SDF-1α/CXCL12 and dendritic cells in ovarian cancer microenvironment

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1 Ginekol Pol. 2011, 82, P R A C E O R Y G I N A L N E SDF-1α/CXCL12 and dendritic cells in ovarian cancer microenvironment SDF-1α/CXCL12 i komórki dendrytyczne w mikroêrodowisku raka jajnika Wertel Iwona 1, Polak Grzegorz 1, Tarkowski Rafał 1, Kotarska Maria 2 1 I Chair and Department of Oncological Gynaecology and Gynaecology, Medical University, Lublin, Poland 2 University of Nevada, Las Vegas, USA Abstract Aim: To evaluate the α-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 levels in the peritoneal fluid (PF) and plasma of patients with epithelial ovarian cancer (EOC, n=101), serous cyst (n=34) or normal controls (n=20). SDF-1α concentrations were correlated to myeloid (M) and lymphoid (L) dendritic cells (DC). Material and methods: SDF-1α levels were analysed using a sensitive enzyme-linked immunosorbent assay (ELISA). DC were evaluated by flow cytometry. Results: The SDF-1α concentrations in peritoneal fluid were elevated in the EOC patients in comparison to the patients with serous cyst (the reference group). The PF SDF-1α levels were significantly higher in FIGO III and IV patients than in FIGO stage I. There were no significant differences in the PF SDF-1α levels with respect to tumor grade or histology. Additionally, there were no significant differences in the plasma SDF-1α levels in EOC patients in comparison with the reference and control groups. The SDF-1α levels in the plasma of FIGO stage III patients were significantly higher than that of stage I. Patients with undifferentiated carcinoma had significantly higher plasma SDF-1α levels than patients with serous cystadenocarcinoma. No significant differences were noted in the plasma SDF-1α levels within different tumor grades. There was no significant correlation between PF and plasma SDF-1α levels and the percentage of myeloid and lymphoid DC in the PB and PF of EOC patients. Conclusion: SDF-1α production in EOC patients may depend on the clinical stage of the tumor. Increased levels of SDF-1α and lymphoid DC in the PF of EOC patients may play an important role in the pathogenesis of this disease. Key words: SDF-1α/CXCL12 / ovarian cancer / dendritic cells / Corresponding author: Iwona Wertel I Chair and Department of Oncological Gynaecology and Gynaecology, Medical University, Lublin Poland, Lublin ul. Staszica 16; tel.: , fax: iwonawertel@wp.pl Otrzymano: Zaakceptowano do druku: Polskie Towarzystwo Ginekologiczne 421

2 P R A C E O R Y G I N A L N E Ginekol Pol. 2011, 82, Wertel I, et al. Streszczenie Cel: Celem pracy była ocena stężenia chemokiny SDF-1α/CXCL12 w płynie otrzewnowym (PO) oraz osoczu chorych na raka jajnika oraz ustalenie czy istnieje zależność pomiędzy stężeniem SDF-1α a odsetkiem mieloidalnych (M) i limfoidalnych (L) komórek dendrytycznych (DC). Materiał i metody: Stężenie SDF-1α/CXCL12 oceniono u chorych na raka jajnika (n=101), z torbielą surowiczą jajnika (grupa referencyjna, n=34) oraz grupie kobiet zdrowych (n=20) metodą ELISA. Odsetek MDC i LDC wśród komórek jednojądrzastych PO i krwi obwodowej oceniano w cytometrze przepływowym. Wyniki: Stężenie SDF-1α w PO chorych na raka jajnika okazało się znamiennie wyższe niż w grupie referencyjnej jak też od odnotowanego w osoczu. Ponadto stężenie SDF-1α w PO chorych z III lub IV stopniem zaawansowania nowotworu wg FIGO było wyższe niż w grupie z I stopniem choroby. Nie odnotowano istotnych różnic w stężeniu SDF-1α w PO w zależności od stopnia zróżnicowania czy typu histologicznego nowotworu. Stężenie SDF-1α w osoczu chorych na raka jajnika nie różniło się istotnie od odnotowanego w grupie referencyjnej czy kontrolnej. Stężenie SDF-1α w osoczu chorych z III stopniem FIGO było wyższe niż w grupie z I stopniem choroby. Stężenie SDF-1α w osoczu chorych z rakiem niezróżnicowanym było wyższe niż u chorych z rakiem surowiczym. Nie wykazano istotnych różnic w stężeniu SDF-1α w osoczu w zależności od stopnia zróżnicowania histologicznego nowotworu. Odsetek limfoidalnych DC w PO chorych na raka jajnika okazał się znamiennie wyższy niż w grupie referencyjnej. Nie wykazano istotnej korelacji pomiędzy stężeniem SDF-1α w osoczu i PO a odsetkiem MDC i LDC u chorych na raka jajnika. Wnioski: Odnotowane