CSCs, caracterizadas pela alta atividade da ALDH1 e expressão (ou não) de marcadores de células-tronco embrionárias OCT-4, NANOG, SOX2, KLF4, LIN2, estão presentes em tumores de mama HER2+. Mamosferas são estruturas celulares esferóides in vitro enriquecidas em CSCs. Neste trabalho, buscou-se um melhor entendimento do papel dos miRNAs -221, -222 e -4728-3p na biologia das CSCs. Foi observado alto percentual de células ALDH1+ (citometria) em mamosferas das linhagens MCF-7, SKBR3 e BT-474; alta expressão (qRT-PCR) de miR-221 e -222 em mamosferas da linhagem MCF-7 e de tumores de pacientes, além de alta expressão de HER2 em mamosferas das linhagens MCF-7 e SKBR3. A MFE de células MCF-7 mostrou-se aumentada após a indução (com lentivetores) da superexpressão dos miRNAs -221 e -222. A resistência ao quimioterápico paclitaxel estava aumentada em mamosferas que superexpressavam o miRNA-222. A superexpressão de miR-4728-3p, localizado em um intron de HER2, levou a um aumento das subpopulações ALDH1+ em duas linhagens celulares reforçando seu envolvimento com a biologia de CSCs. / CSCs, characterized by high activity of ALDH1 and high (or not) expression of embryonic stem cell markers OCT-4, NANOG, SOX2, KLF4, LIN28 are present in tumors with HER2 amplification. Mammospheres are spheroid cell structures in vitro enriched by CSCs. In this study, we aim to contribute to the better understanding the role of miRNAs -221, 222, 4728-3p in the the biology of CSCs. We observed high percentage of ALDH+ cells (cytometry) in mammospheres of MCF-7, SKBR3 and BT-474 cell lines; high expression (qRT-PCR) of miR-221 and -222 in mammospheres of MCF-7 cell line and cells derived from patients, moreover HER2 is upregulated in mammospheres from MCF-7 and SKBR3 cell lines. MFE is higher in MCF-7 cell line after induction (with lentivectors) of miR-221 and -222 expression. The resistance to paclitaxel was increased in mammopheres that were overexpressing miRNA-222. Overexpression of miR-4728-3p, located in an intron of HER2 gene, induced the increment of ALDH1+ subpopulations in two cell lines, reinforcing its role in the biology of CSCs.

Dans ce travail, nous avons pu sélectionner par la méthode SELEX un aptamère à ARN modifié, appelé Apt1, qui se lie avec une haute affinité au récepteur CD44. L'aptamère sélectionné a été modifié avec par des 2'-F-pyrimidines afin d’augmenter sa stabilité vis-à-vis des nucléases pour une application thérapeutique. Cet aptamère a été ensuite greffé sur des liposomes contenant des séquences de siRNA dirigées contre un gène rapporteur, dans le but d’un ciblage actif des cellules tumorales exprimant le récepteur CD44. Cette fonctionnalisation a été réalisée par la conjugaison d’un dérivé 3'-thiol de Apt1 et un dérivé maléimide de phospholipides, directement à la surface des liposomes, ou bien séparément puis par post-insertion sur les liposomes. Les liposomes ainsi formulés présentent une forte affinité pour les cellules exprimant le CD44 sans déclencher de réponse inflammatoire au sein de ces cellules. En outre, nous montrons que l'inhibition du gène rapporteur est augmentée et prolongée lorsque l’aptamère est couplé aux liposomes chargés aux siRNA, in vitro ainsi qu’in vivo sur un modèle murin orthotopique de cancer du sein. De tels vecteurs de siRNA constituent donc un outil prometteur pour le ciblage actif de tumeurs exprimant le récepteur CD44. L'étape suivante consistera charger ces vecteurs par des séquences de siRNA permettant de réprimer des oncogènes. / In this work we succeeded to select a modified RNA aptamer, named Apt1, to bind the human CD44 receptor protein with high affinity using the Systemaic Evolution of Ligands by EXponential enrichment (SELEX) method. The selected aptamer was modified with 2'-F-pyrimidines to increase its stability against nucleases for therapeutic applications. Furthermore, we designed and characterized aptamer-functionalized liposomes loaded with siRNA molecules against a reporter gene as a model drug delivery system for the active targeting CD44-expressing tumor cells in vitro and in vivo. Such functionalization was performed by conjugation of 3'-thiol-modified Apt1 to maleimide-modified phospholipids, either on the surface of liposomes, or separately, followed by post-insertion onto liposomes. The targeted liposomes displayed high affinity for CD44-positive cells without triggering any inflammatory response within these cells. Moreover, we show that a higher and prolonged inhibition of the targeted gene can be achieved when siRNA-loaded liposomes are functionalized by the aptamer, both in vitro and in vivo on a murine orthotopic breast cancer model. Such a delivery system may thus be a useful tool for the active targeting of CD44-expressing tumors and silencing oncogenes in vivo.

