This report presents a deep learning based approach for segmenting and characterizing tumor cell deaths using images provided by the Önfelt lab, which contain NK cells and HL60 leukemia cells. We explore the efficiency of convolutional neural networks (CNNs) in distinguishing between live and dead tumor cells, as well as different classes of cell death. Three CNN architectures: MobileNetV2, ResNet-18, and ResNet-50 were employed, utilizing transfer learning to optimize performance given the limited size of available datasets. The networks were trained using two loss functions: weighted cross-entropy and generalized dice loss and two optimizers: Adaptive moment estimation (Adam) and stochastic gradient descent with momentum (SGDM), with performance evaluations based on metrics such as mean accuracy, intersection over union (IoU), and BF score. Our results indicate that MobileNetV2 with cross-entropy loss and the Adam optimizer outperformed other configurations, demonstrating high mean accuracy. Challenges such as class imbalance, annotation bias, and dataset limitations are discussed, alongside potential future directions to enhance model robustness and accuracy. The successful training of networks capable of classifying all identified types of cell death, demonstrates the potential for a deep learning approach to identify different types of cell deaths as a tool for analyzing immunotherapeutic strategies and enhance understanding of NK cell behaviors in cancer treatment.

Aim: To develop an immunomagnetic assay for the isolation of circulating tumor cells (CTCs) followed by the analysis of a multimarker panel, which will enable the characterization of these malignant cells with high accuracy. Patients and Methods: Peripheral blood (PB) was collected from 32 metastatic breast cancer patients and 42 negative controls. The antibodies BM7 and VU1D9 were used for immunomagnetic tumor cell enrichment. A real-time reverse transcription-polymerase chain reaction (RT-PCR) approach for the markers KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 and ERBB2 was used for CTC detection and characterization. Results: The positivity rates for each marker were as follows: 46.9% for KRT19, 25.0% for SCGB2A2, 28.1% for MUC1, 28.1% for EPCAM, 21.9% for BIRC5, and 15.6% for ERBB2. After the creation of individualized cutoffs, the sensitivity and specificity of the combined marker gene panel increased to 56.3% and 100%, respectively. Interestingly, 27.0% of the HER2-negative tumor patients showed ERBB2 mRNA-positive CTCs. Conclusions: The described technique can be used to measure CTCs with great accuracy. The use of a multimarker panel for the characterization of CTCs may provide real-time information and be of great value in therapy monitoring. / Ziel: Entwicklung eines immunomagnetischen Verfahrens zur Isolierung zirkulierender Tumorzellen (CTCs) in Kombination mit einer molekularen Multimarkeranalyse für die hochspezifische Identifizierung maligner Zellen. Patientinnen und Methoden: Peripheres Blut (PB) von 32 Patientinnen mit metastasiertem Mammakarzinom und von 42 gesunden Kontrollen wurde für die immunomagnetische Tumorzellanreicherung mit den Antikörpern BM7 und VU1D9 genutzt. Eine Real-Time Reverse Transkription Polymerase-Kettenreaktion (RT-PCR)-Methodik mit den Markern KRT19, SCGB2A2, MUC1, EPCAM, BIRC5 und ERBB2 wurde für den CTC-Nachweis und die Tumorzellcharakterisierung entwickelt. Ergebnisse: Für die einzelnen Marker wurden die folgenden Positivitätsraten ermittelt: 46,9% für KRT19, 25,0% für SCGB2A2, 28,1% für MUC1, 28,1% für EPCAM, 21,9% für BIRC5 und 15,6% für ERBB2. Nach der Bestimmung individualisierter Cut-off-Werte ergab sich für den kombinierten Multimarkernachweis eine Sensitivität und Spezifität von 56,3% bzw. 100%. Bemerkenswert war der Befund, dass 27,0% der HER2-tumornegativen Patientinnen ERBB2-mRNA-positive CTCs aufwiesen. Schlussfolgerung: Die hier beschriebene Methodik bestimmt CTCs mit hoher Spezifität. Die molekulare Multimarkeranalyse liefert wertvolle Real-Time-Informationen für personalisierte Behandlungsmodalitäten. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle. / Dissertation

Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire. / Background: Circulating tumor cells (CTCs) have been detected in many cancers and are used in multiple clinical applications including disease prognosis, tumor recurrence prediction and prediction of tumor sensitivity to chemotherapeutic drugs. Studies in most major solid cancer(s) (breast, lung, colon and prostate) are progressing rapidly, but there has been very little progress concerning uterine cervical cancer (UCC).Objective: our aim is to optimize enrichment processes and the molecular biomarker-based detection of human circulating tumor cells (CTCs) in uterine cervical cancer (UCC). Material & Methods: To mimic CTCs in patients, we designed an experimental spiking model where the CaSki HPV16-positive UCC cell line was serially diluted and spiked into human blood collected from healthy volunteers. CaSki CTCs were enriched using either Ficoll density centrifugation, red blood cell (RBC) lysis or RBC lysis combined with cell surface markers negative or positive enrichment. CTCs were detected using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure the gene expression of human papillomavirus (HPV) viral oncogenes (E6 and E7), cytokeratin 19 (CK19), or the cyclin dependent kinase inhibitor p16INK4A. Finally, ten HPV16- positive UCC patients and six healthy controls were recruited to validate CTCs detection in vivo. Result: In the spiking model, RBC lysis alone or combined with negative or positive enrichment suggests detection limits close to 1 CTC per mL of blood for all molecular biomarkers used. The sensitivity of detection increased when using positive and negative enrichment probably by reducing the peripheral blood mononuclear cell-derived RNA background. Unlike HPV oncogenes, CK19 and p16INK4A were detected in normal individuals, thus appropriate basal expression levels need to be accurately determined compared to cancer patients. Alternatively, Ficoll density gradient had a detection limit of only about 1000 cells per mL of blood. Finally CTCs were detected in 2/10 patients using CK19. None of the patients had E6/E7 transcripts and p16INK4A was expressed at similar level across all samples (cancer and healthy). Conclusion: qRT-PCR of HPV16 E6 and E7 is the most sensitive and specific biomarker used to detect CTCs in the spiking model. In early disease UCC patients, only CK19 revealed the presence of CTCs suggesting that these cells are rare at that stage of the disease. Keywords: uterine cervical cancer, circulating tumor cells, qRT-PCR, E6 and E7 oncoprotein, CK19, p16INK4A, immune-magnetic enrichment, molecular detection.

Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l’espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l’oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l’ajout d’un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l’inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l’association de l’inhibiteur de mTOR everolimus et de l’inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l’injection orthotopique d’une lignée bioluminescente de CBP-NPC chez des souris nude a permis d’établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L’association d’un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d’envisager un transfert en clinique. / Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Activation of phosphatidylinositol-3-kinase (PI3K)-AKT and Kirsten rat sarcoma viral oncogene homologue (KRAS) can induce cellular immortalization, proliferation, and resistance to anticancer therapeutics such as epidermal growth factor receptor inhibitors or chemotherapy. This study assessed the conséquences of inhibiting these two pathways in tumor cells with activation of KRAS, PI3K-AKT, or both. We investigated whether the combination of a novel RAF/vascular endothelial growth factor receptor inhibitor, RAF265, with a mammalian target of rapamycin (mTOR) inhibitor, RAD001 (everolimus), could lead to enhanced antitumoral effects in vitro and in vivo. To address this question, we used cell lines with different status regarding KRAS, PIK3CA, and BRAF mutations, using immunoblotting to evaluate the inhibitors, and MTT and clonogenic assays for effects on cell viability and proliferation. Subcutaneous xenografts were used to assess the activity of the combination in vivo. RAD001 inhibited mTOR downstream signaling in all cell lines, whereas RAF265 inhibited RAF downstream signaling only in BRAF mutant cells. In vitro, addition of RAF265 to RAD001 led to decreased AKT, S6, and Eukaryotic translation initiation factor 4E binding protein 1 phosphorylation in HCT116 cells. In vitro and in vivo, RAD001 addition enhanced the antitumoral effect of RAF265 in HCT116 and H460 cells (both KRAS mut, PIK3CA mut); in contrast, the combination of RAF265 and RAD001 yielded no additional activity in A549 and MDAMB231 cells. The combination of RAF and mTOR inhibitors is effective for enhancing antitumoral effects in cells with deregulation of both RAS-RAF and PI3K, possibly through the cross-inhibition of 4E binding protein 1 and S6 protein. We then focus on animal models. Preclinical models of NSCLC require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.