w prezentowanej pracy wysokie stężenie chemokiny SDF-1α i wysoki odsetek limfoidalnych DC w PO może odgrywać istotną rolę w rozwoju choroby. Słowa kluczowe: SDF-1α/CXCL12 / rak jajnika / komórki dendrytyczne / Introduction It has been shown that chemokines and their receptors are implicated in the development of ovarian cancer [1, 2]. In 2001, Zou et al. and Scotton at al. reported that human ovarian epithelial tumor cells express high levels of the SDF- 1 α/cxcl12 chemokine [3, 4]. This 8 kda protein is produced mainly by stromal and endothelial cells [5, 6, 7]. Studies showed a significant correlation between SDF-1α expression, bone marrow and lymph node metastasis of breast and prostate cancer [5, 8]. The investigators further found that the expression of the SDF-1α and its receptor CXCR4 by malignant ovarian epithelium influences development and spread of cancer [4, 9, 7]. SDF-1α has an impact on tumor cell proliferation and survival [3, 9, 10]. Additionally, it may also promote angiogenesis [11]. SDF-1α also influences migration of lymphoid/plasmacytoid DC into the ovarian tumor microenvironment and may be important for the suppression of the host immune response [3,12]. Related studies showed that LDC accumulate in the malignant ascites and inhibit anti-tumor immunity in EOC patients [12]. It was found that lymphoid DC produce high levels of angiogenic cytokines (TNF-alpha and IL-8) in vivo and induce IL-10- expressing T cells [3, 12]. Experimental data indicate that tumor SDF-1α protects tumor LDC from interleukin-10 induced apoptosis [3]. Consequently, lymphoid DC support tumor vascularization [12]. It was also shown in vitro that SDF-1α may be a chemoattractant for myeloid dendritic cells [13]. In this study we investigated the concentrations of SDF-1α in the peritoneal fluid and plasma of EOC patients. The SDF- 1α levels were correlated with PF and peripheral blood (PB) myeloid and lymphoid DC. To our knowledge, SDF-1α levels in patients with different histological types and grades of advanced ovarian cancer have not been previously reported. Material and methods Patients The study group consisted of 101 women with histologically confirmed ovarian carcinomas that were classified into the following types: serous (n=47), mucinous (n=14), endometrioid cystadenocarcinoma (n=13), clear cell carcinoma (n=4) and undifferentiated carcinoma (n=23). According to International Federation of Gynecologists and Obstetricians (FIGO), 17 cases were of stage I, 11 of stage II, 64 of stage III and 9 of stage IV. The tumor grade showed 5 cases of grade 1 (G1), 41 of grade 2 (G2) and 55 of grade 3 (G3). None of the women received chemotherapy before surgery. The reference group consisted of 34 women with serous cyst of the ovary. As a control, peripheral blood of 20 healthy donors was taken. The study was approved by the Lublin University School of Medicine Ethics Committee. Methods Peripheral blood samples were collected into heparinized tubes before the surgery. Peritoneal fluid specimens were obtained at the time of surgery. Plasma and PF samples were centrifuged at rpm for 10 minutes to remove cells and debris, and stored at -80 C before being tested by ELISA. SDF-1α concentrations in plasma and PF were determined by the Immunoassay kit (Research and Diagnostic Systems, Minneapolis, Minnesota, USA), manufacturer s protocol was followed. Sensitivity of the SDF-1α ELISA was 18 pg/ml. All samples were assayed in duplicate. 422 Nr 6/2011

3 Ginekol Pol. 2011, 82, P R A C E O R Y G I N A L N E SDF-1α/CXCL12 and dendritic cells in ovarian cancer microenvironment. Flow cytometric analysis of myeloid (M) and lymphoid (L) DC Flow cytometric analysis of DC was performed with a FacsCanto flow cytometer (Becton Dickinson, San Jose, California, USA). Myeloid DC as BDCA-1 (CD1c) + CD19 - cells, and lymphoid DC as BDCA-2 + CD123 + cells were identified, as previously described [14]. Results are expressed as percentages of MDC and LDC in mononuclear cells. Statistical analysis Results were analyzed using Statistica 9.0. software. Data were presented as medians and range. The Wilcoxon paired test was used to compare results obtained from PF and plasma. The Mann-Whitney U test was applied in order to compare studied groups. Spearman s rank test was used to