Detecting CXCR4 in Pancreatic NET Cells

In order to develop the assay for CXCR4 detection using the CellSearch platform, the pancreatic NET (pNET) cell line BON1 was used. This has previously been reported to express both EpCAM and CXCR4.^{12 (link)} Five hundred BON1 cells were spiked into 7.5 mL of healthy donor blood collected in CellSave preservative tubes and processed through the CellSearch platform. A fluorescein-conjugated antibody can be added for detection by the fourth fluorescence channel to further characterise the cells for an additional marker of interest. For this study, the AF488-conjugated anti-CXCR4 antibody [UMB2, Abcam, Ab208128] was used and cells were defined as positive for CXCR4 expression when staining was present in the fourth channel (Fig. 1a, b). Patient samples were analysed using the protocol optimised on cells (antibody concentration 1:10 and exposure time 5 s), and evaluations regarding the expression of CXCR4 on CTCs were made by two independent operators having no knowledge of the clinical status of the patients.Fig. 1

CellTracks Analyzer II example images. a Spiked BON1 cells, with no CXCR4 antibody used. b BON1-spiked blood samples run with CXCR4 antibody. i CXCR4-positive BON1 cell. ii CXCR4-negative BON1 cell. c Clinical validation in patient samples assayed with CXCR4 antibody. i CXCR4-positive CTC in patient ID1. ii CXCR4-negative CTC in patient ID1