Huiling Qiu, Lili Cao, Dan Wang, Huicheng Xu and Zhiqing Liang

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1 bs_bs_banner doi: /jog J. Obstet. Gynaecol. Res. Vol. 39, No. 7: , July 2013 High levels of circulating CD34+/VEGFR3+ lymphatic/ vascular endothelial progenitor cells is correlated with lymph node metastasis in patients with epithelial ovarian cancer Huiling Qiu, Lili Cao, Dan Wang, Huicheng Xu and Zhiqing Liang Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University, Chongqing, China Abstract Aim: Lymph node metastasis is one of the predictive factors associated with poor prognosis of epithelial ovarian cancer. To clarify the role of CD34 and vascular endothelial growth factor receptor-3-positive (CD34+/ VEGFR3+) lymphatic/vascular endothelial progenitor cells (LVEPC) in patients with lymph node metastasis and epithelial ovarian cancer progression, the levels of circulating CD34+/VEGFR3+ LVEPC in epithelial ovarian cancer patients were detected. We also tested the plasma protein levels of VEGF and stromal cellderived factor to find out their possible relationships with lymph node metastasis in our epithelial ovarian cancer cohort. Material and Methods: Peripheral blood samples were collected from 54 patients diagnosed as epithelial ovarian cancer, and 31 normal samples as control. The circulating levels of LVEPC were carried out by flow cytometry, and blood protein levels of biomarkers were measured by enzyme-linked immunosorbent assay (ELISA). Results: The level of circulating LVEPC was significantly higher in patients with ovarian cancer compared with that of healthy controls. There was also a statistically significant correlation between LVEPC levels and surgical staging of epithelial ovarian cancer (P < 0.01). Conclusion: The circulating levels of bone marrow-derived LVEPC are significantly increased in epithelial ovarian cancer patients and these levels correlate with lymph node metastasis too. Key words: epithelial ovarian cancer, lymph node metastasis, lymphangiogenesis, lymphatic/vascular endothelial progenitor cell. Introduction Epithelial ovarian cancer (EOC), the most lethal gynecologic malignancy, accounts for 3% of new cancer cases diagnosed in women. 1 The high mortality rate of ovarian cancer is largely attributed by the late diagnosis of this tumor. In spite of aggressive therapy associating surgery and intensive chemotherapy, the cure rate for these patients is lower than 20%. 2 Novel therapeutic approaches are urgently needed to improve prognosis. The most common way of ovarian cancer metastasis is direct extension to adjacent organs, especially to the abdominal peritoneum and omentum. Besides, lymph node metastasis is one of the key prognostic predictors for ovarian cancer. 3 Lymphatic node metastasis results Received: February Accepted: December Reprint request to: Dr Zhiqing Liang, Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University, Chongqing , China. zhiqing.liang@hotmail.com The Authors