assess the correlation between SDF-1α level and percentage of DC. A p value of less than 0.05 was considered as statistically significant. Results Concentration of SDF-1α in the PF and plasma of women with EOC, in the reference and control group The concentrations of SDF-1α in the peritoneal fluid and plasma of patients with ovarian tumors and control group are presented in Table I. The SDF-1α levels detected in the PF of women with EOC were significantly higher (p<0.001) when compared to the reference group. In the EOC group, such as in the reference group, SDF-1α levels were significantly higher in the PF (p<0.001) than in the plasma. (Table I). There were no significant differences in the plasma SDF- 1α levels among women with ovarian cancer, serous cyst or controls. (Table I). Concentration of SDF-1alfa in patients with different stage, grade and histologic type of ovarian cancer Plasma The SDF-1α levels in the plasma of stage III FIGO patients were significantly higher than that of stage I. There were no significant differences in the plasma SDF-1α levels within different tumor grades. However, patients with undifferentiated carcinoma had significantly higher (p<0.05) plasma SDF-1α levels than patients with serous cystadenocarcinoma. (Table I). PF The peritoneal fluid SDF-1α levels were significantly higher in FIGO III and IV patients than that of stage I. There were no significant differences in the PF SDF-1α levels with respect to tumor grade or histology. (Table I). The percentage of DC in the PB and PF of EOC patients The percentage of MDC in the PF was significantly higher (p<0.001) than in the PB. (Table II). Also, the percentage of LDC in EOC patients was higher in peritoneal fluid (p<0.001) compared with PB. (Table III). Table I. Levels of SDF-1α (pg/ml) in the PF and plasma of patients with ovarian cancer, the reference and control groups. Polskie Towarzystwo Ginekologiczne 423

4 P R A C E O R Y G I N A L N E Ginekol Pol. 2011, 82, Wertel I, et al. Table II. The percentage of myeloid DC in the PF and PB of women with ovarian cancer, in the reference and control groups. Table III. The percentage of lymphoid DC in the PF and PB of women with ovarian cancer, in the reference and control groups. Table IV. Correlations between SDF-1α (pg/ml) and MDC and LDC in patients with EOC. The percentage of DC in the PB and PF of the reference group The percentage of MDC in the PF of patients with serous cyst was significantly higher (p<0.001) than in the PB (Table II). However, the percentage of lymphoid DC was significantly higher (p<0.05) in the PB than in the PF. (Table III ). The percentage of DC in the PF and PB of women with ovarian cancer, serous cyst and in the control group The percentage of MDC was significantly lower (p<0.001) in the PF of patients with ovarian cancer in comparison to women with serous cyst. (Table II). In contrary, the percentage of LDC was higher (p<0.001) in the PF of patients with ovarian cancer than in the reference group. (Table III). The percentage of MDC and LDC in the PB was significantly higher (p<0,05) in the reference group and in the control group than in patients with ovarian cancer. (Table II, III). Correlation between concentration of SDF-1α and dendritic cell subsets in EOC patients There was no significant correlation between PF and plasma SDF-1α levels and the percentage of PB and PF myeloid and lymphoid DC in EOC patients. The statistical data is presented in Table IV. Discussion In this study we have evaluated the concentration of SDF-1α in the PF and plasma of women with different stage, grade and histological types of ovarian cancer. We detected that SDF-1α levels in the PF from EOC patients were significantly higher than in the plasma. Moreover, we found levels of SDF-1α in the PF to be significantly higher in patients with ovarian cancer versus patients with ovarian cyst. This observation may be useful in differentiating between benign and malignant ovarian tumors. 424 Nr 6/2011

