Magnetbasierte Anreicherung von zirkulierenden epithelialen tumorverdächtigen Zellen und deren Genotypisierung

Walther, Katharina

Introduction: Cancer is one of the main causes of death and 90% of patients die from distant metastases. The circulating epithelial tumor cells (CETCs) are able to form metastases. The separation of CETCs from healthy leukocytes and their characterization is a major challenge. Methods: Whole peripheral blood samples from 390 breast cancer patients were analyzed. Peripheral blood leukocytes were prepared by erythrocyte lysis. The isolated cells were incubated with carboxymethyldextran-coated magnetic nanoparticles (CMD-NP) and separated by using a SuperMACS Separator (Miltenyi-Biotech, Germany). The magnetic labeled cells were analyzed for EpCAM-positive cells by fluorescence microscopy. In addition, the separation of rare tumor cells from leukocytes allows further molecular study. The evaluation of the mutational status of single cell was established on human cell culture and on patient samples. Results: Using magnetic CMD-NP we can enrich CETCs selectively. A median of 80±20% was collected from the entire CETCs in the enriched fraction. We observed a difference in the enrichment efficiency of CETCs with regard to the individual therapy status of the patient (patients in follow-up care: 83±17%, n=65; patients undergoing chemotherapy: 54±24%, n=39). Apart from increasing the sensitivity the enriched cells can be characterized by specific genomic analysis. That the magnetically enriched cells are actual tumor cells was demonstrated in a colorectal carcinoma patient. One of the isolated single cells from the NP-enriched fraction showed a genetic modification in the KRAS gene which was also present in the corresponding tumor. Conclusion: Molecular characterization of tumor cells on single cell level is feasible and a selective enrichment of CETCs by CMD-NP is an effective enrichment method. This noninvasive method is may be a tool to effectively eliminate CETCs from the periphery and prerequisite to evaluate the mutation status of rare CETCs from cancer patients.


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