<p>There is increasing evidence that prostate tumours are organized as a hierarchy with rare cancer stem cells (CSCs) implicated in initiating and maintaining the tumour. However, prospective prostate cancer stem cells (PCSCs) have not been thoroughly characterized from primary tissue specimens. Using the DU145 cell line, PCSCs have been propagated as non-adherent spheres <em>in vitro</em>. Approximately 1.25% of monolayer DU145 cells formed primary spheres while 26% of sphere cells formed subsequent spheres; a measure of PCSC self-renewal capacity. Spheres are enriched for cells expressing prostate basal and luminal cytokeratins and CSC markers (CD44, CD24, integrin alpha2beta1). PCSCs initiate xenograft tumours with enhanced capacity compared to monolayer cells. While epidermal growth factor (EGF) promoted PCSC propagation, basic fibroblast growth factor (bFGF) inhibited these events. Activation of EGF receptor (EGFR) signalling, following EGF treatment or expression of constitutively-active EGFR (EGFRvIII), increased sphere formation. Conversely, attenuation of EGFR signalling inhibited PCSC self-renewal. Consistent with the MEK-ERK pathway being a major target of EGFR signalling, the MEK-ERK pathway contributes to EGFR-facilitated PCSC propagation. Inhibition of ERK activation following MEK inhibitor treatment, expression of dominant-negative MEK1(K97M), or knockdown of ERK1 or ERK2 reduced PCSC propagation. Therefore, EGFR signalling promotes PCSC self-renewal by activating the MEK-ERK pathway.</p> <p>SOX2 is an essential transcription factor for stem cells, however, its role in PCSCs remains unclear. SOX2 protein is upregulated in PCSCs propagated as spheres, and its expression is regulated by EGFR signalling. EGFR activation, following EGF treatment or expression of constitutively-active EGFRvIII, increased SOX2 expression and PCSC self-renewal, while being attenuated by EGFR inhibitor treatment. Ectopic SOX2 expression enhanced EGF-induced PCSC self-renewal, while SOX2 knockdown renders PCSCs non-responsive to EGF-induced self-renewal and reduced their anchorage-independent growth. Furthermore, SOX2 expression is associated with the ability of PCSCs to form aggressive xenograft tumours. Collectively, SOX2 regulates EGFR-mediated PCSC self-renewal.</p> / Doctor of Philosophy (PhD)

<p dir="ltr">Embryonic development relies on the complex interplay of epigenetic regulation, timely expression of genes, signal transduction pathways, and diverse morphological changes. The heart is the first organ to form during mammalian embryonic development. The proper development of the heart is critical to supply nutrients and oxygen to other cell types of the organism. Most cells that comprise the heart originate from the mesoderm post-gastrulation. Cardiomyocytes are the predominant cell type and confer function to the heart via contractile activity. The development and proliferation of cardiomyocytes ceases shortly after birth, where cardiomyocytes only nucleate and increase in size. Consequently, cardiomyocyte insufficiency underlies most cardiovascular diseases, a leading cause of death globally.</p><p dir="ltr">Vascular endothelial zinc finger 1 (VEZF1) is a transcription factor expressed predominantly in mesoderm during development. Previous studies from our lab show that the loss of VEZF1 impairs the differentiation of embryonic stem cells into endothelial cells, a cell type derived from mesoderm. Other published studies also show that Vezf1 loss impairs cardiomyocyte growth in Zebrafish and hematopoietic cell differentiation. Our work here describes a detailed investigation of the role of Vezf1 in the differentiation of mesoderm and cardiomyocytes using mouse embryonic stem cell (ESC) differentiation as a mammalian model system. We initially developed an efficient method, known as the Wnt Switch method, to differentiate ESCs into cardiomyocytes. Our technique relies on the treatment of differentiating ESCs with small molecule inhibitors: i) CHIR99021, which induces mesoderm development via the activation of Wnt signaling in the first 48 hours of differentiation, followed by ii) XAV939, which inhibits Wnt signaling and drives mesoderm cells toward cardiomyocyte differentiation pathway. The Wnt Switch method significantly increases the efficiency of cardiomyocyte derivation (86%) from ESC compared to published methods (56%).