Das Tumormikromilieu beeinflusst maßgeblich Tumorwachstum und -progression sowie das Ansprechen von Tumorzellen auf Strahlen- und Chemotherapie. Weiterhin ist bekannt, dass Wachstumsfaktoren, Sauerstoffgehalt und extrazelluläre Matrix (EZM) als Resistenzfaktoren das Zellüberleben nach Exposition mit ionisierender Strahlung oder zytotoxischen Substanzen bestimmen. Weitere zelluläre Parameter, wie Zellmorphologie, Zytoskelettarchitektur und Chromatinkondensation, werden ebenfalls in Abhängigkeit der Wachstumsbedingungen moduliert, wie vergleichende Untersuchungen an physiologischeren drei- (3D) mit herkömmlichen zwei-dimensionalen (2D) Zellkulturen zeigen. Veränderungen der Chromatindichte beeinflussen zudem die Genexpression, wodurch wichtige zelluläre Prozesse, wie Überleben, Proliferation und Differenzierung der Zellen, reguliert werden. Außerdem ist die Chromatinkondensation für eine effektive Reparatur strahleninduzierter DNA-Schäden, wie DNA-Doppelstrangbrüche (DSB), von großer Bedeutung. Das Ziel der vorliegenden Arbeit bestand darin, die zelluläre Strahlenempfindlichkeit unter Berücksichtigung der Chromatinkondensation in humanen Bronchial- (A549) und Plattenepithelkarzinomzellen (UTSCC-15) in Abhängigkeit der Wachstumsbedingungen zu analysieren. Da die molekularen Mechanismen der Wechselwirkung zwischen Chromatindichte und Reparatur strahleninduzierter DSB bis heute unklar sind, war die Untersuchung dieser Zusammenhänge unter 2D, 3D und in vivo Wachstumsbedingungen von besonderem Interesse. Die Ergebnisse dieser Arbeit zeigen, dass das Zellwachstum in einer physiologischen 3D Matrix im Vergleich zur herkömmlichen 2D Zellkultur zu einer geringeren Anzahl an strahleninduzierten residuellen DSB (rDSB) und letalen Chromosomenaberrationen führen kann, was wiederum für ein verbessertes Zellüberleben nach Bestrahlung verantwortlich sein könnte. Des Weiteren konnte in 3D im Zusammenhang mit einer höheren Chromatinkondensierung eine Erhöhung der zellulären Strahlenresistenz gezeigt werden. Auf molekularer Ebene zeigen die Ergebnisse dieser Arbeit außerdem, dass eine siRNA-vermittelte Hemmung chromatinmodifizierender Histondeacetylasen (HDAC 1, 2 und 4) zu keiner Strahlensensibilisierung führt, während durch die Behandlung mit dem pharmakologischen HDAC-Inhibitor Panobinostat (LBH589) neben der Chromatindekondensierung auch eine erhöhte Strahlenempfindlichkeit der Zellen erreicht werden konnte. In Abhängigkeit der untersuchten Wachstumsbedingungen konnten Unterschiede in der Verteilung strahleninduzierter DSB zwischen hetero- und euchromatischen DNA-Bereichen nachgewiesen werden. Interessanterweise nimmt in 2D dosisabhängig der prozentuale Anteil der Heterochromatin (HC)-assoziierten Foci ab, wohingegen in 3D und im Xenografttumormodell dosisunabhängig etwa die Hälfte der Foci mit heterochromatischen DNA-Bereichen assoziiert sind. Diese Daten zeigen, dass Tumorzellen in 3D und in vivo in Abhängigkeit der veränderten Zellmorphologie und Chromatinkondensierung deutlich mehr HC-assoziierte rDSB besitzen als in 2D, was die Hypothese einer beeinträchtigten Reparatur im HC unterstützt. Dennoch zeigt die Korrelation zwischen der deutlich geringeren rDSB Gesamtanzahl und dem erhöhtem Zellüberleben in 3D, dass neben dem Anteil an kondensiertem Chromatin auch die Gesamtanzahl rDSB ein wichtiger Einflussfaktor der zellulären Strahlenempfindlichkeit zu sein scheint. Die Ergebnisse dieser Arbeit liefern somit wichtige Erklärungsansätze für einen direkten Zusammenhang zwischen Zellmorphologie, Chromatinkondensation und zellulärer Strahlenempfindlichkeit. Des Weiteren unterstreichen diese Untersuchungen das verwendete 3D Zellkulturmodell als Annäherung an die in vivo Situation. Damit sind diese Daten von großer Relevanz für ein besseres Verständnis der zellulären Strahlenempfindlichkeit auf molekularer Ebene und können entscheidend dazu beitragen die Behandlung von Tumorerkrankungen sowie die Heilungschancen der Patienten zu verbessern.