2 Cell level detection by flow cytometry in a development from stage I to stage IIIC, and 5-year survival rate decreases from more than 90% to 20 60%. 4,5 Increased lymphatic vessel density is associated with a worse prognosis for survival. 6,7 Lymphangiogenesis, the formation of the lymphatics, plays a crucial role in the spread of ovarian cancer. However, the role of lymphangiogenesis in the growth and dissemination of ovarian cancer has not been clearly established. It is well established that sprouting of new lymphatics from the preexisting lymphatic vessels appears not to be the only mechanism for the tumor lymphangiogenesis. 8 Rare circulating CD34 and vascular endothelial growth factor receptor-3-positive (CD34+/ VEGFR3+) lymphatic/vascular endothelial progenitor cells (LVEPC), the newly identified as bone marrowderived progenitor cells, are reported to contribute to de novo lymphangiogenesis in experimental tumor systems. 8 It has been reported that peripheral blood levels of LVEPC significantly increased in patients with small cell lung cancer and correlated with lymphatic involvement. 9 More importantly, high level of circulating LVEPC is associated with poor prognosis in patients with human small cell lung cancer. 9 Furthermore, circulating levels of VEGFR2+ bone marrow-derived endothelial progenitor cells are shown to have clinical significance in human ovarian cancer. 10 Therefore, it is of great interest to study clinical relevance of circulating LVEPC in human ovarian cancer, especially their influence on lymphangiogenesis. The aim of the present study was to provide the first evaluation of levels of circulating LVEPC in patients with EOC by flow cytometry and to determine the correlation between LVEPC level and lymphangiogenesis and disease progression. In addition, plasma protein levels of vascular endothelial growth factor (VEGF)-C and stromal cell-derived factor (SDF)-1 were also determined. Material and Methods Study subjects This study was approved by the Institutional Review Board and local ethics committee. Informed consent was obtained from all study participants and healthy volunteers. 54 patients (median age, 48 years; age range, years) were histologically diagnosed as EOC, including serous cancer (n = 26), mucinous cancer (n = 17), endometrioid cancer (n = 8), and clear cell cancer (n = 3). Among the mucinous cancers, 4 of them were categorized into FIGO I stage, 4 were in FIGO II stage, 7 were in FIGO III stage and the rest were in FIGO IV stage. The ratio of lymph node metastasis of the mucinous cases was 29.4% (5/17). The detailed information could be found in supplemental information (Table S1). Patients with additional malignant or inflammatory diseases, pulmonary fibrosis, wounds or ulcers, cardiovascular risk including diabetes mellitus, chronic renal failure, chronic obstructive lung disease, untreated hypertension, deep venous thrombosis, and rheumatoid arthritis were excluded. 11 The cases conducted with bone marrow transplantation were also excluded from the study. 12 Relevant data were also collected on the age of patients, parity and menopausal status, initial complaints, physical examination, complete blood counts, CA125 levels, comprehensive chemistry panels, ultrasonographic appearance, chest radiographs, magnetic resonance image or computed tomography scans of the abdomen, intraoperative findings and final pathological reports. All patients were subjected to a primary surgical staging procedure by the surgical team led by one of the authors and then evaluated by the pathologist, according to the federation international of gynecology and obstetrics (FIGO) recommendations. The control group included 31 healthy volunteers matched for age, gravida, parity and menopausal status (Table 1). They were recruited from a local medical examination center. The control group s median age was 47 (range: 19 78) years. They were free of inflammatory, infectious, autoimmune, or vascular diseases. Measurement of circulating LVEPC by flow cytometry All patients were sampled before surgery. The control blood samples were collected during routine medical examination. After discarding the first 2 ml after venepuncture, peripheral blood samples were drawn: 10 ml of whole blood was collected in standard heparin tubes for circulating LVEPC analysis. Flow cytometry analysis was based on the expression of cell surface markers CD34 and VEGFR3 in the mononuclear gate where LVEPCs were commonly found. 8 The samples were coped with density centrifugation, following erythrocyte lysis. The remaining peripheral blood mononuclear cell fraction was resuspended in 100 ml of cell-sorting buffer containing PBS and 0.1% bovine albumin and then incubated for 30 min at 4 C with fluorescent isothiocyanate(fitc)- conjugated antihuman CD34 (ebioscience) and phycoerythrin(pe)-conjugated antihuman VEGFR The Authors 1269

