CXCL12 expression by healthy and malignant ovarian epithelial cells

Epithelial ovarian cancer (EOC) has one of the highest mortality rates of all gynecologic malignancies. It is the sixth most common cancer and the fifth most common cause of cancer-related death among women in developed countries [1]. Due to the silent nature of early-stage disease, most women with EOC have disseminated disease (i.e. expansion in the peritoneum and metastasis in the omentum) at the time of diagnosis and present an advanced stage of the disease, with a five-year survival rate below 30% [2]. Despite the high incidence and mortality rates, the etiology of EOC and the molecular pathways underlying its progression remain poorly understood. According to the International Federation of Gynecology and Obstetrics (FIGO), clinical stage, histologic grade and postoperative residual tumor mass are the most important prognostic factors in patients with EOC [3]. However, clinical factors and derivative prognostic models remain inadequate for the accurate prediction of outcome for a specific patient, indicating a need for the identification of biological factors to improve prognostic assessment. This aspect has recently been addressed with the identification of several biomarkers for the identification of histologic subtypes and the more accurate prediction of patient outcome [4‐9]. Chemokines and their receptors have been known for many years to influence the development of primary epithelial tumors, in which they regulate the proliferation and survival of tumor cells, tumor-infiltrating leukocytes, angiogenesis and metastasis [10‐12]. In epithelial cancers, these molecules play a key role in controlling both autocrine and paracrine communication between the different cell types of the tumor microenvironment [13]. Thus, chemokines and their receptors may constitute new biomarkers of potential prognostic value in various cancers, including EOC.

In this study, we focused on the α-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12, which, together with its receptors CXCR4 and CXCR7, constitutes the chemokine/receptor axis attracting the greatest level of interest in oncology [11, 14]. In EOC, CXCL12 products (i.e. protein and mRNA) have been detected in tumor cells [15, 16]. We previously showed that CXCL12 orchestrates the recruitment of pre-DC2s and protects them from tumor macrophage IL-10-promoted apoptosis, thereby contributing to the immunosuppressive network within the tumor microenvironment [15]. In addition, CXCL12 regulates tumor angiogenesis, a critical step in tumor growth. Indeed, we have shown that hypoxia triggers the production of CXCL12 and vascular endothelium growth factor (VEGF) by EOC, with these two molecules acting in synergy to enhance tumor angiogenesis in vivo [17]. CXCL12 also acts on tumor cell proliferation and survival and, through its main receptor CXCR4, governs the migration of malignant cells and their invasion of the peritoneum, a major route for ovarian cancer spread [16, 18‐20]. Other factors must also been considered, but previous observations strongly suggest that CXCL12 provides the autocrine and paracrine signals controlling malignant progression in EOC [11]. Some recent studies have investigated CXCL12 status in EOC, and reported no prognostic significance of CXCL12 production [11, 21, 22]. However, the results were obtained with ovarian cancer specimens from patients undergoing chemotherapy via heterogeneous protocols, with a follow-up period of less than four years. The prognostic significance of CXCL12 production by ovarian cancer cells remains to be clearly assessed in larger cohorts of EOC patients undergoing the same type of chemotherapy and followed up for longer periods. Furthermore, the pattern of CXCL12 expression in healthy ovaries and in benign and borderline ovarian tumors has scarcely been investigated. Elucidation of these points is required to determine whether CXCL12 production is associated with the malignant process and whether it constitutes a valuable prognostic factor in EOC.

In this study, we investigated CXCL12 status in the reproductive tracts of healthy women. We studied the ovarian surface epithelium (OSE) and fallopian tubes, both of which are considered probable sources of EOC [23, 24]. We also investigated CXCL12 status in benign and borderline epithelial tumors, and in a series of 183 patients with advanced primary EOC enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin/gemcitabine (TCG)-based chemotherapy [ClinicalTrials.gov Identifier: NCT00052468]. We quantified CXCL12 by immunohistochemistry (IHC) in EOC specimens and further assessed its potential association with clinical and pathologic features, including staging parameters and tumor histotypes, and with the expression of HER2, a tyrosine kinase receptor that may influence outcome when overexpressed [21, 25]. Finally, we investigated whether the production of CXCL12 within the tumor affected progression-free survival (PFS) and the overall survival (OS) of patients with advanced primary EOC.

In this study, we demonstrate the previously unappreciated constitutive expression of CXCL12 by healthy ovarian epithelial cells and ovarian epithelial tumor cells, whether benign or malignant. CXCL12 was recovered from both the OSE and the epithelium of the fallopian tubes, both of which are considered possible origins of EOC. By contrast, it was not detected in follicles and oocytes or in malignant tumor cells arising from them, granulosa tumors or dysgerminomas. Furthermore, CXCL12 expression correlated neither with clinical parameters nor with HER2 status in specimens from 183 patients with advanced primary EOC enrolled in a multicenter clinical trial of first-line TCG-based chemotherapy (GINECO study). The intratumoral production of CXCL12 does not reflect the morphological heterogeneity of EOC and has no impact on PFS or OS after adjustment for established prognostic factors. Thus, CXCL12 is expressed by ovarian epithelial cells before tumorigenesis and does not constitute a valuable prognostic factor in EOC patients.

