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

Abstract

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-1alpha (HIF-1alpha) 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-1alpha 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-1alpha or pFAK antibodies, respectively.

Results: pFAK expression in circulating tumor cells was detected in 92% of patients whereas expression of VEGF, VEGF2 and HIF-1alpha was observed in 62%, 47% and 76% of patients, respectively. VEGF, VEGF2, HIF-1alpha 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-1alpha and VEGF2.

Conclusions: The expression of pFAK, HIF-1alpha, 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.

Figure 1

Vascular endothelial growth factor expression in breast cancer cell lines and in circulating tumor cells. A: VEGF, VEGFR2 HIF-1α and pFAK expression in cultured breast cancer cell lines, 1: MCF7, 2:T47D, 3: SKBR3 4:MDA MB-453, 5: MDA MB-231 (western blot analysis of equal protein amount of cell lysates). B (I): Representative images of confocal laser scanning microscopy of PBMCs cytospins stained with pancytokeratins A45 B/B3 (green), and VEGF anti-goat (red) antibodies. B (II): Representative images of PBMCs cytospins stained with pancytokeratins A45 B/B3 (green), VEGF anti-goat (red) antibodies and Dapi (blue). B (III): Quantification of VEGF and CK co-expression in CTCs of 34 metastatic breast cancer patients. All the examined patients (100%) presented CTCs in their blood, while 62% also presented double positive cells (VEGF⁺CK⁺). (C) Quantification of double positive CTCs/total CTCs for each examined molecule in 34 metastatic breast cancer patients. For pFAK testing cytospins from 12 patients were evaluated. indicatively. Seventy-three percent of the examined CTCs were VEGF⁺CK⁺ positive, 71% were VEGFR2⁺CK⁺, 56% were HIF-1α⁺CK⁺ and 81% were pFAK⁺CK⁺.

Figure 2

Vascular endothelial growth factor receptor 2 and hypoxia-inducible factor-1α in circulating tumor cells of patients with breast cancer. A (I): Representative images of cytospin double stained with monoclonal pancytokeratin (A45-B/B3) (green) and polyclonal VEGFR2 anti-rabbit (red) antibodies. A (II): Representative images of PBMCs cytospins stained with pancytokeratins A45 B/B3 (green), VEGFR2 anti-rabbit (red) antibodies and Dapi (blue). A (III): Quantification of VEGFR2 and CK co-expression in CTCs of 34 patients with metastatic breast cancer. CTCs were detected in all (100%) patients while in 47% double positive cells (VEGFR2⁺CK⁺) were identified. B (I): Representative image of a cytospin double stained with monoclonal pancytokeratin (A45-B/B3) (green) and polyclonal HIF-1α (red) antibodies. B (II): Representative images of PBMCs cytospins stained with pancytokeratins A45 B/B3 (green), HIF-1α anti-rabbit (red) antibodies and Dapi (blue). B (III): Quantification of HIF-1α and CK co-expression in CTCs of 34 patients with metastatic breast cancer. All the examined patients (100%) harvested CTCs in their blood, while 76% also presented double positive cells (HIF-1α⁺CK⁺).

Figure 3

Phosphorylated-focal adhesion kinase expression in circulating tumor cells and co-expression of vascular endothelial growth factor/vascular endothelial growth factor receptor or vascular endothelial growth factor/hypoxia-inducible factor-1α in circulating tumor cells of patients with breast cancer. A (I): Representative image of a cytospin double stained with polyclonal pancytokeratin (red) and (monoclonal) pFAK (green) antibodies. A (II): Quantification of pFAK and CK co-expression in CTCs of 12 patients with metastatic disease. All the examined patients (100%) presented CTCs in their blood, while 92% also presented double positive cells (pFAK⁺CK⁺). B: Representative confocal laser scanning micrographs of CTCs cytospins after negative immunomagnetic separation in 10 patients with metastatic breast cancer. Cells cytospins were double stained with VEGF and either (I) VEGFR2 or (II) HIF-1α antibodies, respectively. C: Representative image of a cytospin, triple stained with monoclonal pancytokeratin (A45-B/B3) (green) and VEGF antibodies (blue) along with either (I) VEGFR2 or (II) HIF-1α (B) (red) polyclonal antibodies (red).

Figure 4

Vascular endothelial growth factor, vascular endothelial growth factor receptor 2 and hypoxia-inducible factor-1α expression in MCF7, Human umbilical vein endothelial cells and MDA-MB-231 respectively. A: MCF7 cells stained with pancytokeratin/FITC (green) and VEGF/Alexa555 (red) or MCF7 cells stained with pancytokeratin/FITC (green) and just Alexa555 antibody without VEGF antibody (negative control). B: HUVEC cells stained with pancytokeratin/FITC (green) and HIF1-α/Alexa555 (red) or HUVEC cells stained with pancytokeratin/FITC (green) and just Alexa555 antibody without HIF1-α antibody (negative control). C: MDA-MB-231 cells stained with pancytokeratin/FITC (green) and VEGFR2/Alexa555 (red) or MDA-MB-231 cells stained with pancytokeratin/FITC (green) and just Alexa555 antibody without VEGFR2 antibody (negative control).