3 H. Qiu et al. Table 1 General clinical characteristics of patient and control groups Patients (n = 54) Controls (n = 31) P-value Mean of ages SD * (years) Mean of gravida SD * Mean of parity SD * Menopausal status 0.255** Postmenopausal Premenopausal *Derived from a Student s t-test; **Derived from Pearson s chi-square test. (R&D Systems) according to the manufacturer s protocol. Fluorescent isotype matched antibodies IgG1-FITC (ebioscience)/igg1-pe (R&D Systems) were used as control, the exact same quantity isotype was used. The suspension was then incubated with fluorescenceactivated cell sorter (FACS) lysing solution for 10 min, according to the manufacturer s instructions. Results were described as the percentage of CD34+/VEGFR3+ cells among peripheral blood using the CyFlow SL flowcytometer and the FACSDIVA software (both from BD Bioscience). Serum levels of VEGF-C and SDF-1 Serum samples from all patients and controls were collected and stored at -70 C until further analysis. Blood levels of VEGF-C and SDF-1 were measured by enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems) according to the manufacturer s protocol. All serum samples were assayed in duplicates. Statistical analysis All statistical analyses were performed with Statistical Package for Social Sciences version 13.0 software (SPSS Inc. Chicago, IL, USA). Continuous variables were estimated using Student s t-test when the sample distribution was normal, and estimated using Mann-Whitney U test when the sample distribution was asymmetrical. Categorical data were estimated using c 2 test. Correlations were measured with Spearman s rank correlation test. Significant was determined as P < Results Levels of circulating LVEPC progenitors We measured the number of LVEPC in the peripheral blood using flow cytometry. As shown in Figure 1b, the level of LVEPC per ml in the peripheral blood of ovarian cancer patients (median level 0.98%, range %) were higher than that of healthy controls (median level 0.15%, range %) (P < 0.01). There was a statistically significant correlation between LVEPC levels and FIGO stage (P < 0.01). Among patients, the median level of LVEPC was 0.81% (range, %) for those with stage I and stage II but was 1.69% (range, %) for those with stage III and stage IV, the LVEPC levels were significantly higher in patients with high stage tumor (Fig. 1c). The level of LVEPC was significantly higher in patients with distant metastasis compared with that with localized metastasis. Relationship between circulating LVEPC levels and clinical behavior of EOC We compared LVEPC levels in different groups of patients with EOC subdivided based on clinical characters. The results are summarized in Table 2. No significant differences in patient age, gravida, parity, menopausal status, or histological grade were observed between patient groups (P > 0.05). There was a statistically significant correlation between LVEPC levels and lymph node involvement; the LVEPC levels from patients with lymph node metastasis were significantly higher than from those without lymph node metastasis (P < 0.01). Correlation with CA125 values and tumor diameter was also analyzed using Pearson s correlation. There was no significant association between LVEPC levels and CA125 value or tumor diameter could be found. For CA125, the Pearson correlation was and the P-value was 0.353; for tumor diameter, the Pearson correlation was and the P-value was Plasma levels of VEGF-C and SDF-1 The plasma levels of VEGF-C and SDF-1 in the EOC patients were significantly higher (P < 0.01) compared to the control (Fig. 2a). For those EOC patients, the The Authors

4 Cell level detection by flow cytometry Figure 1 (a) Flow cytometric analysis from a patient with serous ovarian cancer. Left, flow cytometry gating; middle, isotype negative control for flow-cytometry; right, flow cytometric analysis determined the number of CD34/VEGFR3 double double-positive cells with a value of 1.30%. (b) Comparison of circulating LVEPC levels in ovarian cancer patients and healthy controls (P < 0.01). (c) Comparison of circulating LVEPC levels in patients with ovarian cancer in different stages (P < 0.01). Box plots show median (central dots), 25 75% quartile ranges (boxes), and minimum and maximum levels in ovarian cancer patients and healthy controls. VEGF-C and SDF-1 levels were pg/ml and pg/ml, respectively. However, the levels in healthy controls were pg/ml and pg/ml, respectively. Plasma concentrations of SDF-1 and circulating LVEPC levels were significantly correlated in EOC patients (P < 0.01, Fig. 2b). But there was no significant correlation between serum concentrations of VEGF-C and circulating LVEPC levels. Discussion Lymph node metastasis is one of the predictive factors associated with poor prognosis of ovarian cancer. However, the mechanism of lymph node metastasis remains unclear. Recent studies have demonstrated that lymphangiogenesis is an essential issue in the development of different solid tumors. 13,14 In the present study, we investigated the potential utility of circulating progenitor cells in ovarian lymph node metastasis from the perspective of lymphangiogenesis. Lymphangiogenesis is the development of new lymph ducts, associated with enhanced metastatic processes. 15 A series of studies have presented that lymphangiogenesis relied on both the sprouting of nearby blood vessels, with migration and differentiation of existing mature endothelial cells, and the recruitment of mobilized bone marrow-derived endothelial progenitor cells. 8,16 As part of the lymphangiogenic machinery, the newly identified circulating CD34+/VEGFR3+ cell, 2013 The Authors 1271