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The analysis of potentially harmful biological particles is imperative for the mitigation of disease. As a result, there is a growing need for tools which can characterize, detect, and separate biological particles for the alleviation of a multitude of disease. One powerful technique for the analysis of cells is the use of dielectrophoresis (DEP) forces for the manipulation of particle movement. DEP is a particle transport phenomenon, induced by the presence of non-uniform electric fields. The dependence on intrinsic electrical properties of cells, have enabled DEP force to be utilized for numerous biological analyses. This thesis presents the investigation of breast cancer, pathogen, neuronal and glial cells and their DEP profiles. The drug response of various breast cancer cell lines when exposed to a variety of chemical stimuli were analyzed using shifts in their DEP profiles in relation to control groups. These results were supplemented with gene expression analysis to identify biophysical changes which could contribute to the DEP shifts. Additional experiments were conducted for the monitoring of pathogens. Live/dead bacteria mixtures were evaluated using an integrated system with DEP enrichment and impedance spectroscopy. Another application of DEP which was investigated was the separation of heterogeneous mixtures. Through the use of a novel microfluidic channel design, the separation of simulated circulating tumor cells (CTCs) from diluted blood and neuron cells from glial cells was demonstrated. The wide range of applications examined in this thesis highlights the versatility of DEP and the flexibility of the reported devices. / MS / Microscale technology can be utilized for the identification, characterization and sorting of biological material in a plethora of biomedical applications. One promising technique which is capable of cell manipulation is dielectrophoresis (DEP). DEP is a microscale force which causes particles to be attracted or repelled by specific geometries in microchannels. The DEP force is produced by the application of electric fields and can be utilized to analysis biological cell populations. This is because biological particles have unique electrical properties based on their cell morphology. Distinctions in their external protrusions and internal structures contribute to their electrical properties and can be identified in their DEP profiles. Based on this concept a variety of biomedical applications of DEP was explored. Chapter 2 and 3 describe the investigation of cells when exposed to various drugs. Drug induced responses were characterized based on their shifts in their DEP profiles. Chapter 4 presents the a rapid and low-cost live/dead assay for bacteria in aqueous samples through DEP and impedance spectroscopy. In chapter 5, the development of a DEP platform for cell sorting is reported. The wide range of biomedical applications which were explored demonstrate the useful nature of the DEP phenomenon.

