Dissertation Janis Stiefel
Automated Isolation of Circulating Tumor Cells and their Molecular Biological Analysis
We warmly congratulate Janis Stiefel on the successful defense of his dissertation in applied research in liquid biopsy. Stiefel's thesis deals with detecting tumor features based on the automated isolation of circulating tumor cells from liquid biopsies and thus contributes significantly to establishing a platform technology of Fraunhofer IMM in applied research of liquid biopsy.
Janis Stiefel is a research associate in the Diagnostics Division at Fraunhofer IMM. The defense of his dissertation on "Automated Isolation of Circulating Tumor Cells and their Molecular Biological Analysis" took place on January 30, 2023 at the Johannes Gutenberg University Mainz.
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, biofunctionaliza-tion 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) demon-strating up to 87 Percent enrichment, 73 percent optical detection and dispensing efficiency. 40 to 56.7 percent 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. Addition-ally, 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.