Coexpression of CD140b (PDGFRb) and CD146 has been used to isolate endometrial mesenchymal stem-like cells (eMSCs), which have a perivascular location. This study aims to evaluate a single marker for purifying eMSCs. Using an antibody panel with novel specificities, we screened human endometrial tissues and stromal cell suspensions by flow cytometry and immunohistochemistry to identify perivascular markers. Sorted subpopulations were examined for colony-forming unit (CFU), self-renewal, and differentiation assays for mesenchymal stem cell (MSC) function. We also transplanted sorted eMSCs under the kidney capsule of superimmunodeficient NSG mice. Magnetic bead selection was compared with flow cytometry sorting (flow sorting) using CFU assay. One novel marker (W5C5) was particularly effective in selecting eMSCs. W5C5 + cells comprise 4.2 ± 0.6% (n = 34) of endometrial stromal cells and reside predominantly in a perivascular location in both basal and functional layers of endometrium. + cells gave rise to significantly higher CFU numbers compared to flow-sorted W5C5 + cells. This study identified W5C5 as a single marker capable of purifying eMSCs possessing MSC properties and reconstituting endometrial stromal tissues in vivo. W5C5 enriches eMSCs to high purity and provides a simple protocol for their prospective isolation using magnetic bead selection rather than flow sorting. W5C5 selection may provide an alternate, readily available autologous source of MSC, obtainable with minimal morbidity using an office endometrial biopsy procedure for future cell-based therapies.

Patients with high-grade, serous epithelial ovarian carcinoma (HGSOC) are generally diagnosed with extensive peritoneal metastases, and exhibit 5-year survival rates <30%. A subset of these tumours, defined as "immunoreactive," overexpress mRNA encoding the T-cell-recruiting chemokine CXCL10 (10-kDa interferon gamma-induced protein; C-X-C motif chemokine 10). Tumour-infiltrating CD41 CD8 1 T-cells are a well-documented, positive prognostic indicator for HGSOC patients; paradoxically, however, patients diagnosed with HGSOC (overexpressing CXCL10 and therefore theorised to recruit T-cells) typically exhibit poor survival. Recently, an "antagonistic" CXCL10 variant was identified that inhibited leucocyte recruitment to inflamed liver in vivo (Casrouge et al., J Clin Invest 2011;121:308-17). We hypothesised that "immunoreactive" HGSOC might also express antagonistic CXCL10, interfering with leucocyte recruitment and contributing to poor patient prognosis. CXCL10 expression was analysed in HGSOC tissues grouped according to pathology, grade and FIGO stage at diagnosis, and its localisation and association with T-cells established by immunohistochemical staining in tissue microarrays. CXCL10 expression was increased in a subset of serous epithelial tumour samples; however, it did not correlate well with CD45-positive tumour infiltrate. Immunoprecipitation and de novo sequence analysis of CXCL10 identified the N-terminally cleaved, "antagonistic" variant of CXCL10 specifically in malignant tumours, and not in benign ovarian disease. The data demonstrate the presence of the antagonistic form of CXCL10 in HGSOC for the first time, and provide a partial explanation for reduced leucocyte infiltration observed in these tumours. We suggest that CXCL10 cleavage and subsequent antagonism of immune cell recruitment may be a feature of the "immunoreactive" HGSOC subtype, leading to early impairment of the immune response and subsequently worsening patient prognosis.Epithelial ovarian cancers account for between 80 and 95% of ovarian tumours diagnosed, and remain the fourth highest cause of cancer-related mortality for women with an estimated 22,000 new cases and 14,000 deaths in the United States in 2010. 1 Despite a relatively low population incidence and incremental improvements in surgery and chemotherapy, the 5-year survival rate for patients diagnosed with ovarian neoplasms has improved only marginally over time. Although traditionally classified as serous, mucinous, clear cell or endometroid based on their histological appearance, epithelial ovarian tumours are increasingly considered as different disease subtypes with characteristic molecular genetic alterations (reviewed in Ref.2). In particular, high-or low-grade serous epithelial tumours are now believed to represent distinct molecular diseases (termed Type I or Type II) with different underlying pathologies. Low-grade (Type I) tumours are believed to progress through benign and borderline stages to malignancy, typically exhibit mutations in KRAS and BRAF (amongst othe...