No / Dystroglycan is a ubiquitously expressed cell adhesion molecule frequently found to be altered or reduced in adenocarcinomas, however the mechanisms or consequences of dystroglycan loss have not been studied extensively. We examined the consequence of overexpression or RNAi depletion of dystroglycan on properties of in vitro growth migration and invasion of LNCaP, PC3, and DU145 prostate cancer cell lines. RESULTS: Using LNCaP cells we observed cell density-dependent changes in beta-dystroglycan with the appearance of several lower molecular weight species ranging in size from 43 to 26 kDa. The bands of 31 and 26 kDa were attributed to proteolysis, whereas bands between 43 and 38 kDa were a consequence of mis-glycosylation. The localization of beta-dystroglycan in LNCaP colonies in culture also varied, cells with a mesenchymal appearance at the periphery of the colony had more pronounced membrane localization of dystroglycan. Whereas some cells demonstrated nuclear dystroglycan. Increased dystroglycan levels were inhibitory to growth in soft agar but promoted Matrigel invasion, whereas reduced dystroglycan levels promoted growth in soft agar but inhibited invasion. Similar results were also obtained for PC3 and DU145 cells. This study suggests that changes in beta-dystroglycan distribution within the cell and/or the loss of dystroglycan during tumorigenesis, through a combination of proteolysis and altered glycosylation, leads to an increased ability to grow in an anchorage independent manner, however dystroglycan may need to be re-expressed for cell invasion and metastasis to occur.

Introduction: Simultaneous detection of disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) was shown to be associated with an especially poor prognosis and increased incidence of disease-related deaths in non-metastatic breast cancer patients. We analyzed the occurance of DTCs in bone marrow and CTCs in peripheral blood in patients with primary breast cancer, we evaluated the correlation of their presence with other prognostic markers and we investigated the changes in DTCs/CTCs number at different time points during treatment. Materials and methods: Blood of 50 patients with primary breast cancer were used for immunomagnetic separation and detection of circulating tumor cells using the commercial available system the AdnaTest Breast Cancer™ (AdnaGen GmbH, Langenhagen, Germany). Bone marrow aspirates from 50 patients were analyzed for DTCs by immunocytochemistry using the pancytokeratin antibody conjugated with FITC (Monoclonal Anti-Cytokeratin antibody F3418, Sigma Aldrich, USA). Results: DTCs were identified in 30% (15/50) and CTCs in 22% (11/50) of patients. We found that DTC positivity could point to a significantly high risk of larger primary tumor size (p- value 0.011) and significantly higher risk of lymph node involvement (p- value 0.002). For CTC positivity, no such...

Introduction: Simultaneous detection of disseminated tumor cells (DTCs) and circulating tumor cells (CTCs) was shown to be associated with an especially poor prognosis and increased incidence of disease-related deaths in non-metastatic breast cancer patients. We analyzed the occurance of DTCs in bone marrow and CTCs in peripheral blood in patients with primary breast cancer, we evaluated the correlation of their presence with other prognostic markers and we investigated the changes in DTCs/CTCs number at different time points during treatment. Materials and methods: Blood of 50 patients with primary breast cancer were used for immunomagnetic separation and detection of circulating tumor cells using the commercial available system the AdnaTest Breast Cancer™ (AdnaGen GmbH, Langenhagen, Germany). Bone marrow aspirates from 50 patients were analyzed for DTCs by immunocytochemistry using the pancytokeratin antibody conjugated with FITC (Monoclonal Anti-Cytokeratin antibody F3418, Sigma Aldrich, USA). Results: DTCs were identified in 30% (15/50) and CTCs in 22% (11/50) of patients. We found that DTC positivity could point to a significantly high risk of larger primary tumor size (p- value 0.011) and significantly higher risk of lymph node involvement (p- value 0.002). For CTC positivity, no such...