Nous avons abordé dans l’ensemble de nos travaux deux paramètres importants pour l’immunomonitoring des patients atteints d’un cancer : les cellules tumorales circulantes (CTC) comme un indicateur de la « charge antigénique tumorale » et la réponse immune lymphocytaire T spécifique d’antigènes associés aux tumeurs (AAT). Nous avons évalué d’abord la valeur diagnostique et pronostique des CTC dans les cancers des voies aérodigestives supérieures (« HNSCC » en anglais) par une revue systématique et meta-analyse de la littérature. Les preuves actuelles identifient le test de détection de CTC comme un test extrêmement spécifique, mais de faible sensibilité dans les HNSCC. En outre, la présence de CTC indique une DFS (« disease free survival ») inférieure. Nous rapportons également pour la première fois un cas rare d’énumération extrêmement élevée de CTC détectées par le système CellSearch® chez un patient présentant un carcinome épidermoïde de la cavité buccale en utilisant. Le nombre absolu de CTC pourrait donc prédire une phase particulière de développement du cancer ainsi qu'une mauvaise survie, contribuant potentiellement à une prise en charge médicale personnalisée. De plus, nous décrivons une adaptation de la méthode CellSearch® qui nous avons développée pour détecter les cellules tumorales dans le liquide céphalo-rachidien de patients atteints de méningites carcinomateuses. Cette nouvelle approche permet une sensibilité nettement améliorée en comparaison avec la cytologie conventionnelle. La technologie CellSearch®, appliquée à des volumes limités des échantillons et permettant une augmentation du temps pré-analytique, pourrait ainsi avoir un grand intérêt dans le diagnostic de métastases leptoméningées chez les patients atteints d’un cancer d’origine épithéliale. Par une évaluation concomitante des CTC et des réponses lymphocytaires spécifiques aux AAT chez 24 patients avec HNSCC, nous avons trouvé que les CTC pourraient être un indicateur indépendant de la charge tumorale immunogène. L'absence de CTC, la présence de lymphocytes T spécifiques aux AAT, ou la combinaison de ceux-ci, étaient tous des paramètres montrant une tendance pour une meilleure survie globale ou une survie sans maladie. L’amplitude et les signatures fonctionnelles des lymphocytes T spécifiques aux AAT chez les patients atteints de HNSCC étaient associées à la présence de CTC. Ces résultats suggèrent qu’une évaluation concomitante de ces deux paramètres pourrait être plus informative sur le pronostic et potentiellement sur l’impact des traitements (notamment dans la perspective d’un traitement par des « immune checkpoints ») / We have evaluated herein two important parameters in the immunomonitoring of cancer patients: circulating tumor cells (CTC) as an indicator of “tumoral antigenic load” and tumor-associated antigens (TAA) specific T-cells. We firstly evaluated the diagnostic and prognostic value of CTC in Head and Neck Squamous Cell Carcinoma (HNSCC) by a systematic review and meta-analysis. We came to the conclusion that current evidence identifies the CTC detection test as an extremely specific but low sensitive test in HNSCC. In addition, the presence of CTC indicates a worse disease-free disease (DFS). Also, we report for the first time a rare case of extremely high enumeration of circulating tumor cells detected in a patient with squamous cell carcinoma of the oral cavity using the CellSearch® system. The absolute number of CTC could therefore predict a particular phase of cancer development as well as a poor survival, potentially contributing to personalized health. In addition, we describe an adaptation of the CellSearch® method that we have developed for detecting tumor cells in the cerebrospinal fluid of patients with carcinomatous meningitis. This new approach reaches a significantly improved sensitivity compared to conventional cytology. CellSearch® technology, applied to limited sample volumes and