</p><p dir="ltr">Interestingly, the Wnt Switch method showed that despite the external stimulation of Wnt signaling, Vezf1 KO cells are unable to differentiate into cardiomyocytes and show reduced expression of mesodermal genes 48 hrs post-differentiation. To better understand the stage-specific role of Vezf1 in cardiomyocyte development, we generated doxycycline-inducible Vezf1 knockdown clones that significantly reduce Vezf1 protein levels upon treatment with doxycycline. We found that the knockdown of Vezf1 prior to mesoderm induction significantly impaired ESC differentiation but had no significant effect on cardiomyocyte development after mesoderm induction. These data indicate that Vezf1 expression is crucial for proper mesoderm and, thus, mesodermal lineage development. Further, FACS analysis showed reduced mesoderm cell populations derived from Vezf1 null post-differentiation. We used high throughput sequencing methods to determine genome-wide Vezf1 binding by ChIP-SEQ and compared gene expression in WT and Vezf1 null cells using RNA-SEQ. The data indicated that VEZF1 binds near the promoters of numerous Wnt signaling genes after differentiation and that the expression of Wnt pathway genes decreases when Vezf1 is lost. Interestingly, supplementing WNT3A protein in culture media of Vezf1 null cells rescues the expression of Wnt target genes necessary for mesoderm formation.</p><p dir="ltr">Differentiating Vezf1 KO cells to endothelial or cardiomyocyte lineages also resulted in massive cell death. The surviving cells interestingly stained positive for alkaline phosphatase (AP) staining, indicating retention of the pluripotency in Vezf1 KO cells. Whereas, re-culturing of WT ESC in LIF media, after differentiating them for five days in the absence of LIF, results in cell death, Vezf1 KO cells proliferate and form AP-positive and SSEA-positive colonies. We further show the retention of pluripotency gene expression post-differentiation using RNA sequencing and RT-qPCR. Moreover, we show that the continued expression of pluripotency genes post-differentiation was not a consequence of reduced global DNA methylation in Vezf1 KO cells.</p><p dir="ltr">Interestingly, our data show that Vezf1 is a transcriptional activator and binds to key pro-differentiation pathways like the MAPK signaling and WNT signaling pathways. The loss of Vezf1 correlates with reduced expression of genes in the pro-differentiation pathways. We show that CTCF, an insulator-binding protein, opportunistically binds to VEZF1 sites on genes in the pro-differentiation signaling pathways in VEZF1 KO cells. Therefore, we hypothesized that this opportunistic CTCF binding is the mechanism that drives the repression of pro-differentiation signaling genes or compensates for the loss of Vezf1 binding to support basal gene expression in the absence of VEZF1. Given the dire consequences of pluripotency in cancer stem cells, we investigated the expression of Vezf1 in cancers. We found that Vezf1 expression is reduced in many cancers and is correlated with poor prognosis. We also show that MAPK3, a prominent member of the MAPK signaling pathway, is reduced in these cancers, highlighting a strong correlation between Vezf1 expression and Mapk3 gene expression in cancers. The data extend our observation of pluripotency in ESCs to cancers. To gain further insights into the role of Vezf1 in cancer, we utilized F9 embryonic carcinoma cells. F9 cells have been reported to retain pluripotency expression post-differentiation. Interestingly, the ectopic and transient expression of Vezf1 in F9 cells significantly reduced the expression of pluripotency genes, suggesting that Vezf1 is sufficient to repress pluripotency gene expression in F9 carcinoma cells. These data highlight the significant role of Vezf1 in pluripotency gene repression and provide an excellent avenue for treating cancer relapse caused by the occurrence of cancer stem cells.</p><p dir="ltr">In conclusion, our research elucidates the critical role of Vezf1 in cardiomyocyte formation and pluripotency regulation during embryonic development. Understanding the molecular mechanisms underlying Vezf1-mediated pathways provides insights into developmental processes and holds promise for therapeutic interventions for cardiomyocyte regeneration and against cancers.</p>

