Dokument: Workflow zur Anreicherung und Isolierung einzelner zirkulierender Tumorzellen (CTC) von Mammakarzinom-Patientinnen zur immunzytochemischen Charakterisierung und Amplifikation ihrer genomischen DNA

Titel:	Workflow zur Anreicherung und Isolierung einzelner zirkulierender Tumorzellen (CTC) von Mammakarzinom-Patientinnen zur immunzytochemischen Charakterisierung und Amplifikation ihrer genomischen DNA
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=62656
URN (NBN):	urn:nbn:de:hbz:061-20230530-081619-2
Kollektion:	Dissertationen
Sprache:	Deutsch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Link, Yvonne [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 13,06 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 13.05.2023 / geändert 13.05.2023
Beitragende:	Prof. Dr. Hans Neubauer [Gutachter] Prof. Dr. med. Nikolas Stoecklein [Gutachter]
Stichwörter:	CTC, zirkulierende Tumorzellen, Mammakarzinom, CellCelector
Dewey Dezimal-Klassifikation:	600 Technik, Medizin, angewandte Wissenschaften » 610 Medizin und Gesundheit
Beschreibungen:	Das Mammakarzinom ist bekannt für seine frühe lymphogene und hämatogene Metastasierung, sodass schon bei der Erstdiagnose möglicherweise eine systemische Erkrankung vorliegen kann und zirkulierende Tumorzellen (CTCs) im peripheren Blut nachweisbar sind. Cristofanilli et al. (2004) erläuterten im Rahmen einer prospektiven Studie, dass bei Patientinnen mit metastasiertem Brustkrebs ein Nachweis von mehr als 5 CTC in 7,5 ml Blut mit einem signifikant kürzeren Überleben korreliert. Diese Tumorzellen sind eine sehr heterogene Population von Tumorzellen und weisen unterschiedlichste Eigenschaften auf. Ein besseres Verständnis dieser heterogenen CTC-Biologie ermöglicht die Entwicklung prognostischer und prädiktiver Marker, die Auswahl der optimalen individuellen Therapie und somit auch die Prognose von Mammakarzinom-Patientinnen zu verbessern. Durch die Kombination der Vorteile des CellSearch®-Systems, des VyCap- und ISET-Filters und des CellCelector-Systems konnte ein Verfahrensprotokoll entwickelt werden, welches die Detektion, Isolierung und Charakterisierung EpCAM-positiver und -negativer CTCs als Einzelzellen bei Blutproben von Mammakarzinom-Patientinnen ermöglicht. Insgesamt konnten mit dem neu entwickelten Arbeitsweg zur Einzelzellisolierung bei 32 Patientinnen 1038 CTCs detektiert und 893 dieser nach dem Transfer der Probe durch den CellCelector wiedergefunden werden. Dies entspricht einer Transfer- und Detektionseffizienz von 86 %. Anschließend konnten 98 %, also 872 CTCs, in PCR Tubes oder auf Objektträger als Einzelzellen abgelegt werden. Zur immunzytochemischen Charakterisierung der CTCs auf das, mit einer Zellinvasivität in Verbindung stehende, Protein CapG wurden aus 16 Cartridges des CellSearch-Systems 167 CTCs als Einzelzellen auf Objektträger isoliert und anschließend für den Marker CapG gefärbt. Bei 143 CTCs, 86 % der gesamten CTCs, konnte eine CapG- Expression durch die Antikörperfärbung nachgewiesen werden. 24 CTCs zeigten keine immunzytochemisch nachweisbare Expression von CapG. Zum Beweis der guten DNA-Qualität der Einzelzellproben für eine mögliche genomische Analyse der CTCs wurden aus 32 Cartridges des CellSearch-Systems 95 CTCs in PCR Tubes abgelegt und eine „Whole genome amplification“ durchgeführt. In der Qualitätskontrolle des Ampli1-WGA-Kit (silicon biosystems) hatten 24 % der WGA-Produkte eine gute DNA-Qualität mit > 3 Banden und 76 % hatten < 3 Banden. Die entwickelte Technologie für die Einzelzellisolierung von CTCs in der Kombination des CellSearch®-Systems, VyCap- und ISET-Filter und des CellCelector-Systems bietet einen Grundstein für die Verbesserung der Charakterisierung von EpCAM-positiven und -negativen CTCs aus Blutproben von Mammakarzinom-Patientinnen. Breast cancer is known for its early lymphogenic and hematogenic metastasis, so that systemic disease may already be present at initial diagnosis and circulating tumor cells (CTCs) are detectable in the peripheral blood. Cristofanilli et al (2004) explained in a prospective study that in patients with metastatic breast cancer, detection of more than 5 CTCs in 7,5 ml blood correlates with significantly shorter survival. These tumor cells are a very heterogeneous population of tumor cells and exhibit a wide variety of characteristics. Understanding this heterogeneous CTC biology will enable the development of new prognostic and predictive markers, the selection of optimal individualized therapy, and thus improve the prognosis of breast cancer patients. By combining the advantages of the CellSearch-system, the VyCap- and ISET-filter, and the CellCelector-system, a procedural protocol has been developed that enables the detection, isolation, and characterization of EpCAM-positive and -negative CTCs as single cells in blood samples from breast cancer patients. In total, 1038 CTCs were detected from 32 patients using the newly developed workflow for single cell isolation and 893 of these were recovered after transfer of the sample through the CellCelector. This corresponds to a transfer and detection efficiency of 86 %. Subsequently, 98 %, i.e. 872 CTCs, could be deposited in PCR tubes or on slides as single cells. For immunocytochemical characterization of CTCs for the protein CapG, which is associated with cell invasiveness, 167 CTCs were isolated from 16 cartridges of the CellSearch system as single cells on slides and subsequently stained for the marker CapG. CapG expression was detected in 143 CTCs, 86 % of the total CTCs, by antibody staining. 24 CTCs showed no immunocytochemically detectable expression of CapG. To prove the good DNA-quality of the single cell samples for a possible genomic analysis of the CTCs, 95 CTCs were deposited into PCR tubes from 32 cartridges of the CellSearch- system and a whole genome amplification was performed. In the quality control of Ampli1-WGA-kit (silicon biosystems), 24 % of WGA-products had good DNA-quality with > 3 bands and 76 % had < 3 bands. The developed technology for single cell isolation of CTCs in the combination of the CellSearch® System, VyCap- and ISET-filter and the CellCelector™ -System provides a cornerstone for improving the characterization of EpCAM-positive and -negative CTCs from blood samples of breast cancer patients.
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Fachbereich / Einrichtung:	Medizinische Fakultät
Dokument erstellt am:	30.05.2023
Dateien geändert am:	30.05.2023
Promotionsantrag am:	06.01.2023
Datum der Promotion:	11.05.2023