allowing an increased pre-analytical time, may be of great interest in the diagnosis of leptomeningeal metastases in patients with epithelial cancer. By a concomitant evaluation of CTC and TAA-specific lymphocyte responses in 24 HNSCC patients, we describe that CTC could be an independent indicator of immunogenic tumor burden. The absence of CTC, the presence of TAA-specific T-cells, or the combination of these, were all parameters showing a trend for a better overall survival or DFS. The amplitude and functional signatures of TAA-specific T-lymphocytes in patients with HNSCC were associated with the presence of CTC. These results suggest that a concomitant evaluation of these two parameters may be more pertinent for prognosis assessment as well as for treatment impact, especially in “checkpoint-inhibitors” new immunotherapies

O uso da LDE como veículo para quimioterápicos tem mostrado ser uma boa estratégia para aumentar a eficácia terapêutica dos mesmos. Nesse estudo, a LDE foi empregada como veículo para um derivado lipofílico do metotrexato (MTX), o di-dodecil metotrexato, que foi obtido com rendimento elevado através de reação de esterificação do MTX. O aumento na lipofilicidade do derivado possibilitou incorporação na LDE com rendimento e estabilidade elevados. O IC50 de LDE-di-dodecil MTX foi cerca de 100 vezes menor em relação ao MTX comercial, sua captação celular mais elevada nas linhagens leucêmicas estudadas e sua toxicidade animal reduzida, mostrando que a LDE é um veículo promissor para este fármaco. / The use of LDE as vehicle to drugs is a great strategy to improve the therapeutic index and reduce the side effects. In this study LDE was used as vehicle to di-dodecyl methotrexate, a lipophilic derivative of MTX, obtained through an esterification reaction with a high yield. The increased lipophilicity of the derivative allowed a high association to LDE and good stability. The IC50 of LDE-di-dodecyl MTX was lower than that of the MTX and the uptake was higher in leukemic cells. The MTX toxicity in mice was reduced after association to LDE, showing that LDE is a promising vehicle to this drug.

Sabe-se que TRAIL é capaz de matar células tumorais de forma seletiva e que TRAIL tem sua expressão reduzida em diversos tumores, porém pouco se sabe sobre os mecanismos responsáveis pela sua inibição. Tendo em vista que a expressão de TRAIL pode ser regulada pelo Ácido Retinóico; que PRAME é capaz de inibir a via do ácido retinóico através da proteína EZH2 e que nós observamos anteriormente que a expressão de TRAIL esta diminuída em pacientes com LMC, nós decidimos investigar a associação entre PRAME, EZH2 e TRAIL na LMC. Nós demonstramos que PRAME, mas não EZH2, tem sua expressão aumentada em células BCR-ABL+ e sua expressão está associada com a progressão da LMC. Alem disto, existe uma correlação positiva entre PRAME e BCR-ABL e negativa entre PRAME e TRAIL nestes pacientes. A inibição da expressão de PRAME ou EZH2 por RNAi induziu um aumento da expressão de TRAIL. Estes dados revelam um novo mecanismo de regulação responsável por diminuir a expressão de TRAIL, e geram novos possíveis alvos para a terapia da LMC e, possivelmente, também para outros tumores. / TRAIL was shown to selectively kill tumor cells. Not surprisingly, TRAIL is down-regulated in a variety of tumor cells, but the mechanism responsible for TRAIL inhibition remains elusive. Because TRAIL can be regulate by retinoic acid; PRAME was shown to inhibit transcription of retinoic acid receptor target genes through the polycomb protein EZH2; and we have found that TRAIL is inversely correlated with BCR-ABL in CML patients, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is up-regulated in BCR-ABL cells and is associated with the progression of disease in CML patients. In addition, PRAME expression is positively correlated with BCR-ABL and negatively with TRAIL in these patients. Importantly, knocking down of PRAME or