DNA Methylierung ist ein zentraler epigenetischer Prozess, welcher entscheidend an der Organisation von Genregulation beteiligt ist. Dieser Vorgang ist wichtig für die Funktion sowohl von embryonalen als auch von Gewebs-Stammzellen. Krebszellen weisen häufig veränderte DNA Methylierungsmuster auf, was auf eine ähnlich wesentliche Rolle der DNA Methylierung in Krebsstammzellen (KSZ) hindeutet. Diese These wurde hier mit Hilfe eines Mausmodells mit verringerter Expression der DNA Methyltransferase Dnmt1 anhand verschiedener Leukämiemodelle untersucht. In einem bi-linearen B-lymphatischen/myeloischen Leukämiemodell konnte gezeigt werden, dass hypomethylierte leukämieinitiierende (Stamm-)zellen (LSZ) myeloische Krebszellen hervorbringen, allerdings nicht zur Bildung von B-lymphatischen Leukämiezellen befähigt sind. Darüber hinaus konnte in einem T-Zell-spezifischen Leukämiemodell gezeigt werden, dass reduzierte Dnmt1 Expression nicht mit der Bildung von T-Zelllymphomen vereinbar ist. Detaillierte Analysen eines myeloischen Leukämiemodells ergaben, dass hypomethylierte LSZs ein vermindertes Selbsterneuerungspotenzial aufweisen. Im Gegensatz zu den starken Funktionseinschränkungen hypomethylierter LSZs, hatten hypomethylierte Knochenmarks-Stromazellen keinen Effekt auf die Entwicklung von Leukämien. Außerdem führte die Behandlung leukämischer Zellen mit demethylierenden Agenzien zu einer teilweisen Aufhebung methylierungsvermittelter Genrepression. Die dadurch verstärkte Expression von Differenzierungsfaktoren verminderte das Leukämiewachstum, was einen möglichen Erklärungsansatz für das eingeschränkte Potenzial hypomethylierter Leukämien darstellt. Diese Ergebnisse demonstrieren eine zentrale Rolle der DNA Methylierung für die Selbsterneuerung und Linienwahl von LSZs, und erlauben somit neue Einblicke in die epigenetische Regulation von KSZs. Diese Erkenntnisse implizieren, dass KSZs möglicherweise ein geeignetes Ziel für epigenetische Therapieansätze darstellen. / DNA methylation is one of the major epigenetic processes which is crucially involved in orchestrating gene regulation primarily by repression of gene expression. DNA methylation plays an important role in controlling functional programs of embryonic and tissue stem cells. As altered DNA methylation patterns are a hallmark of cancer, we hypothesized that DNA methylation might be equally important for cell fate determinations of cancer stem/initiating cells (CSC). To test this, I analyzed a genetic knockdown mouse model of the main somatic DNA methyltransferase Dnmt1 in the context of three different leukemia models. In a bilinear B-lymphoid/myeloid leukemia model hypomethylated bi-potential leukemia stem/initiating cells (LSCs) were shown to be capable of forming a myeloid leukemia, whereas the generation of B-lymphoid blasts was almost entirely abrogated. Moreover, failure of hypomethylated cells to develop T-cell lymphomas in a Notch1-based leukemia model demonstrated their profound lack of T-lineage commitment capacities. Furthermore, detailed analyses of a myeloid leukemia model revealed a severely impaired self-renewal potential in LSCs with reduced Dnmt1 expression. However, contrasting the drastic cell-intrinsic impairments of LSC function by reduced DNA methylation, leukemia development was found to be unaffected by hypomethylated bone marrow stroma. Mechanistically, treatment of cell lines with a demethylating drug led to enhanced expression of differentiation factors due to loss of methylation mediated gene silencing. This was followed by inhibition of leukemia cell growth, thus providing a potential mechanism for impaired functions of hypomethylated leukemias. Collectively, this thesis revealed a critical role for DNA methylation levels in malignant self-renewal and lineage fate choices. These new insights into epigenetic regulation of CSCs suggest that epigenetic therapy displays a potential treatment concept specifically targeting CSCs.

