Circulating endothelial progenitor cells in gynaecological cancer

Abstract

Objectives

To compare the frequency and absolute numbers of circulating endothelial progenitor cells (EPCs) in healthy control subjects and patients with gynaecological cancer, and to test the hypothesis that cancer treatment lowers EPC numbers.

Methods

Patients with cervical or ovarian cancer and healthy control subjects provided peripheral blood samples for the isolation of mononuclear cells. EPCs were identified by quadruple immunofluorescence staining and flow cytometry as CD45^–/CD34⁺/CD133⁺/vascular endothelial growth factor receptor 2 (VEGFR2)⁺ cells.

Results

In total, 28 participants were enrolled. Circulating EPCs were present at higher frequencies (and in greater absolute numbers) in patients with cervical or ovarian cancer (n = 14) than in controls (n = 14). Concurrent chemoradiation therapy or surgery significantly reduced the frequency and number of EPCs in patients with gynaecological cancer, compared with pretreatment levels.

Conclusions

EPC levels decline throughout cancer treatment; their measurement may therefore be a useful surrogate marker to monitor treatment response.

Introduction

Tumour growth and metastasis are closely related to abnormal angiogenesis and neovascularization. The mechanisms of tumour angiogenesis are unclear, but studies have shown that circulating endothelial progenitor cells (EPCs) play an important role in this process.^1,2

Circulating EPCs have been suggested as potential surrogate markers of angiogenesis in cancer and in other vascular diseases. ³ Increases in circulating EPC levels might reflect the abnormally high turnover of tumour endothelium in invasive cancer and the disordered nature of angiogenesis, and may relate to tumour vascular volume. Tumours encourage the mobilization of circulating progenitor cells from the bone marrow, which may be important in tumour vascularity.^1,2,4,5

Circulating EPCs can be detected and quantified in peripheral blood samples by flow cytometric analysis, using cell surface markers of hematopoietic stem cells including CD34, CD133 and vascular endothelial growth factor receptor 2 (VEGFR2). ⁶ The numbers of circulating EPCs have been shown to be increased in malignant diseases such as breast cancer, ovarian cancer, nonsmall cell lung cancer and lymphoma.^7–12

The kinetics of circulating EPCs and endothelial cells in response to antiangiogenic treatment have been studied in animal models. ² There is evidence that metronomic chemotherapy and inhibitors of the VEGFR signalling pathway can suppress circulating EPC numbers.^13–18 In addition, there may be a correlation between circulating EPC numbers and disease status or treatment. ¹⁹

The aim of the current study was to compare circulating EPC numbers and frequencies in healthy individuals with those in patients with gynaecological cancer, in order to test the hypothesis that cancer treatment (such as tumour debulking surgery or concurrent chemoradiation therapy [CCRT]) may reduce circulating EPC numbers.

Patients and methods

Study population

The study recruited patients first diagnosed with cervical or ovarian cancer between December 2011 and April 2012, who underwent treatment at the Department of Obstetrics and Gynaecology, Korea University Anam Hospital, Seoul, Republic of Korea. Tumours were classified according to the 1987 staging criteria of the International Federation of Gynecology and Obstetrics (FIGO). ²⁰ Healthy women with no previous cancer diagnosis (who were attending the Department of Obstetrics and Gynaecology, Korea University Anam Hospital for routine screening) were recruited as control subjects. No attempt was made to age match the study groups.

Written informed consent was obtained from all participants prior to blood collection. The study was approved by the Institutional Review Board, Korea University Anam Hospital, Seoul, Republic of Korea.

Blood collection

All study participants provided peripheral blood samples. Patients with cervical cancer who were undergoing CCRT provided blood samples before treatment and after every third cycle of chemotherapy (40 mg/m² cisplatin, once per week). Patients with cervical cancer who underwent surgery instead of CCRT, and those with ovarian cancer (all of whom underwent surgery only) provided blood samples before and after surgery. Control subjects provided blood samples on enrolment.

PBMC isolation

Peripheral blood mononuclear cells (PBMCs) were isolated from 15–20-ml whole blood samples, which had been collected in ethylenediaminetetra-acetic acid-coated sterile tubes. Blood was mixed with an equal volume of phosphate buffered saline (PBS, pH, 7.4), layered on Ficoll® reagent (1077 g/ml) and centrifuged at 400  g for 20 min at 15–20°C. The buffy coat containing mononuclear cells was recovered, mixed with ≤ 50 ml PBS and centrifuged at 300  g for 10 min at 15–20°C. The supernatant was discarded and the cell pellet was resuspended in 1 ml PBS. A further 19 ml of PBS was added and samples were centrifuged at 300  g for 10 min at 15–20°C. Viable cells were counted by trypan blue staining.