EZH2 by RNA interference restores TRAIL expression. Our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Les phénomènes cicatriciels et de carcinogenèse partagent des points communs et peuvent être définis comme des processus complexes et adaptatif régulés par les interactions entre l'hôte et le microenvironnement tissulaire. Après la chirurgie, la radiothérapie est la seconde modalité la plus efficace dans le traitement du cancer et une approche thérapeutique multimodale impliquant chirurgie, radiothérapie et chimiothérapie est aujourd’hui classiquement utilisée. Des résultats récents suggèrent que, en plus des effets létaux sur les cellules tumorales, la radiothérapie modifie le microenvironnement tissulaire. Ces modifications affectent le phénotype cellulaire, le métabolisme des tissus, et les événements de signalisation entre les cellules.Les interactions complexes entre les cellules stromales et les cellules cancéreuses suscitent beaucoup d’intérêt et dans la première partie de ma thèse, j’ai exploré les interactions entre stroma et cellules de carcinome en réponse à la radiothérapie par modulation génétique du stroma après irradiation. J’ai constaté que les fibroblastes, indépendamment de leur statut RhoB, ne modulaient pas la radiosensibilité intrinsèque des TC- 1, mais produisaient des facteurs diffusibles capables de modifier le devenir des cellules tumorales. Ensuite, j’ai constaté que fibroblastes sauvages et RhoB déficients stimulaient la migration des TC-1 par des mécanismes distincts, impliquant respectivement TGF- β1 et MMP. J’ai également constaté que la co-irradiation, des fibroblastes et des TC- 1, abrogait le phénotype pro-migratoire des TC-1 par répression de la sécrétion du TGF- β et des MMP. Alors que le protocole de co-irradiation utilisé mime la situation clinique, mes résultats sont en désaccord avec les publications récentes et suggèrent que le stroma irradié ne renforce pas la migration des cellules tumorales mais au contraire pourrait être manipulé pour promouvoir une réponse immunitaire anti-tumorale.Deuxièmement, mes expériences in vivo, semblent confirmer les données obtenues in vitro et montrent que l’irradiation préalable du lit tumoral ne stimule ni croissance de la tumeur et ni sa dissemination. Nos résultats semblent montrer que l’irradiation du stroma ne favorise pas la migration des cellules de carcinome et ceci indépendamment de leur génotype. La Troisième Partie De Mon Projet, a été consacrée à l’étude des cellules tumorale circulantes (CTC) après la radiothérapie. En accord avec les résultats rapportés après la chirurgie, le nombre de CTC augmente dans la circulation sanguine après radiothérapie probablement à cause des lésions vasculaires radio-induites ou/et par induction d’EMT dans les cellules tumorales. Néanmoins ces CTC semblent être piégées dans la cavité cardiaque. La signification de la présence de ces CTC pour le développement métastatique n’est pas élucidée mais on peut suspecter un effet promoteur de métastase. Ainsi le microenvironnement pourrait avoir des effets antagonistes promoteurs ou inhibiteurs de malignité. / Wound healing and carcinogenesis are defined as complex, adaptive processes which are controlled by intricate communications between the host and the tissue microenvironment. A number of phenotypic similarities are shared by wounds and cancers in cellular signaling and gene expression. Radiotherapy is the second most effective modality of cancer treatment after surgery and can be used, either alone or in combination with chemotherapy. Recent findings suggest that radiotherapy apart from tumor cell death also rapidly and persistently modifies the tissue microenvironment. These modifications affect cell phenotype, tissue metabolism, bidirectional exchanges and signaling events between cells. The complex interactions between stromal cells and cancer cells are of immense interest and in The First Part of My Thesis, I tried to