Hypoxia-inducible factor-1 alpha and vascular endothelial growth factor expression in circulating tumor cells of breast cancer patients

Introduction The detection of peripheral blood circulating tumor cells (CTCs) and bone marrow disseminated tumor cells (DTCs) in breast cancer patients is associated with a high incidence of disease relapse and disease-related death. Since hypoxia-inducible factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) play an important role in angiogenesis and tumor progression, the purpose of the current study was to investigate their expression in CTCs. Methods The expression of cytokeratins (CK), VEGF, vascular endothelial growth factor receptor-2 (VEGF2), HIF-1 alpha and phosphorylated-focal adhesion kinase (pFAK) in CTCs from 34 patients with metastatic breast cancer who had detectable CK-19 mRNA-positive CTCs was assessed using double staining experiments and confocal laser scanning microscopy. Peripheral blood mononuclear cells (PBMCs) were stained with a monoclonal A45-B/B3 pancytokeratin antibody in combination with either VEGF or VEGFR2 or HIF-1 alpha or pFAK antibodies, respectively. Results pFAK expression in circulating tumor cells was detected in 92% of patients whereas expression of VEGF, VEGF2 and HIF-1 alpha was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGF2, HIF-1 alpha and pFAK were expressed in 73%, 71%, 56% and 81%, respectively, of all the detected CTCs. Vascular endothelial growth mRNA was also detected by quantitative real-time RT-PCR in immunomagnetically-separated CTCs. Double and triple staining experiments in cytospins of immunomagnetically-isolated CTCs showed that VEGF co-expressed with HIF-1 alpha and VEGF2. Conclusions The expression of pFAK, HIF-1 alpha, VEGF and VEGF2 in CTCs of patients with metastatic breast cancer could explain the metastatic potential of these cells and may provide a therapeutic target for their elimination.