Flow cytometry

Endothelial progenitor cells were identified by quadruple immunofluorescence staining and flow cytometry. In brief, 1 × 10⁶ PBMCs (in 200 µl) were incubated for 30 min at 4°C with 2 μl each of fluorescein isothiocyanate-conjugated mouse antihuman CD45, phycoerythrin-conjugated mouse antihuman VEGFR2, peridinin chloropyll protein-conjugated mouse antihuman CD34, and allophycocyanin-conjugated mouse antihuman CD133 (all from BD Biosciences, Heidelberg, Germany). Cells were washed three times in BD FACS^TM Buffer (BD Biosciences) at 4°C (5 min each wash) then fixed in 1% paraformaldehyde. Parallel cell samples were stained with 1 μl propidium iodide for 5 min to validate the live cell population. A FACSCaliber™ flow cytometer (BD Biosciences) was used to acquire ≥ 2 × 10⁵ region-one events, and data were analysed with WinMDI software, version 2.9 (Scripps Research Institute, San Diego, CA, USA). A representative flow cytometric gating strategy for the identification of EPCs is shown in Figure 1. EPCs were identified as CD45^–/CD34⁺/CD133⁺/VEGFR2⁺ cells.

Figure 1.

Representative flow cytometric plots indicating the strategy for identification of circulating endothelial progenitor cells (EPCs). EPCs were defined as CD45^–/CD34⁺/CD133⁺/vascular endothelial growth factor receptor 2 (VEGFR2)⁺ cells. Flow cytometry was performed using sequential gatings: (A) forward (FSC) and side scatter (SSC) plot of cells and gating region G1 to include all mononuclear cells while excluding cell debris; (B) CD45^– gating to exclude lymphocytes; (C) Region R7 comprising CD45^–/CD34⁺ cells; (D) Double-positive CD133⁺/VEGFR2⁺ gating identifies ECPs in region R8.

Statistical analyses

Data were expressed as mean ± SD; between-group comparisons were made using Student’s t-test. Statistical analyses were performed with SPSS® software, version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. A P-value < 0.05 was considered statistically significant.

Results

The study included six patients with cervical cancer (CCRT n = 4; surgery n = 2), eight patients with ovarian cancer, and 14 healthy control subjects. Demographic and clinical characteristics of patients are shown in Table 1.

Table 1.

Demographic and clinical characteristics of patients with cervical or ovarian cancer and healthy control subjects, included in a study to examine the effect of cancer and cancer treatment on frequency and numbers of circulating endothelial progenitor cell.

Characteristic	Cervical cancer group	Ovarian cancer group	Control group
	n = 6	n = 8	n = 14
Age, years	58.5 ± 8.85	54.0 ± 19.55	26 ± 5.73
Parity	2.7 ± 0.82	2.3 ± 1.49	0 (0)
Cancer type
SCC	5 (83.3)	–	–
Adenocarcinoma	1 (16.7)	–	–
Serous carcinoma	–	7 (87.5)	–
Mucinous carcinoma	–	1 (12.5)	–
FIGO stage 20
IB	2 (33.3)	0	–
IIA	1 (16.7)	0	–
IIB	3 (50.0)	0	–
IIIC	0	8 (100)	–
Treatment
CCRT	4 (66.7)	0	–
Surgery	2 (33.3)	8 (100)	–

Data presented as mean ± SD or n (%) of patients.

SCC, squamous cell carcinoma; FIGO, International Federation of Gynecology and Obstetrics; CCRT, concurrent chemoradiation therapy.

The frequency (but not the numbers) of circulating EPCs was significantly lower in control subjects than in patients with cervical cancer (P = 0.004, Table 2). CCRT (three and six cycles) resulted in a significant decrease in EPC frequency from baseline (baseline vs three cycles P = 0.009, baseline vs six cycles P = 0.016, three cycles vs six cycles P = 0.025; Table 2). Surgical removal of cancer tissue significantly reduced the frequency of circulating EPCs compared with preoperative values (P = 0.044; Table 2). There were no significant differences in EPC numbers associated with any cervical cancer treatment.

Table 2.

Frequency and numbers of circulating endothelial progenitor cells (EPCs) in patients with cervical cancer before and after concurrent chemoradiation therapy (CCRT) or surgical treatment, and in healthy control subject.