Le carcinome hépatocellulaire (CHC) est un cancer au pronostic sombre, car il est souvent diagnostiqué trop tardivement pour entreprendre un traitement curatif. Il se développe dans 80-90% des cas sur fond de cirrhose. On connait mal comment la fibrose, étape préliminaire à la cirrhose, et son principal constituant, le collagène de type 1 (COL1), peuvent jouer un rôle dans le processus du CHC. Nous avons tout d’abord étudié le développement de la fibrose dans un modèle utilisant la souris nue. Nous avons déterminé qu’après 16 semaines d’administration de thioacétamide dans l’eau de boisson, il est possible d’obtenir une fibrose suffisante pour induire une hépatoprotection en présence de différents hépatotoxiques (AST dans le sérum de souris fibrotiques vs non-fibrotiques : Anti-Fas JO2 (4665 ± 2596 vs. 13953 ± 2260 U/L; P<0.05), acétaminophène (292 ± 66 vs. 4087 ± 2205 U/L; P<0.01) et CCL4 (888 ± 268 vs. 15673 ± 2782 U/L; P<0.001)). Ces résultats confirment que la présence de COL1 et de fibrose favorise la survie des hépatocytes normaux tel qu’observé précédemment au laboratoire. Par la suite, nous avons sélectionné in vivo, par injection intrasplénique de la lignée de CHC Hepa1-6, une lignée à forte tumorigénicité nommée dt-Hepa1-6 (28±12 lésions vs. 0±0 lésions à 21 jours). Cette lignée était composée d’une sous-population cellulaire arborant la protéine de surface EpCAM (34.0±0.1%). Par tri cellulaire, nous avons démontré que ces cellules étaient partiellement responsables de la tumorigénicité accrue (EpCAM + (86.7±2.3%) :1093±74 lésions vs. EpCAM- (15.3±1.0%) :473±100 lésions; P<0.01). Nous avons alors démontré que la présence de fibrose favorise le développement de la lignée dt-Hepa1-6 in vivo (604±242 vs 22±9 lésions; P<0.05). De plus, la présence de fibrose réduit l’efficacité du traitement au cisplatin in vivo (44.5±4.9 vs. 78.7±6.9%; P<0.01) confirmant les résultats obtenus in vitro (Apoptose : COL1 13.75±0.44% vs. plastique 31.45±1.37%; P<0.001). En conclusion, la présence de fibrose et de son principal constituant, le COL1, favorise la survie et la progression du CHC. / Hepatocellular carcinoma (HCC) is a dreadful pathology, often diagnosed too late to be cured. In 80-90% of cases, it arises in the context of liver cirrhosis. Little is known on the implication of liver fibrosis, one of the key elements of cirrhosis, and its major constituent, type I collagen (COL1), on the development of HCC. We first studied the development of fibrosis in a nude mouse model. We determined that, after 16 weeks of thioacetamide administration in drinking water, we obtained a sufficient degree of fibrosis to reach a hepatoprotective state when animals were exposed to different hepatotoxic agents (Serum AST of fibrotic vs non-fibrotic mice : Anti-Fas JO2 (4665 ± 2596 vs. 13953 ± 2260 U/L; P<0.05), acetaminophen (292 ± 66 vs. 4087 ± 2205 U/L; P<0.01) et CCL4 (888 ± 268 vs. 15673 ± 2782 U/L; P<0.001)). This confirmed that COL1 and the presence of fibrosis protects normal hepatocytes as observed previously in our laboratory. Next, we selected in vivo, by intrasplenic injection of the murine HCC cell line Hepa1-6, a highly tumorigenic cell line that we named dt-Hepa1-6 (28±12 lesions vs. 0±0 lesions at 21 days). This cell line was constituted of cell subsets expressing EpCAM protein at their surface (34.0±0.1%). Through cell sorting, we demonstrated that these cells were partially responsible for the enhanced tumorigenicity observed (EpCAM + (86.7±2.3%) :1093±74 lesions vs. EpCAM- (15.3±1.0%) :473±100 lesions; P<0.01). We then showed that the presence of liver fibrosis increases the development of dt-Hepa1-6 cell line in vivo (604±242 vs 22±9 lesions; P<0.05). Moreover, fibrosis reduced the anti-neoplastic efficacy of cisplatinum in vivo (44.5±4.9 vs. 78.7±6.9%; P<0.01) confirming in vitro results (Apototic index : COL1 13.75±0.44% vs. plastic 31.45±1.37%; P<0.001). In conclusion, fibrosis and its major constituent, COL1, favor the survival and progression of HCC.

