University of Twente Student Theses


Identification of circulating tumour cell subpopulations

Deelstra, F. (2017) Identification of circulating tumour cell subpopulations.

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Abstract:Circulating tumour cells (CTC) can be found in blood of cancer patients. A high number of CTC in blood indicates a poor prognosis. These CTC can have an epithelial like, more adhesive phenotype, can be more motile, like cells with a mesenchymal phenotype, or have a hybrid state. The state of cells depends on the degree of epithelial-to-mesenchymal transition (EMT) they underwent. Cells express different markers, depending on their EMT state. The CellSearch system isolates CTC based on EpCAM expression, which are subsequently identified by the expression of cytokeratin. Both of these markers are indicative of an epithelial phenotype and are not expressed in mesenchymal cells. Cells that do not express these markers are either not isolated or not detected by the CellSearch system. The goal of this project is to identify markers that can be used next to EpCAM and cytokeratin for the identification of CTC subpopulations. Therefore, immunostaining of PSMA, E-cadherin, N-cadherin, cadherin-11, SLUG and EGFR was tested on cancer cell lines and blood cells. For low abundance proteins, the amplification methods proximity ligation assay (PLA) and tyramide signal amplification (TSA) were developed and applied. To determine the cancerous origin of EpCAM negative cancer cells in filtered blood of the CellSearch system, microRNA-21 detection with fluorescent in situ hybridisation and TSA was investigated. After optimisation of the staining protocol, cancer cells could be discriminated from white blood cells with 2 μL anti-PSMA per 100 μL cell suspension, 2 μg/mL E-cadherin or 2 μg/mL cadherin-11 staining. Even though the staining protocol for N-cadherin was optimised to 2 μg/mL it could only be detected in cell-cell contact areas of adhering cells. Spiked tumour cells and white blood cells could not be discriminated based on used anti-SLUG and anti-EGFR staining. With PLA and TSA low abundance proteins could be detected. Compared to TSA PLA showed better spatial resolution, enabling single molecule quantification. MicroRNA-21 expression could neither be detected in cells nor when hybridising the anti-microRNA-21 probe against the amplified microRNA-21 gene. It is concluded that suitable markers for identification of CTC are epithelial marker E-cadherin, mesenchymal marker cadherin-11 and PSMA which is expressed by CTC in different phenotypic states and that low abundance proteins are detected with signal amplification. Keywords: CTC, EMT, EpCAM, MET, microRNA-21, PLA, TSA, tumour markers
Item Type:Essay (Master)
Faculty:TNW: Science and Technology
Subject:42 biology, 44 medicine
Programme:Biomedical Engineering MSc (66226)
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