5 H. Qiu et al. Table 2 Comparison of LVEPC levels in different groups of patients with EOC subdivided based on clinical characters Clinical characteristics Subgroup n Mean of LVEPC P-value level (%) SD Age > Gravida > Parity > Menopausal status Postmenopausal Premenopausal Lymph node metastasis Metastasis None Histologic grade Histologic type Serous Mucinous Endometrial Clear cell Cutoff value is median value; Derived from Student s t-test; Derived from one way anova. called LVEPC, has been shown to contribute to neovessels in cancer-bearing animals 8 and as well as in human renal transplants. 12 Some novel studies have identified circulating LVEPC progenitor cells, which are derived from the bone marrow and peripheral blood, are believed to contribute to tumor microvascular architecture in a variety of human cancers. 17,18 A study by Bogos et al. has demonstrated that patients with small cell lung cancer have a significantly larger number of peripheral blood circulating CD34+/VEGFR3+ cells than tumorfree control subjects, and that the levels of these cells correlated to lymphatic progression and some clinical behavior. 9 Similarly, we observed that levels of LVEPC in the peripheral blood, characterized by CD34+/ VEGFR3+ cells, were significantly higher in patients with EOC than in healthy control subjects. Moreover, among patients, strikingly elevated levels of LVEPC correlated with lymph node metastatic disease. These findings supported the results that the recruitment and differentiation of bone marrow-derived LVEPCs formed new lymphatic capillaries via blood circulation. 16 Despite the small number of patients in our study, we observed significant correlations between circulating LVEPCs levels and tumor stage in EOC patients. Recent studies indicate that some lymphangiogenic cytokines like VEGF-C play an important role in cancer progression by inducing lymphangiogenesis and enhancing metastatic spread via the lymphatics. 19,20 The expression of VEGF-C is significantly associated with lymphatic vessel density. 21 Peripheral blood VEGF-C levels predicted lymph node status in various types of tumor Therefore, we measured peripheral blood levels of VEGF-C to determine lymph node metastasis in ovarian cancer. Similar results were obtained in our study; the expression of VEGF-C was associated with lymph node metastasis in EOC. However, we failed to detect a significant correlation between serum VEGF-C levels and LVEPC in patients with EOC. We also analyzed chemokine SDF-1 in EOC patients. It is known that SDF-1 is highly expressed in the lymph nodes and attracts lymphocytes to this organ. 25 Chemokine SDF-1 is the ligand for chemokine (C-X-C motif) receptor 4 (CXCR4) and the SDF-1/CXCR4 signaling plays a critical role in cancer metastasis. It is reported that the receptor for SDF-1 is involved in tumor progression of ovarian cancer 26 and contributes to lymph nodal metastasis in various types of cancer Our study demonstrated that serum SDF-1 level correlated with lymph node metastasis, and SDF-1 also had close relationships with serum LVEPC levels in EOC. These results suggested that high SDF-1 expression might induce lymphangiogenesis, and then led to increase lymphatic invasion and lymph node metastasis. These findings led us to hypothesize that The Authors

6 Cell level detection by flow cytometry Figure 2 (a) Measurement of levels of plasma VEGF-C, and plasma SDF-1 in 54 patients with EOC and 31 healthy subjects. (b) Significant correlation was found between plasma SDF-1 and circulating LVEPC levels for patients with EOC (r = 0.707, P < 0.01). perhaps SDF-1 induces releasing of LVEPC from the bone marrow that are required for lymph node metastasis to occur in EOC. In conclusion, the current study demonstrates that the circulating levels of bone marrow-derived LVEPC are significantly increased in EOC patients and these levels are correlated with lymph node metastasis. It is not clear how LVEPC plays a role in the lymphatic spread of the tumor, further elucidation of the mechanism of lymph node metastasis is necessary The Authors 1273

7 H. Qiu et al. Acknowledgments We would like to thank for the Department of Obstetrics and Gynecology, Southwest Hospital, Third Military Medical University, Chongqing, China, for providing samples. We also thank Dr Jun Chen and Yun Cai for their advice and assistance. Disclosure This study has no relationship with any industry, and no author has relationships with companies. Grant Support This study was supported by the National Basic Research Program of China (973 Program, No. 2010CB529403). Conflicts of Interest The authors declare that they have no competing interests. References 1. Jemal A, Siegel R, Xu J et al. Cancer statistics, CA Cancer J Clin 2010; 60: Roett MA, Evans P. Ovarian cancer: An overview. Am Fam Physician 2009; 80: Naora H, Montell DJ. Ovarian cancer metastasis: Integrating insights from disparate model organisms. Nat Rev Cancer 2005; 5: Benedet J, Bender H, Jones H et al. 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8 Cell level detection by flow cytometry metastasis of head and neck cancer in xenograft mouse models. Cancer Res 2007; 67: Tan CT, Chu CY, Lu YC et al. CXCL12/CXCR4 promotes laryngeal and hypopharyngeal squamous cell carcinoma metastasis through MMP-13-dependent invasion via the ERK1/2/AP-1 pathway. Carcinogenesis 2008; 29: Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher s web-site: Table S1 Summary of clinical characteristics of 54 EOC cases The Authors 1275