Specific Detection of Circulating Tumor Cells in Peripheral Blood by Targeting Gastrin Releasing Peptide Receptors

Objectives The gastrin releasing peptide receptor (GRPR) is an emerging target for imaging and therapeutic applications for prostate and breast cancer. However, the use of GRPR for detecting circulating tumor cells remains under-explored. The present study focuses on the specific labeling of prostate cancer cells with a fluorescent bombesin peptide (Fluor-BBN) and the quantitation of prostate cancer cells in blood by flow cytometry from peripheral blood white blood cells.

Methods Fluor-BBN was synthetized, purified and characterized according to the published procedure (Cai et al, Prostate, 2013 73(8):842-54). Prostate cancer PC-3 cells and mouse white blood cells (WBCs) were incubated with the Fluor-BBN with varies time and concentration to optimize labeling condition. Detection and quantitation of the PC-3 cells in the white blood cells were performed by flow cytometry and fluorescence microscope. A gating protocol based on Fluor-BBN labeling of GRPR and autofluorescence on PC-3 cells was developed for quantitation of the PC-3 cells in the presence of white blood cells. Nonfluorescent beads were added to the labeled cell mixture and served as an internal standard for precise cellular quantification of the PC-3 cells by flow cytometry.

Results The binding of Fluor-BBN is highly specific to PC-3 cells with no binding observed to WBCs. The minimum time and concentration of Fluor-BBN for effective labeling of the PC-3 cells in WBCs was 30 min and 50 nM at 37 ºC. Pre-incubation of PC-3 cells with unlabeled BBN resulted in a complete inhibition of Flour-BBN labeling, confirming that the Flour-BBN labeling was specific to GRPR. In blood samples in which PC3 cells were added, an average of 4.6 cells was detected in the most dilute sample examined (10 cells / ml); 3.9 cells were expected theoretically.

Conclusions The results suggest that the GRPR is a potentially effective marker to detect and quantify prostate cancer cells in blood in vitro. These results support the use of Fluor-BBN compounds in the application of fluorescence flow cytometry or fluorescence microscope to detect, quantify, and characterize circulating tumor cells. Research Support: DoD prostate cancer research program (LM).