Circulating endothelial cells and angiogenic serum factors during neoadjuvant chemotherapy of primary breast cancer

Abstract

Circulating endothelial cells (CECs) as well as bone-marrow-derived endothelial precursor cells (EPC) play an important role in neovascularisation and tumour growth. To study the impact of neoadjuvant chemotherapy on the amounts of CEC and their precursor cells, mature CEC and their progenitors were quantified by flow cytometry in peripheral blood of breast cancer patients during anthracycline and/or taxane based neoadjuvant chemotherapy and subsequent surgery in comparison to age-matched healthy controls. Cell numbers were tested for correlation with serum levels of angiopoietin-2, erythropoietin, endostatin, endoglin, VEGF and sVCAM-1 as well as clinical and pathological features of breast cancer disease. Circulating endothelial cells were significantly elevated in breast cancer patients and decreased during chemotherapy, whereas EPC (CD34+/VEGFR-2+) as well as their progenitor cell population CD133+/CD34+ and the population of CD34+ stem cells increased. Concomitantly with the increase of progenitor cells an increase of VEGF, erythropoietin and angiopoietin-2 was observed. These data suggest that chemotherapy can only reduce the amounts of mature CEC, probably reflecting detached cells from tumour vessels, whereas the EPC and their progenitors are mobilised by chemotherapy. Since this mobilisation of EPC may contribute to tumour neovascularisation an early antiangiogenic therapy in combination with chemotherapy could be beneficial for the success of cancer therapy.

Figure 1

Flow cytometric quantification of CEC. The amounts of CEC were quantified in peripheral blood samples of breast cancer patients and matched controls by four colour flow cytometry analysis. (A) Comparison of CEC amounts in blood samples from patients (group A) at the time of diagnosis and matched controls. ^**P<0.01 vs matched controls. (B) CEC levels during the course of neoadjuvant chemotherapy and subsequent surgery. ^*P<0.05, ^**P<0.01 vs values at time of diagnosis. (C) Comparison of CEC amounts in blood samples from breast cancer patients (Groups A+B) with oestrogen receptor negative and oestrogen receptor positive tumours at the time of diagnosis. ^*P<0.05 vs values of oestrogen receptor negative patients. (D) Comparison of CEC amounts in blood samples from breast cancer patients (Groups A+B) with progesterone receptor negative and progesterone receptor positive tumours at the time of diagnosis. P=0.067 vs values of progesterone receptor negative patients.

Figure 2

Flow cytometric quantification of CD34⁺ and CD34⁺/CD133⁺ progenitor cells. The amounts of CD34⁺ and CD34⁺/CD133⁺ progenitor cells were quantified in peripheral blood samples of breast cancer patients and matched controls by four colour flow cytometry analysis. (A) Comparison of the amounts of CD34⁺ and CD34⁺/CD133⁺ progenitor cells in blood samples from patients (Group A) at the time of diagnosis and matched controls. P=0.094 vs values of matched controls. (B) CD34⁺ levels during the course of neoadjuvant chemotherapy and subsequent surgery. ^*P<0.05 vs values at time of diagnosis. (C) CD34⁺/CD133⁺ levels during the course of neoadjuvant chemotherapy and subsequent surgery. ^*P<0.05 vs values at time of diagnosis. (D) Comparison of CD34⁺ amounts in blood samples from breast cancer patients (Groups A+B) with different clinical tumour stages at the time of diagnosis. ^*P<0.05 vs values of patients with clinical tumour stage 1. (E) Comparison of CD34⁺/CD133⁺ amounts in blood samples from breast cancer patients (Groups A+B) with different clinical tumour stages at the time of diagnosis.

Figure 3

Flow cytometric quantification of EPC (CD34⁺/VEGFR-2⁺). The amounts of EPC were quantified in peripheral blood samples of breast cancer patients and matched controls by four colour flow cytometry analysis. (A) Comparison of the amounts of EPC in blood samples from patients at the time of diagnosis and matched controls. P=0.09 vs values of matched controls. (B) EPC levels during the course of neoadjuvant chemotherapy and subsequent surgery. ^*P<0.05 vs values of matched controls.