As imagens de ressonância magnética (RM) ponderadas em Difusão são conhecidas como uma técnica funcional capaz de refletir alterações estruturais e celulares de neoplasias. No câncer de mama, a difusão e sua quantificação através dos valores do coeficiente de difusão aparente (CDA) têm sido utilizados para avaliar resposta tumoral após quimioterapia neoadjuvante (QTN). Os variados desfechos clínicos do câncer de mama, incluindo as diferentes respostas ao tratamento quimioterápico podem estar relacionados à heterogeneidade da doença. A presença das células tronco tumorais (CTT) é uma das hipóteses aceitas para explicar os diferentes comportamentos biológicos dos tumores. Este estudo buscou avaliar uma possível associação entre os valores de CDA nas neoplasias invasivas da mama e a presença de marcadores de CTT e os principais marcadores prognósticos da doença em pacientes tratadas com QTN. Foram avaliadas prospectiva e consecutivamente as imagens de RM pré-tratamento de 27 pacientes com câncer da mama que realizaram QTN seguida de cirurgia. Os valores de CDA média, p10, p25 e p50 foram obtidos através de duas mensurações, uma com único ROI e outra com múltiplos ROIs envolvendo toda extensão tumoral. Esses valores de CDA foram correlacionados: à quantificação por citometria de fluxo de CTT com fenótipos ESA+/CD44+/CD24-, células ESA+ com alta atividade ALDH1 e células ESA+/ABCG2+, à capacidade de formação de mamoesferas, e aos principais fatores prognósticos do câncer de mama, incluindo estágio clínico, doença axilar linfonodal, grau tumoral, receptores de estrógeno (RE), receptores de progesterona (RP) e superexpressão do HER2. Também foi realizada correlação dos valores de CDA com a resposta patológica completa após QTN. A presença de CTT, a capacidade de formação de mamoesferas e a resposta patológica completa não se correlacionaram aos valores de CDA. Para ambas as medidas e todos os parâmetros avaliados de CDA (x10-3mm2/s), os valores foram significantemente menores nos tumores com estágio clínico III e IV vs II (0,90±0,16; 1,02±0,18); com doença linfonodal após QTN vs axila livre (0,89±0,16; 1,01±0,17); RE+ vs RE- (0,90±0,16; 1,00±0,18); RP+ vs RP- (0,91±0,16; 0,98±0,18) e HER2+ vs HER2- (0,92±0,17;0,97±0,18). Tumores grau 1 apresentaram CDA com valores significativamente maiores em relação aos tumores grau 2 (diferença 0,18; CI: 0,03-0,33, p=0,02). Os valores de CDA dos tumores de mama pré-QTN não predizem a presença de CTT, a capacidade de formação de mamoesferas ou a resposta patológica completa, porém se correlacionam com o estágio clínico da doença, doença linfonodal axilar após QTN, grau tumoral e expressão das proteínas RE, RP e HER2, sendo um promissor marcador de agressividade tumoral / The diffusion-weighted magnetic resonance imaging (DWMRI) is a functional technique able to reflect structural and cellular changes in the tumors. In the breast cancer, the diffusion-weighted images and its numeric value known as the apparent diffusion coefficient (ADC) has been applied to evaluate pathologic response in patients treated with neoadjuvant chemotherapy (NC). The difference in the clinical results after breast cancer treatment, including different rates of responses to the NC has been associated to the heterogeneity of the disease. The presence of the breast cancer stem cells (BCSC) is an accepted hypothesis to explain the different biologic breast cancers behaviors. The aim of this study was to correlate the ADC value of invasive breast cancer with the presence of cancer stem cells markers and the major prognostic factors in patients treated with neoadjuvant chemotherapy. Prospectively, the MRI pre-treatment of twenty-seven consecutive patients with invasive breast cancer posteriorly treated with NC followed by surgery were evaluated. The ADC values mean, 10th percentile, 25th percentile, 50th percentile were obtained from two measurements, one of them with a unique ROI and the other with multiple ROIs encompassing the entire lesion. The ADC values were correlated to: presence of BCSCs (cell surface markers CD44+/CD24-, ABCG2 and ALDH1) identified by flow cytometric analysis, tumor grade, breast cancer staging, lymph nodal involvement, expression of estrogen receptors (ER), expression of progesterone receptors (PR) and expression of HER2. The assay mammospheres (Mammocult ®) were analyzed in 18 samples. Additionally, the ADC values were correlated to the pathologic complete response after QN treatment. There were no correlations between ADC values and breast cancer stem cells markers or mammospheres formation efficiency. For all parameters calculated, the ADC values (x10- 3 mm 2 /s) were lower in: breast cancer stage III and IV than stage II (0,90±0,16; 1,02±0,18), tumors with lymph node metastasis than without lymph node metastasis (0,89±0,16; 1,01±0,17), ER expression than ER negative (0,90±0,16; 1,00±0,18), PR expression than PR 10 negative (0,91±0,16; 0,98±0,18) and HER2 expression than HER2 negative (0,92±0,17; 0,97±0,18). The ADC values were significantly higher in grade-1 tumors (difference 0,18; CI: 0,03-0,33) compared to grade-2 tumors (p=0,02). The tumors values of ADC pretreatment were not correlated to the pathologic complete response after NC. The ADC values in pre-treatment invasive breast cancers are not a predictor of BCSC presence, mammospheres formation efficiency or pathologic complete response to QN. However it is correlated to the tumor grade, breast cancer staging, lymph nodal involvement, expression of ER, PR and HER2 and may represent a promising marker of tumor aggressiveness

As imagens de ressonância magnética (RM) ponderadas em Difusão são conhecidas como uma técnica funcional capaz de refletir alterações estruturais e celulares de neoplasias. No câncer de mama, a difusão e sua quantificação através dos valores do coeficiente de difusão aparente (CDA) têm sido utilizados para avaliar resposta tumoral após quimioterapia neoadjuvante (QTN). Os variados desfechos clínicos do câncer de mama, incluindo as diferentes respostas ao tratamento quimioterápico podem estar relacionados à heterogeneidade da doença. A presença das células tronco tumorais (CTT) é uma das hipóteses aceitas para explicar os diferentes comportamentos biológicos dos tumores. Este estudo buscou avaliar uma possível associação entre os valores de CDA nas neoplasias invasivas da mama e a presença de marcadores de CTT e os principais marcadores prognósticos da doença em pacientes tratadas com QTN. Foram avaliadas prospectiva e consecutivamente as imagens de RM pré-tratamento de 27 pacientes com câncer da mama que realizaram QTN seguida de cirurgia. Os valores de CDA média, p10, p25 e p50 foram obtidos através de duas mensurações, uma com único ROI e outra com múltiplos ROIs envolvendo toda extensão tumoral. Esses valores de CDA foram correlacionados: à quantificação por citometria de fluxo de CTT com fenótipos ESA+/CD44+/CD24-, células ESA+ com alta atividade ALDH1 e células ESA+/ABCG2+, à capacidade de formação de mamoesferas, e aos principais fatores prognósticos do câncer de mama, incluindo estágio clínico, doença axilar linfonodal, grau tumoral, receptores de estrógeno (RE), receptores de progesterona (RP) e superexpressão do HER2. Também foi realizada correlação dos valores de CDA com a resposta patológica completa após QTN. A presença de CTT, a capacidade de formação de mamoesferas e a resposta patológica completa não se correlacionaram aos valores de CDA. Para ambas as medidas e todos os parâmetros avaliados de CDA (x10-3mm2/s), os valores foram significantemente menores nos tumores com estágio clínico III e IV vs II (0,90±0,16; 1,02±0,18); com doença linfonodal após QTN vs axila livre (0,89±0,16; 1,01±0,17); RE+ vs RE- (0,90±0,16; 1,00±0,18); RP+ vs RP- (0,91±0,16; 0,98±0,18) e HER2+ vs HER2- (0,92±0,17;0,97±0,18). Tumores grau 1 apresentaram CDA com valores significativamente maiores em relação aos tumores grau 2 (diferença 0,18; CI: 0,03-0,33, p=0,02). Os valores de CDA dos tumores de mama pré-QTN não predizem a presença de CTT, a capacidade de formação de mamoesferas ou a resposta patológica completa, porém se correlacionam com o estágio clínico da doença, doença linfonodal axilar após QTN, grau tumoral e expressão das proteínas RE, RP e HER2, sendo um promissor marcador de agressividade tumoral / The diffusion-weighted magnetic resonance imaging (DWMRI) is a functional technique able to reflect structural and cellular changes in the tumors. In the breast cancer, the diffusion-weighted images and its numeric value known as the apparent diffusion coefficient (ADC) has been applied to evaluate pathologic response in patients treated with neoadjuvant chemotherapy (NC). The difference in the clinical results after breast cancer treatment, including different rates of responses to the NC has been associated to the heterogeneity of the disease. The presence of the breast cancer stem cells (BCSC) is an accepted hypothesis to explain the different biologic breast cancers behaviors. The aim of this study was to correlate the ADC value of invasive breast cancer with the presence of cancer stem cells markers and the major prognostic factors in patients treated with neoadjuvant chemotherapy. Prospectively, the MRI pre-treatment of twenty-seven consecutive patients with invasive breast cancer posteriorly treated with NC followed by surgery were evaluated. The ADC values mean, 10th percentile, 25th percentile, 50th percentile were obtained from two measurements, one of them with a unique ROI and the other with multiple ROIs encompassing the entire lesion. The ADC values were