Recent studies on the origin and histogenesis of EOC have proposed that type I tumors, which are believed to include all major histotypes, originate from the OSE, whereas type II tumors, which are thought to consist almost exclusively of high-grade serous carcinomas, arise from the distal region of fallopian tubes [4, 24, 34]. Our findings clearly demonstrate that CXCL12 is constitutively produced by the epithelial cells of the OSE, whereas such expression was not previously suspected [11, 16]. This apparent discrepancy may result from differences between our experimental protocol and those used in previous studies. For example, we used an anti-CXCL12 mAb rather than a polyclonal Ab and an additional microwave pretreatment for antigen retrieval, both of which would have increased the sensitivity of immunostaining. CXCL12 was also recovered from the epithelial cells of fallopian tubes, which were recently identified as a possible origin of high-grade serous EOC and which have a Müllerian duct-derived embryologic origin in common with the OSE [23, 28, 35]. By contrast, CXCL12 was undetectable in follicles, oocytes and their malignant non epithelial counterparts. Thus, CXCL12 is a chemokine constitutively produced by epithelial ovarian cells, from both healthy and malignant tissues. CXCL12 is present in ovarian epithelial cells before they become malignant and is therefore not useful as a marker of malignancy in EOC.

Scotton and coworkers reported a trend toward stronger CXCL12 expression in higher grade tumors [16]. However, Pils and coworkers recently found that the abundance of CXCL12 did not differ between borderline and malignant tumors [21]. In the present work, CXCL12 expression was detected in benign tumors as well as in borderline and malignant tumors. Although CXCL12 is unevenly distributed in low-grade (i.e. borderline and stage I) and in more advanced stage tumors, we have no evidence that its expression level is weaker in low-grade tumors. Among CXCL12-positive EOC specimens, we observed no significant differences in the fraction of CXCL12^high-producing tumors for the four histotypes examined (i.e. serous, clear-cell, endometrioid and mucinous), despite previous reports of differences in epidemiologic and genetic changes, tumor markers and response to treatment (reviewed in [11]). We suggest that CXCL12 production levels overlap with EOC histotype differentiation and staging. Consistent with previous findings [16, 22], CXCL12 was detected in more than 90% of patients with advanced primary EOC. However, it was barely detectable in the remaining cases (<10%), suggesting that CXCL12 expression might have been silenced, possibly through epigenetic mechanisms, such as promoter hypermethylation, a phenomenon already reported for colon carcinoma and breast cancer [36, 37]. Indeed, further in-depth studies are required to determine whether transcriptional regulatory mechanisms account for heterogeneous CXCL12 production in EOC.

Recent studies have assessed the prognostic significance of CXCL12 expression in various cancers, including colorectal carcinoma [38], pancreatic ductal adenocarcinoma [39], breast cancer [40], esophageal squamous cell carcinoma [41], endometrial cancer [42], germ cell tumors [43] and EOC [21, 22]. The study reported here was based on a large, homogeneous cohort of 183 patients, all given standard TCG-based chemotherapy. IHC showed that CXCL12 abundance was not correlated with any of the clinical parameters tested or with the HER2 status. Patients with CXCL12^high-producing tumors had a PFS and OS similar to those of patients with CXCL12^low/moderate-producing tumors. Consistent with the findings of smaller cohorts of patients given heterogeneous treatments [21, 22], we therefore suggest that there is no evidence that CXCL12 production by malignant epithelial ovarian cells is of prognostic significance in EOC.

This lack of prognostic value for CXCL12 in EOC is somewhat puzzling, as this chemokine has been reported to enhance tumor cell proliferation and survival [16, 18‐20, 44], to promote angiogenesis [17], to inhibit the host immune response [15] and to mediate resistance to hyperthermic intraperitoneal chemotherapy [45], which may favor tumor growth and spread. This apparent paradox may be explained by the cellular expression of CXCL12 not providing a true reflection of its bioavailability, which depends principally on the presence in the tumor microenvironment of factors capable of disrupting CXCL12 from glycosaminoglycans [46]. Moreover, CXCL12 activity may be mediated by two receptors, CXCR4 and CXCR7, and these receptors may also be rate-limiting elements. For many years, CXCL12 and CXCR4 were thought to act as an exclusive non redundant pair. However, the recent identification of RDC1/CXCR7 as a second receptor for CXCL12 has challenged this view, and we now need to determine the respective contributions of CXCR4 and CXCR7 to the homeostatic and pathological activities of CXCL12 [47‐49]. The emerging possibility that CXCR7 acts as a decoy receptor provides further support for a potential role in EOC. Finally, the lack of influence of CXCL12 may reflect the unusual characteristics of metastases, predicting the occurrence of which is one of the major challenges in efforts to improve the clinical outcome of EOC. By contrast to breast cancer, in which distant metastases to the liver, lung and bone marrow are favored by high levels of CXCL12 expression in target organs and lower levels within the tumor [50], EOC spreads by the direct seeding of tumor cells into the peritoneal cavity, with preferential metastasis to local lymph nodes. In EOC, CXCL12 mRNA and protein have been detected mostly in the tumor cells themselves, and this feature has been reported for other cancers, including follicular lymphoma, pancreatic cancer, glioma and astrocytoma [10]. Ovarian epithelial tumor cells constitute a potent source of CXCL12. CXCL12 may therefore retain tumor cells at the site of production, rather than encouraging them to disseminate and to form secondary tumors in organs at some distance from the original tumor.

Our findings highlight the previously unappreciated constitutive expression of CXCL12 by healthy epithelia of the ovary surface and the fallopian tubes, both these epithelia having been identified as probably sources of EOC. Thus, CXCL12 is expressed by epithelial cells before they become malignant. We also show that the level of CXCL12 expression in cancer cells is not a valuable prognostic factor in patients with advanced EOC. These findings do not exclude the possibility that CXCL12 contributes to tumor growth and spread via autocrine and/or paracrine action. There is therefore a need to determine whether CXCL12 status in EOC depends on its bioavailability and on the CXCR4/CXCR7 ratio in tumor cells, which would support an effect of CXCL12.