5 Ginekol Pol. 2011, 82, P R A C E O R Y G I N A L N E SDF-1α/CXCL12 and dendritic cells in ovarian cancer microenvironment. Our results are consistent with previous reports showing that EOC patients had elevated PF levels of SDF-1α [4,10,11]. In agreement with our data, Kajiyama et al. reported that SDF- 1α levels in malignant ascites from patients with stage III FIGO were higher when compared with stage I of EOC [10]. In contrast, Nowak et al. [15] did not find correlation between SDF-1α expression and clinicopathologic data in ovarian cancer patients. Interestingly, we did not detect significant differences in plasma SDF-1α levels among women with ovarian cancer, serous cyst or the controls. Our results suggest that SDF-1α may be produced locally by ovarian tumor cells. This is in agreement with findings by Zou et al. and Scotton et al. [3, 9], who reported that ovarian epithelial tumor cells express high levels of SDF-1α. Scotton et al. hypothesized that elevated levels of SDF-1α found in the ascites could create a chemokine gradient aiding in tumor cells migration into the peritoneal cavity [4]. Previous studies have demonstrated that the chemokine SDF-1α is a chemoattractant for specific immune cell subsets like regulatory T cells and lymphoid/plasmacytoid dendritic cells [12, 16]. The results from Zou et al. and Curiel et al., as well as from our group, showed that ascites from EOC patients contain more dendritic cells than the PB [12, 13, 14]. The reason for the lymphoid DC accumulation in the ascites remains unclear. Zou et al. showed that the chemokine SDF-1 α induces migration of plasmacytoid DC to the ovarian tumor microenvironment [3]. Also, in vitro studies have demonstrated that LDC, in contrary to MDC, migrated only in response to SDF-1α [13]. In our study we demonstrated the presence of SDF-1α gradient between ascites and plasma. Also, an elevated percentage of LDC in the PF of EOC patients was recorded; however, we did not find a correlation between ascites SDF-1α levels and the percentage of lymphoid DC. This is in agreement with studies done by Vicari et al. who did not observe a correlation between SDF-1α levels and lymphoid DC in breast carcinoma [17]. Such observation is difficult to explain. It is likely that multiple chemokines and factors cooperate to attract lymphoid DC population into the ovarian cancer microenvironment. Future studies will be necessary to examine this complicated relationship. In our study we show that plasma and ascites SDF-1α levels differ significantly between patients with FIGO stage I and III of ovarian cancer. Our results led us to conclude that SDF-1α production in peritoneal cavities of EOC patients depends on the clinical stage of the tumor. We surmise that the increased level of SDF-1α and lymphoid DC in the PF of EOC patients may play an important role in the pathogenesis of this disease, especially in induction of neoangiogenesis. References: 1. Wilson J, Balkwill F. The role of cytokines in the epithelial cancer microenvironment. Semin Cancer Biol. 2002, 12, Barbieri F, Bajetto A, Florio T. Role of chemokine network in the development and progression of ovarian cancer: a potential novel pharmacological target. J Oncol. 2010, Zou W, Machelon V, Coulomb-L Hermin A, [et al.]. Stromal-derived factor-1 in human tumors recruits and alters the function of plasmacytoid precursor dendritic cells. Nat Med. 2001, 7, Scotton C, Wilso J, Milliken D, [et al.]. Epithelial cancer cell migration: a role for chemokine receptors? Cancer Res. 2001, 61, Taichman R, Cooper C, Keller E, [et al.]. Use of the stromal cell-derived factor-1/cxcr4 pathway in prostate cancer metastasis to bone. Cancer Res. 2002, 62, Kryczek I, Wei S, Keller E, [et al.]. Stroma-derived factor (SDF-1/CXCL12) and human tumor pathogenesis. Am J Physiol Cell Physiol. 2007, 292, C987-C Jiang Y, Wu X, Xing H, Du X. Role of CXCL12 in metastasis of human ovarian cancer. Chin Med J (Engl). 2007, 120, Muller A, Homey B, Soto H, [et al.]. Involvement of chemokine receptors in breast cancer metastasis. Nature. 2001, 410, Scotton C, Wilson J, Scott K, [et al.]. Multiple actions of the chemokine CXCL12 on epithelial tumor cells in human ovarian cancer. Cancer Res. 2002, 62, Kajiyama H, Shibata K, Terauchi M, [et al.]. Involvement of SDF-1α/CXCR4 axis in the enhanced peritoneal metastasis of epithelial ovarian carcinoma. Int J Cancer. 2008, 122, Kryczek I, Lange A, Mottram P, [et al.]. CXCL12 and vascular endothelial growth factor synergistically induce neoangiogenesis in human ovarian cancers. Cancer Res. 2005, 65, Curiel T, Cheng P, Mottram P, [et al.]