16-F10 à former des métastases pulmonaires. Nous avons cherché à comprendre si cette perte de tumorigénicité, suite à l'inhibition du facteur IGF-1, était due à une modification de l'immunogénicité/antigénicité des cellules tumorales et/ou à une modification du potentiel tumorigène intrinsèque des cellules tumorales métastatiques.1/ L’immunisation de souris C57BL/6 à l'aide de cellules B16-F0mod induit la formation d’effecteurs humoraux lytiques en présence de complément dirigés contre la lignée parentale, mais également d’effecteurs cellulaires CD8+ capables d’induire la lyse des cellules tumorales in vitro et d’inhiber la croissance tumorale in vivo. Bien que l'analyse des voies humorale et cellulaire n'ait pas permis de démontrer les mécanismes IGF-1-dépendants mis en jeu avec les cellules B16-F10, l'immunisation des souris C57BL/6 à l'aide de cellules B16- F0mod conduit à une inhibition de la croissance des tumeurs sous-cutanées et du nombre de métastases pulmonaires, confirmant l'implication du facteur IGF-1 dans des mécanismes d'échappement tumoral au système immunitaire.2/ Nos résultats montrent par ailleurs que le facteur IGF-1 joue un rôle direct sur le potentiel tumorigène intrinsèque des cellules tumorales. Outre son action sur la prolifération des cellules tumorales, IGF-1 est impliqué dans le processus de transition épithélio-mésenchymateuse (augmentation des marqueurs N-cadhérine, vimentine, CD44 et CD29), favorisant le maintien de populations tumorales présentant un caractère souche (Sox2, Oct3/4, CD44, CD24, activité ALDH, side population, capacité à former des sphéroïdes). Par ce mécanisme, IGF-1 favorise à la fois les propriétés migratoires et d'efflux de drogues, comme la mitoxantrone, par les transporteurs ABC, qui explique en partie la forte résistance des mélanomes aux thérapies conventionnelles.Ces travaux montrent que l’inhibition de la voie IGF1/IGF1-R pourrait être une bonne stratégie pour le développement de traitements anti-tumoraux contre le mélanome. Outre le développement de stratégies d'immunothérapie, le blocage de la voie IGF-1 permettrait également de sensibiliser les cellules de mélanome aux traitements conventionnels et de diminuer le potentiel métastatique des cellules tumorales. / Metastatic melanoma is the least common (5-7 %), but is responsible for most skin cancer deaths by its strong resistance to conventional anti-cancer treatments. Although immunogen, no effective treatment currently exists against this aggressive form, making urgent to find new therapeutic targets. In this context, we assessed whether the Insulin-like Growth Factor-1 (IGF-1) could represent a target of therapeutic interest in melanoma inhibiting the expression of IGF-1 by means of an episome-based vector encoding antisense IGF-1, in two cellular models: primary melanoma cells B16-F0 and metastatic B16-F10 (designated B16-F0mod and B16-F10mod when IGF-1 expression is inhibited).In experimental models in vivo, our results show that the reduction of IGF-1 expression induced a decrease of the melanoma cells tumorigenicity, generating smaller tumors under the skin (B16-F0 and B16-F10 in the C57BL/6 mice) and inhibiting totally (C57BL/6) or strongly (NSG mice) the developpment of B16-F10 lung metastases. We sought to understand whether this loss of tumorigenicity, following IGF-1 inhibition, was due to a change of immunogenicity/antigenicity of tumor cells and/or to intrinsic tumorigenic potential modification of metastatic tumor cells.1 / Immunization of mice C57BL/6 mice with B16-F0mod cells induces the formation of humoral lytic effectors in the presence of complement against the parental line, but also CD8+ effector cells capable of inducing tumor cells lysis in vitro and inhibiting tumor growth in vivo. Although the analysis of humoral and cellular pathways did not demonstrate IGF-1- dependent mechanisms involved in B16-F10 cells, immunization of C57BL/6 mice with B16 cells F0mod leads to skin tumor growth inhibition and a reduction in pulmonary metastases number, confirming the involvement of IGF-1 factor in tumor escape mechanisms of the immune system.2 / Our results also show that IGF-1 plays a direct role in the intrinsic tumorigenic potential of tumor cells. In addition to its effect on tumor cells proliferation, IGF-1 is involved in epithelial-mesenchymal transition (increased N-cadherin, vimentin, CD44 and CD29 markers), promoting the maintenance of tumor populations with stemness properties (Sox2, Oct3/4, CD44, CD24, ALDH activity side-population and ability to form spheroids). By this mechanism, IGF-1 promotes both migration properties and drugs efflux such as mitoxantrone, via ABC transporters, which partly explains the strong resistance of melanoma to conventional therapies.This work shows that the inhibition of IGF1/IGF1-R pathway might be a good strategy for the development of anti-tumor treatments against melanoma. In addition to developing immunotherapy strategies, blocking the IGF-1 pathway would also sensitize melanoma cells to conventional therapy and decrease the metastatic potential of tumor cells.