EPCs	Control group	Cervical cancer group	CCRT group			Surgery group
			n = 4			n = 2
	n = 14	n = 6
			Baseline	3 cycles	6 cycles	Preoperative	Postoperative
Frequency, %	0.002 ± 0.002	0.032 ± 0.014 a	0.034 ± 0.014	0.011 ± 0.005b	0.005 ± 0.004c,d	0.0157 ± 0.0003	0.005 ± 0.001e
Cells/ml blood	21 ± 22	543 ± 773	634 ± 976	422 ± 660	85 ± 41	361 ± 174	56 ± 23

Data presented as mean ± SD.

a

P < 0.01 versus control group, ^bP < 0.01 versus CCRT baseline, ^cP < 0.05 versus CCRT baseline, ^dP < 0.05 versus 3 cycles of CCRT, ^eP < 0.05 versus preoperative data; Student’s t-test.

Patients with ovarian cancer were found to have significantly higher EPC numbers and higher frequencies of EPCs than control subjects (P = 0.000035 for frequency, P = 0.01 for cell numbers; Table 3). Surgery significantly lowered both cell numbers and frequency, compared with preoperative values (P = 0.0001 for frequency and P = 0.005 for cell numbers; Table 3). These postoperative reductions in cell numbers and frequency were observed in all patients with ovarian cancer included in the study.

Table 3.

Frequency and numbers of circulating endothelial progenitor cells (EPCs) in patients with ovarian cancer before and after surgical treatment, and in healthy control subject.

EPCs	Control group	Ovarian cancer group
	n = 14	n = 8
		Preoperative	Postoperative
Frequency, %	0.002 ± 0.002	0.012 ± 0.002 a	0.0005 ± 0.0001 b
Cells/ml blood	24 ± 23	146 ± 108 a	62 ± 47 b

Data presented as mean ± SD.

^aP < 0.05 vs control group, ^bP < 0.01 vs preoperative data; Student’s t-test.

Discussion

Endothelial progenitor cells differentiate from haemangioblasts during the development of mesodermal precursors, and further differentiate into mature endothelial cells to form the lining of blood vessels in a process known as vasculogenesis. In the course of vasculogenesis, EPCs mobilize from bone marrow to peripheral tissue sites in response to endogenous or exogenous signals, and autocrine/paracrine activation results in differentiation, proliferation and vascular growth. ⁵ EPCs can be detected in peripheral and cord blood, and play important roles in physiological processes such as neovascularization, wound healing, tissue regeneration following ischaemia (e.g. myocardial infarction) and tissue remodelling. ³ In tumour pathogenesis, however, EPC recruitment is thought to mediate aberrant vasculogenesis, and facilitate tumour growth and metastasis. ²¹

Circulating EPCs cannot be successfully defined with a single surface antigen, instead requiring the use of several markers (including VEGFR2, CD31, CD34 and CD133) for their detection in peripheral blood. ²² The majority of studies concerning the correlation of circulating EPCs with gynaecological cancer have used double-positive flow cytometry analysis (e.g. CD133/VEGFR2 in breast cancer and CD34/VEGFR2 in ovarian or cervical cancer).^8,11,23 The approach of the current study was to use four concurrent markers (CD45^–/VEGFR2⁺/CD34⁺/CD133⁺) to increase the accuracy of EPC detection.

Studies have found high levels of circulating EPCs to be associated with breast, ovarian and pancreatic tumours.^8,11,24 Circulating EPCs (CD133⁺/VEGFR⁺) were shown to be significantly more numerous in advanced breast cancer than in early stage disease, and these EPC numbers declined significantly following chemotherapy. ⁸ EPCs (CD34⁺/VEGFR⁺) were also present at higher levels in more advanced cases of ovarian cancer. ¹¹ Circulating EPCs were present at a greater frequency in patients with either cervical or ovarian cancer than in control subjects, in the present study. In addition, treatment (CCRT or surgery) lowered EPC frequency in all patients.

Endothelial progenitor cells may represent a potential new treatment modality for tumours, as their role in angiogenesis could be a pharmacological target. Studies suggest that circulating EPCs may be useful markers for predicting therapeutic outcome or prognosis. ⁸ In addition, animal studies have shown that circulating EPC numbers decline in response to antiangiogenic therapy such as anti-VEGFR2 antibodies, and chemotherapeutic drug response can be measured, in part, by levels of EPCs in peripheral blood. ²⁵

In conclusion, the present study demonstrates that circulating EPCs are significantly more numerous, and more frequent, in patients with gynaecological cancer than in healthy control subjects. As EPCs decline throughout treatment they may be useful surrogate markers to monitor cancer treatment response. Further research, involving larger patient populations, is needed in order to validate whether EPCs have such clinical and therapeutic potential.