. Dendritic cells subsets differentially regulate angiogenesis in human ovarian cancer. Cancer Res. 2004, 64, Penna G, Sozzani S, Adorini L. Cutting edge: selective usage of chemokine receptors by plasmacytoid dendritic cells. J Immunol. 2001, 167, Wertel I, Polak G, Bednarek W, [et al.]. Dendritic cell subsets in the peritoneal fluid and peripheral blood of women suffering from ovarian cancer. Cytometry B Clin Cytom. 2008, 74, Nowak-Markwitz E, Puła B, Szajnik M, [et al.]. Expression of survivin, SDF and CXCR4 on tumor cells in ovarian cancer. Ginekol Pol. 2010, 81, Zou L, Barnett B, Safah H, [et al.]. Bone marrow is a reservoir for CD4+CD25+ regulatory T cells that traffic through CXCL12/ CXCR4 signals. Cancer Res. 2004, 64, Vicari A, Treilleux I, Lebecque S. Regulation of the trafficking of tumour-infiltrating dendritic cells by chemokines. Semin Cancer Biol. 2004, 14, Acknowledgments The study was supported by the State Committee for Scientific Research, Warsaw, Poland, Grant #N N and NN Polskie Towarzystwo Ginekologiczne 425

6 P R A C E O R Y G I N A L N E Ginekol Pol. 2011, 82, Pro12Ala PPAR gamma2 gene polymorphism in women with polycystic ovary syndrome Polimorfizm Pro12Ala genu PPAR gamma2 u kobiet z zespo em wielotorbielowatych jajników Bidzińska-Speichert Bożena 1, Lenarcik Agnieszka 1, Tworowska-Bardzińska Urszula 1, Ślęzak Ryszard 2, Bednarek-Tupikowska Grażyna 1, Milewicz Andrzej 1, Krępuła Katarzyna 3 1 Department of Endocrinology and Diabetology, Medical University of Wrocław, Poland 2 Department of Genetics, Medical University of Wrocław, Poland 3 Endocrinology Outpatient Clinic, Wrocław, Poland Abstract Introduction: The pathogenesis of PCOS has not been definitively determined and includes a number of genes linked with steroidogenesis, regulation of gonadotropin secretion, actions of insulin, obesity, as well as chronic inflammatory processes. Some authors indicate that PPARγ play a role in insulin sensitivity and are probably involved in hyperandrogenism in PCOS. The aim of the study was to assess the frequency of the Pro12Ala and Pro115Gln PPARγ2 gene polymorphisms in women with PCOS. Subjects and methods: 54 PCOS women and 51 healthy women were recruited. Genetic studies to detect Pro12Ala and Pro115Gln PPARγ2 gene polymorphism were performed. Results: In the whole studied group the Pro115Gln polymorphism of the PPARγ2 gene was not found. The frequency of the Pro12Ala polymorphism was estimated at 26.47% in the controls and at 23.15% in the PCOS patients. Women from the control and PCOS groups with BMI 30 had statistically higher occurrence of the Ala allele than women with BMI <30 (38.80% versus 12.50% and 38.23% versus 18.75%). Conclusions: The frequency of the Pro12Ala polymorphism observed in the sample of women from the Lower Silesian population was significantly higher than in the majority of European populations. Key words: polycystic ovary syndrome / Pro12Ala / Pro115Gln / PPARγ2 / / polymorphism / Streszczenie Wstęp: Podłoże zespołu wielotorbielowatych jajników (PCOS) nie jest jednoznacznie wyjaśnione. W patogenezę PCOS mogą być zaangażowane geny związane ze steroidogenezą, regulacją wydzielania gonadotropin, działaniem insuliny, otyłością oraz przewlekłym procesem zapalnym. Niektórzy autorzy donoszą o związku PPARγ z insulinowrażliwością i prawdopodobnie z hiperandrogenizmem w PCOS. Cel pracy: Celem pracy była ocena częstości występowania polimorfizmów Pro12Ala i Pro115Gln genu PPARγ2 u kobiet z PCOS. Materiał i metody: do badania zrekrutowano 54 kobiety z PCOS oraz 51 zdrowych kobiet. Zostały przeprowadzone badania genetyczne oceniające polimorfizmy Pro12Ala i Pro115Gln genu PPARγ2. Wyniki: W całej zbadanej grupie nie znaleziono polimorfizmu Pro115Gln genu PPARγ2. Częstość występowania polimorfizmu Pro12Ala genu PPARγ2 w grupie kontrolnej oceniono na 26,47%, a u kobiet z PCOS na 23,15%. Zarówno kobiety z grupy kontrolnej, jak i pacjentki z PCOS z BMI 30 wykazywały istotnie większą częstość występowania allela Ala niż kobiety z BMI <30 (odpowiednio 38.80% versus 12,50% i 38,23% versus 18,75%). Corresponding author: Bożena Bidzińska-Speichert Department of Endocrinology and Diabetology, Medical University of Wrocław ul. Pasteura 4, Wrocław, Poland tel.: bobi@kn.pl Otrzymano: Zaakceptowano do druku: Nr 6/2011