correlated to: presence of BCSCs (cell surface markers CD44+/CD24-, ABCG2 and ALDH1) identified by flow cytometric analysis, tumor grade, breast cancer staging, lymph nodal involvement, expression of estrogen receptors (ER), expression of progesterone receptors (PR) and expression of HER2. The assay mammospheres (Mammocult ®) were analyzed in 18 samples. Additionally, the ADC values were correlated to the pathologic complete response after QN treatment. There were no correlations between ADC values and breast cancer stem cells markers or mammospheres formation efficiency. For all parameters calculated, the ADC values (x10- 3 mm 2 /s) were lower in: breast cancer stage III and IV than stage II (0,90±0,16; 1,02±0,18), tumors with lymph node metastasis than without lymph node metastasis (0,89±0,16; 1,01±0,17), ER expression than ER negative (0,90±0,16; 1,00±0,18), PR expression than PR 10 negative (0,91±0,16; 0,98±0,18) and HER2 expression than HER2 negative (0,92±0,17; 0,97±0,18). The ADC values were significantly higher in grade-1 tumors (difference 0,18; CI: 0,03-0,33) compared to grade-2 tumors (p=0,02). The tumors values of ADC pretreatment were not correlated to the pathologic complete response after NC. The ADC values in pre-treatment invasive breast cancers are not a predictor of BCSC presence, mammospheres formation efficiency or pathologic complete response to QN. However it is correlated to the tumor grade, breast cancer staging, lymph nodal involvement, expression of ER, PR and HER2 and may represent a promising marker of tumor aggressiveness

Le glioblastome (GBM) est la tumeur cérébrale primaire la plus agressive comportant une sous-population de cellules souches tumorales (CSG). Elles sont capables d’auto-renouvellement, de prolifération, de différenciation en cellules exprimant les marqueurs neuraux et de trans-différenciation en cellules de types vasculaires. Dans ce contexte, j’ai dérivé et caractérisé plusieurs lignées de CSG à partir de biopsies de patients. Puis j’ai évalué l’impact des peptides thérapeutiques transmembranaires développés au laboratoire, visant les plateformes de récepteurs de neuropiline-1 et de plexine-A1 surexprimées dans les CSG. Les deux peptides diminuent la croissance des CSG in vitro et in vivo. Finalement, j’ai développé un outil génétique fluorescent permettant de suivre le destin des CSG en direct. Basé sur l’expression de 4 rapporteurs fluorescents contrôlés par des promoteurs spécifiques des types cellulaires, il permet d’identifier l’hétérogénéité de ces cellules en différenciation. / The glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and includes a subpopulation of tumoral stem cells (CSG). Those cells can self-renew, proliferate and differentiate by expressing specific neural markers and/or transdifferentiate into vascular-like cells. In this context, my work consisted first to produce and characterize several CSG lines from patient biopsies to constitute a bank of cell lines with different properties. We also evaluated the impact of in house therapeutic transmembrane peptides targeting the neuropilin-1 / plexin-A1 receptor platforms overexpressed in GBM. We thus showed that both targeting peptides decrease the growth of GSC in in vitro and in vivo models. Finally, I developed an inducible mosaic expression system to track the live differentiation of CSG. This system is based on the expression of four different fluorescent reporters controlled by the activity of cell type specific promoters.

CD47 is a so-called "don't eat me" signal, which protects cells from phagocytosis. Its high expresion on tumor cells brings new perspective to the tumor therapy. Monoclonal antibodies, which are these days undergoing clinical trials, prevent CD47 binding to the SIRPA inhibitory receptor on macrophages, and so they enhance their phagocytic functional capacity. In this way they enable phagocytic removal of tumor cells. Overall expression, structural conformation and stoichiometry of CD47 on a particular cell predestine whether it will be phagocytised. The aim of the thesis is to develop and test methods to characterise expression parameters of CD47 via flow cytometry (FCM), quantitative PCR (qPCR) and microscopy. To achieve this goal I performed competition tests of commercially available antibodies in order to characterise their binding epitopes on cell lines. After performing tSNE analysis of primary BCa patient samples I correlated CD47 expression with other cell surface markers. I focused on CD47 expression in various differentiation stages of the tumor. To better understand the relationship between CD47 expression and differentiation status of cells I performed qPCR analysis of particular transcription factors. Using cell lines I examined method for phagocytosis quantification, which will be...

