Dokument: Detection and characterization of circulating and disseminated tumor cells in ovarian cancer to improve personalized therapeutic strategies
| Titel: | Detection and characterization of circulating and disseminated tumor cells in ovarian cancer to improve personalized therapeutic strategies | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=41095 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20170208-101559-5 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Dipl.Biol. Blassl, Christina [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. rer. nat. Neubauer, Hans [Gutachter] Prof. Dr. Czekelius, Constantin [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | High recurrence rates and chemotherapy resistance imply that ovarian cancer might be initiated and maintained by cancer stem cells. These cells may possess an increased metastatic potential, since they are able to self-renew and insensitive to standard chemotherapeutic agents. Besides that, already during the growth of the primary tumor, the process of epithelial-mesenchymal-transition (EMT) enables tumor cells to detach from the primary tumor by transforming their epithelial phenotype into a mesenchymal one. Ovarian cancer cells, which have undergone this process can either directly enter the peritoneum or transit through the lymphatic or blood system to distant organs. Tumor cells within the blood stream are denoted as circulating tumor cells (CTC) and are able to enter distant organs, e.g. the bone marrow (BM), where they are designated as disseminated tumor cells (DTCs). Both, CTCs and DTCs are supposed to be precursors of subsequent metastatic disease and there is evidence that their presence is correlated with a decreased overall survival (OS) and/or progression free survival (PFS) in ovarian cancer patients.
Consequently, it is useful to detect CTCs/DTCs and to characterize them for EMT and stem cell marker expression. Due to their low frequency compared to numerous blood cells, enrichment of CTCs is necessary to allow their subsequent characterization by molecular methods such as multiplex-RT-PCR. To that aim a typical enrichment strategy for ovarian cancer CTCs (AdnaTest) has initially been used in this project. However, it turned out to be insufficient since it unspecifically co-enriched leukocytes and the subsequent characterization is based on cell pools. For instance co-enriched leukocytes due to their mesenchymal phenotype lead to false positive results regarding expression of EMT-associated transcripts (e.g. neuronal (N)-cadherin and vimentin). Moreover, incompletely differentiated leukocytes disturbed the detection of stem cell associated transcripts (e.g. CD44 (cluster of differentiation 44), ALDH1A1 (aldehyde dehydrogenase 1 family member A1) and Notch1). To overcome these limitations, a workflow for an optimized isolation and subsequent gene expression profiling of single CTCs from ovarian cancer patients was established. It comprises of a density gradient-based enrichment for nucleated cells, depletion of CD45-positive cells of hematopoietic origin and immunofluorescent labeling of CTCs for the epithelial proteins EpCAM (epithelial cell adhesion molecule) and Muc-1 (mucin-1, cell surface associated). Single CTCs were then isolated by micromanipulation. For subsequent expression profiling of candidate genes a multiplex-RT-PCR was developed which enables the detection of 19 transcripts (4 epithelial, 7 EMT and 8 stem cell associated) simultaneously from one CTC/cell without RNA pre-amplification. Using this approach, the analysis of 77 single OvCar3 cells resulted in heterogeneous gene expression patterns as well as co-expression of several epithelial, EMT and stem cell-associated transcripts, underlining the necessity of single cell analysis. Subsequently, 15 single CTCs derived from three ovarian cancer patients were characterized: they were positive for stem cell (CD44, ALDH1A1, Nanog, Oct4 (octamer-binding transcription factor 4)) and EMT associated transcripts (neuronal (N)-cadherin, vimentin, Snai2, CD117, CD146). Particularly, inter-cellular and inter-patient heterogeneity as well as co-expression of epithelial, mesenchymal and stem cell associated transcripts within the same CTC were observed. Analysis of DTCs before (BT) and after (AT) implementation of standard chemotherapy (carboplatin and paclitaxel) was performed on BM aspirates from the iliac crest. DTCs were enriched by density gradient centrifugation and identified by immunocytochemistry. DTCs were detected in 42% BT and in 41% AT. The persistence of DTCs was found in 17% of all patients, 25% were only positive BT, 24% AT and 34% harbored no DTCs. DTC-positive patients (BT) had a significantly decreased OS and were more likely to die than DTC-negative patients (BT). Patients who were initially DTC-negative BT but DTC-positive AT had a significant shorter PFS, while DTC persistence was associated with shorter PFS and OS reaching borderline significance. Immunofluorescent labeling demonstrated that the stem cell associated proteins SOX2 and Lin-28 were expressed in some DTCs BT and AT. In summary, the newly established workflows within this work enable for a rapid, cost-efficient and highly sensitive enrichment of CTCs/DTCs with subsequent characterization on the transcriptomic and proteomic level for EMT and stem cell associated markers. The presented data illustrate that single-cell analysis in comparison to cell pool analysis can provide new hints about the biology of CTC. Moreover, the results give further insights into the potential clinical relevance of DTCs, which survive chemotherapy and may contribute to ovarian cancer progression. Finally, these assays constitute promising tools to improve ovarian cancer treatment and therapy monitoring. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 08.02.2017 | |||||||
| Dateien geändert am: | 08.02.2017 | |||||||
| Promotionsantrag am: | 14.06.2016 | |||||||
| Datum der Promotion: | 26.01.2017 |
