Abstract
Background: detailed information about circulating tumor cells (CTCs) as an indicator of therapy response and cancer metastasis is crucial not only for basic research but also for diagnostics and therapeutic approaches. Here, we showcase a newly developed IsoMAG IMS system with an optimized protocol for fully automated immunomagnetic enrichment of CTCs, also revealing rare CTC subpopulations. Methods: using different squamous cell carcinoma cell lines, we developed an isolation protocol exploiting highly efficient EpCAM-targeting magnetic beads for automated CTC enrichment by the IsoMAG IMS system. By FACS analysis, we analyzed white blood contamination usually preventing further downstream analysis of enriched cells. Results: 1 µm magnetic beads with tosyl-activated hydrophobic surface properties were found to be optimal for automated CTC enrichment. More than 86.5% and 95% of spiked cancer cells were recovered from both cell culture media or human blood employing our developed protocol. In addition, contamination with white blood cells was minimized to about 1200 cells starting from 7.5 mL blood. Finally, we showed that the system is applicable for HNSCC patient samples and characterized isolated CTCs by immunostaining using a panel of tumor markers. Conclusion: Herein, we demonstrate that the IsoMAG system allows the detection and isolation of CTCs from HNSCC patient blood for disease monitoring in a fully-automated process with a significant leukocyte count reduction. Future developments seek to integrate the IsoMAG IMS system into an automated microfluidic-based isolation workflow to further facilitate single CTC detection also in clinical routine.
Highlights
The SCL-1 squamous cell carcinoma line was established by Dr Petra Boukamp (DKFZ, Heidelberg, DE, Germany) [33] and kept in GibcoTM DMEM medium supplemented with 10% FCS
HNSCCUM-02T squamous cell carcinoma cell line was established by Welkoborsky et al (UMC Mainz, DE, Germany) [34], and kept in DMEM:F-12 medium supplemented with 10% FCS and 1% L-glutamine
epithelial cell adhesion molecule (EpCAM)-biotin antibodies were immobilized on the streptavidin-biofunctionalized beads
Summary
Cancer is the second major cause of death in modern society. The development of innovative diagnostics is indispensible for long-term improvement of diagnosis and treatment of cancer patients. The lymph-/hematogenous spread of cancer cells into distant organs and their subsequent growth to overt metastases is the most fatal complication of solid tumors, such as squamous cell carcinoma of the head and neck (HNSCC) [1,2]. Therapy resistance and associated relapses are common and associated with high patient morbidity [3,4]. The classical view is that metastatic spread is a late process in malignant progression, but today it is accepted that
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