explore the crosstalk between stromal and carcinoma cells in response to radiotherapy by genetic modulation of the stroma and irradiation. We found that fibroblasts, irrespective of their RhoB status, do not modulate intrinsic radiosensitivity of TC-1 but produce diffusible factors able to modify tumor cell fate. Then we found that Wt and RhoB deficient fibroblasts stimulated TC-1 migration through distinct mechanisms respectively, TGF-β1 and MMP-mediated. We also found that co-irradiation of fibroblasts and TC-1 abrogated the pro-migratory phenotype by repression of TGF-β and MMP secretion. This result is highly relevant to the clinical situation and suggests that conversely to, the current view; irradiated stroma would not enhance carcinoma migration and could be manipulated to promote anti-tumor immune response. Secondly, our in vivo experiments, tends to confirm the in vitro data showing that irradiated tumor bed does not stimulate tumor growth and escape. Our results also challenges the view that irradiated stroma would promote migration of carcinoma cells as we show that independently from their genotype co-irradiation of fibroblasts and carcinoma cells repressed carcinoma cell migration and confirmations studies are currently performed in vivo. The Third Part of My Project, was dedicated to investigate the effect on CTC release after radiotherapy. Consistently with the results reported after surgery , the number of CTC increases in the blood stream after radiotherapy probably due to radiation-induced vascular injury induced or/and by EMT induction in tumor cells but these cells seemed to be entrapped into the cardiac cavity. The significance of these CTC to metastatic development is still under investigation but there is evidence for a metastasis-promoting effect of RT from animal studies.Thus the microenvironment can exert antagonist stimulatory or inhibitory effects on malignant cells.

Hepatocarcinoma (HCC) é o mais comum tumor maligno primário do fígado. Aurora A é importante durante o ciclo celular, atuando na maturação centrossômica e sua separação, entrada em mitose, montagem de fuso bipolar, alinhamento dos cromossomos na placa metafásica e citocinese. Expressão alterada de Aurora A tem sido associada com o desenvolvimento do tumor e sua superexpressão ocorre em 60% dos HCCs. Inibidores de Aurora quinases têm sido desenvolvidos como drogas antitumorais. 4(4`-Benzamidoanilina)-6,7dimetoxiquizanoline, BADIM, é um recém desenvolvido inibidor da Aurora. Nosso estudo investigou os efeitos de BADIM na linhagem celular HepG2, derivada de HCC, quando tratada com 300, 600 e 1200nm de BADIM por 24 e 48h. Observamos inibição de proliferação, aumento de células tetraplóides, binucleadas e gigantes, bloqueio em G2/M do ciclo celular, alterações nos microtúbulos, mitoses atípicas e apoptose. Por conseguinte, este inibidor é um agente promissor para estudos em HCC, pois atua em pontos críticos relacionados com o processo de tumorigênese. / Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of liver. Aurora A is important during cell cycle, including centrosome maturation and separation, mitotic entry, bipolar-spindle assembly, chromosome alignment on metaphase plate and cytokinesis. Altered expression of Aurora A has been associated with tumor development and its overexpression occurs in 60% of HCC. Aurora kinases inhibitors have been developed as antitumoral drugs. 4(4`-Benzamidoanilino)-6,7-dimethoxiquizanolina, BADIM, is a new Aurora inhibitor. Our study aimed investigates the BADIM effects on HepG2 cell line, derivates of HCC, when treated in 300, 600 and 1200nM for 24 and 48h. We observed inhibition of cell proliferation, increase of tetraploids, binucleated and giant cells, arrest in G2/M cell cycle, microtubules alterations, aberrant cell divisions and apoptosis. Therefore this inhibitor is a promising agent for studies in HCC, since it acts at critical points related to tumorigenesis.