Expression analysis of the macrophage-capping protein (CapG) in single circulating tumor cells (CTCs) isolated from blood samples of breast cancer patients

Objective: Detection of CTCs by the CellSearch system is based on immunomagnetic

enrichment of EpCAM+ cells. Supplementary tools are necessary to isolate and

characterize also EpCAM negative CTCs. CapG was shown to be involved in cancer

cell migration and invasiveness of breast cancer (BC). Aim of this work was to

characterize CapG expression in CTCs of BC patients.

Material and methods: CTCs were enriched and detected in blood samples using the CellSearch system.

EpCAM+ cells were isolated from CellSearch cartridges by the CellCelector system

(ALS, Jena, Germany), an automated scanning microscope and micromanipulator

device. EpCAM- CTCs were isolated from the “waste” of CellSearch System by size

selection. Cytokeratin (CK) positive CTCs were isolated from filters with the

CellCelector and characterized performing immunocytochemistry.

Results: Detection and isolation of single EPCAM+ as well as EPCAM- cells was successfully established using blood samples spiked with low cell numbers of known BC cell lines displaying different EPCAM expression. CellSelector re-analysis of CellSearch cartridges revealed comparable CTC detection rates. EPCAM-/CK+ cells were successfully recovered from CellSearch “waste” samples by size selection and isolated from filter membranes by micromanipulation using CellSelector. CapG

expression of CTCs was detectable performing immuno-cytochemistry directly on

filter membranes or after transfer of CTCs on glass slides. Comparable results were

obtained with CTCs isolated from BC patients.

Conclusion: The established workflow is a reliable method for the detection and isolation of EPCAM+ and/or EPCAM- CTCs. This allows CapG expression analysis of single CTCs in different EPCAM+ and EPCAM- CTC populations.