Les cellules cancéreuses ayant des propriétés souches, appelées cellules souches cancéreuses (CSCs), sont au sommet de l'organisation hiérarchique des tumeurs et sont soupçonnées d'être responsable des rechutes et de la formation de métastases. Cependant, nous nous sommes demandé comment ces cellules sont-elles capables de supporter le stress métabolique lié à l'oncogène et de croitre dans un environnement nutritif défavorable distinct de celui d'une tumeur établie ? Nous avons mis en évidence dans ce travail le rôle de la réponse aux glucocorticoïdes dans le contrôle de la plasticité mammaire et l'induction d'une plasticité métabolique nécessaire au maintien du potentiel tumorigène des CSCs. Nous avons montré qu'en régulant la reprogrammation cellulaire via l'induction du gène MAFB, les glucocorticoïdes contrôlent le caractère souche des cellules épithéliales malignes en activant la voie de biosynthèse des hexosamines. L'efficacité anabolique de ces cellules est alors augmentée, favorisant le développement de la tumeur et des métastases. Ensemble, nos résultats suggèrent que l'inhibition de l'activité métabolique des glucocorticoïdes pourrait être une stratégie pour éliminer les CSCs et traiter les tumeurs mammaires / Cancer cells with stemness properties - generally designated cancer stem cells (CSCs) - are at the apex of the hierarchical organisation of tumours and are believed to drive tumour recurrence and metastasis formation. However, how they perform these neoplastic activities in a nutritive environment that is distinct from the one in an established tumour is unknown. Here, we unveil the prominent role of glucocorticoid activity in the control of mammary cancer cell plasticity and the induction of metabolic pliancy necessary for the tumorigenic potential of CSCs. By regulating MAFB-dependent cell reprogramming, glucocorticoids control stemness traits in malignant epithelial cells. As an integral part of this regulation, glucocorticoids activate the hexosamine biosynthetic pathway and rewire the metabolism of CSCs. The anabolic efficiency of these cells increases then, fostering tumour and metastasis development. Together, our findings suggest that inhibition of glucocorticoid metabolic activity could be an original strategy for CSC eradication and tumour treatment

Les glioblastomes sont des tumeurs primaires du cerveau les plus malignes. L’identification des cellules souches cancéreuses de glioblastome (CSGs) a transformé notre vision globale des glioblastomes en révélant une hiérarchie cellulaire au sein de ces tumeurs. Les CSGs sont douées de propriétés d’auto-renouvellement, de différenciation et peuvent entrer en quiescence. Elles sont considérées comme les cellules entretenant les tumeurs, responsables de leur dissémination et des rechutes après traitement. La découverte des CSGs a conduit à un changement de paradigme dans le développement des thérapies anticancéreuses, avec la nécessité de cibler dans le traitement non seulement les cellules de la masse tumorale, mais aussi les CSGs. Un criblage différentiel de la chimiothèque Prestwick réalisé au laboratoire a permis d’identifier le bisacodyl comme une molécule présentant une cytotoxicité spécifique sur les CSGs en quiescence.Cette thèse présente un travail sur la caractérisation des CSGs, la compréhension du mode d’action du bisacodyl, ainsi que l’évaluation de son potentiel thérapeutique sur un modèle 3D in intro et des modèles in vivo. / Glioblastomas are the most malignant primary brain tumors. The identification of glioblastoma stemcells (GSCs) has transformed our comprehension of those tumors by revealing a hierarchical organization. GSCs can self-renew, differentiate and enter into a quiescent state. They are considered as cells which fuel and as the main culprits of tumor relapse. The discovery of GSCs triggered a change in paradigm for cancer therapy. Indeed to gain in efficacy, therapies need to target, not only the cells forming the bulk of the tumor, but also GSCs particularly resistant and endowed with a high tumorigenic potential. Chemical screening of the Prestwick chemical library in our laboratory, unveiled bisacodyl with a specific activity on quiescent GSCs.This thesis presents work on the characterization of GSCs, study of the mode of action of bisacodyl on GSCs, as well as a preclinical evaluation of bisacodyl on a 3D model in vitro and animal models in vivo.