Single Cell Analysis of tumor cells using reverse transcriptase multiplex ligation-dependent probe amplification
Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Circulating tumor cells (CTCs) are cells that have broken away from a solid tumor and entered the blood stream. Some of these cells can have the potentiao to exit the blood stream at another site and form metastes. The number of CTCs has been shown to be a prognostic marker for both metastatic breast cancer as well as metastatic prostate cancer and metastatic colon cancer. The concentration of CRCs in peripheral blood is however usually low, down to less than one CTC per ml blood. This makes them challenging to detect. Several techniques have been developed for the isolation and detection of CTCs, but the only FDA approved method (CellSearch from Veridex) focuses on enumeration of CTCs, although much more information relevant to prognosis and treatment decisions could be obtained from molecular analysis of the CTCs.
This project aims at detectin and molecularly characterizing single tumor cells using reverse transcriptiase multiplexed ligation-dependent probe amplification (RT-MLPA). RT-MLPA is a PCR-based method where gene specific primers are used to convert transcripts into cDNA flowwoed by gene specific hybridization and ligation of DNA probes. The DNA probes are then amplified using only one primer pair. In this report results supporting the single cell sensitivity is presented, along with work to achieve a more reliable and robust gene expression profile for a seven markers panel perfomred on three different breast cancer cell lines. The tumor cell specificity in contamination mononuclear cell material of this panel is also tested. Finally a proof of concept using fixated input material in the form of whole blood spiked with tumor cells is used for an immunomagnetic isolation targeting EpCAM followed by an RT-MLPA.
Remaining work to be done on the seven markers breast cancer panel for RT-MLPA mainly include a redesign of the reverse transcirption step in order to achiee a more reliable gee expresseion profile before the method can be tested in real purified CTCs.
Place, publisher, year, edition, pages
single cell, tumor, mlpa, amplification
Engineering and Technology
IdentifiersURN: urn:nbn:se:kth:diva-173521OAI: oai:DiVA.org:kth-173521DiVA: diva2:853465