Authors:	Stiefel, Janis Hendrik
Title:	Automated isolation of circulating tumor cells and their molecular biological analysis
Online publication date:	16-Feb-2023
Year of first publication:	2023
Language:	english
Abstract:	Cancer metastases descend from proliferative circulating tumor cells (CTCs) which disseminate from the primary tumor via blood and lymph vessels. In the last two decades, CTC analysis from blood samples was established for diagnostic and prognostic purposes and therapy monitoring in the course of liquid biopsy. Nonetheless the small number of CTCs compared to billions of blood cells in a patient sample requires sensitive methods to obtain reproducible tumor cell recovery rates. Against this background, the Fraunhofer Institute for Microengineering and Microsystems has recognized the unmet need of standardized CTC isolation and developed automated microfluidic platforms to enrich, detect and isolate single tumor cells from 7.5 mL whole blood. The aim of this thesis was to realize single cell isolation by means of automated immunomagnetic bead enrichment and antibody-based fluorescence detection in a chip-based microfluidic system and the molecular biological characterization of these cells to assay tumor features from liquid biopsy. Therefore, this work compares manual and automated immunomagnetic enrichment in terms of cell recovery in dependency of cell line and input number, bead characteristics, biofunctionalization protocol and blood cell contamination focusing on head and neck squamous cell carcinoma (HNSCC) and metastatic breast cancer. In a prototype enrichment device (IsoMAG), we established an isolation protocol with EpCAM-targeting 1 μm magnetic beads for automated enrichment tested on different epithelial cancer cell lines. White blood cell contamination, usually hampering further downstream analysis of enriched tumor cells, was measured by FACS and minimized to about 1200 cells starting from 7.5 mL blood. In the microfluidic benchtop platform CTCelect, we characterized each process step with two different carcinoma cell lines (MCF-7, SCL-1) demonstrating up to 87 % enrichment, 73 % optical detection and dispensing efficiency. 40 to 56.7 % of cells were recovered after complete isolation from 7.5 mL untreated whole blood. We highlighted the bottlenecks of microfluidic cell isolation in dependency of bead-binding on the cell surface and implemented a gating function for the cytometer subunit to selectively dispense cells instead of autofluorescent objects. In doing so, CTCelect enabled automated dispensing of single circulating tumor cells from HNSCC patient samples, qPCR-based confirmation of tumor-related biomarkers with a simplified one-step protocol and immunostaining using a panel of tumor markers. Additionally, the platform was compared to commercial CTC isolation technologies to highlight advantages and limitations of CTCelect. In conclusion, the achieved findings substantially contribute to the establishment of the platform technology in the applied research field of liquid biopsy.
DDC:	570 Biowissenschaften 570 Life sciences 600 Technik 600 Technology (Applied sciences) 610 Medizin 610 Medical sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	FB 10 Biologie Externe Einrichtungen
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-8764
URN:	urn:nbn:de:hebis:77-openscience-514976bb-09f0-4fbc-880b-0734a848ed404
Version:	Original work
Publication type:	Dissertation
License:	CC BY
Information on rights of use:	https://creativecommons.org/licenses/by/4.0/
Extent:	VII, 122 Seiten ; Illustrationen, Diagramme
Appears in collections:	JGU-Publikationen