In recent years, chemokines have generated intense investigations due to their involvement in both physiological and pathological processes of inflammation, particularly in ovarian biology. The physiological process of ovulation in the normal ovary involves various chemokines that mediate the healing of the ruptured endometrium. It is now being reported that many of these chemokines are also associated with the cancer of the ovary. Chronic inflammation underlies the progression of ovarian cancer; therefore, it raises the possibility that chemokines are involved in the inflammatory process and mediate immune responses that may favour or inhibit tumour progression. Ovarian cancer is a gynaecological cancer responsible for highest rate of mortality in women. Although there have been several investigations and advances in surgery and chemotherapy, the survival rate for this disease remains low. This is mainly because of a lack of specific symptoms and biomarkers for detection. In this review, we have discussed the emerging role of the CXC chemokines in epithelial ovarian cancer (EOC). The CXC group of chemokines is gaining importance in the field of ovarian cancer for being angiostatic and angiogenic in function. While there have been several studies on the angiogenesis function, emerging research shows that ELR K CXC chemokines, CXCL9 and CXCL10, are angiostatic. Importantly, the angiostatic chemokines can inhibit the progression of EOC. Given that there are currently no biomarkers or specific therapeutic targets for the disease, these chemokines are emerging as promising targets for therapy.

Our results identify novel roles for the adipokine, adiponectin, in β-cells function. Adiponectin upregulates PPARγ expression, insulin content and insulin secretion through PPARγ-dependent mechanisms. Reductions in circulating adiponectin levels in obese individuals could therefore result in negative effects on β-cell function and this may have direct relevance to β-cell dysfunction in type 2 diabetes.

Obesity is associated with reduced levels of growth hormone (GH) and the disruption of pulsatile GH secretion. This results in relative GH deficiency. It is likely that a regulatory relationship between GH secretion and adipose tissue exists as the secretion of GH recovers to normal levels after a reduction in body weight. This report characterise the expression and interaction of adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and adiponectin, respectively, in regulating the activity of GH secreting cells. Polymerase chain reaction analysis of the GH3 cell line, rat anterior pituitary gland and isolated somatotroph cells from transgenic GFP expressing mice confirmed the expression of both AdipoR1 and AdipoR2 in GH secretory cells. Because GH cells expressed both receptors, it is likely that the measured increase in GH secretion, observed in primary cultured rat pituitary cells after 30 min of incubation with full-length murine adiponectin, was mediated by a direct receptor regulated process. Adiponectin induced an increase in intracellular Ca(2+) through both the influx of extracellular Ca(2+) and the release of intracellular Ca(2+) stores resulting in the secretion of GH. Furthermore, results confirm that this increase in GH secretion depended mainly on an increase in Ca(2+) influx through L-type Ca(2+) channels. It is concluded that adiponectin directly regulates GH secretion from somatotrophs by binding to either adiponectin receptor, and that this is mediated via a similar process observed after the stimulation of GH secretion by GH-releasing hormone.

Objective. The high temperature requirement factor A3 (HtrA3) is a serine protease homologous to bacterial HtrA. Four human HtrAs have been identified. HtrA1 and HtrA3 share a high degree of domain organization and are downregulated in a number of cancers, suggesting a widespread loss of these proteases in cancer. This study examined how extensively the HtrA (HtrA1-3) proteins are downregulated in commonly used cancer cell lines and primary ovarian tumors.Methods. RT-PCR was applied to various cancer cell lines (n=17) derived from the ovary, endometrium, testes, breast, prostate, and colon, and different subtypes of primary ovarian tumors [granulosa cell tumors (n=19), mucinous cystadenocarcinomas (n=6), serous cystadenocarcinomas (n=8)] and normal ovary (n = 9). HtrA3 protein was localized by immunohistochemistry.Results. HtrA3 was extensively downregulated in the cancer cell lines examined including the granulosa cell tumor-derived cell lines. In primary ovarian tumors, the HtrA3 was significantly lower in serous cystadenocarcinoma and granulosa cell tumors. In contrast, HtrA1 and HtrA2 were expressed in all samples with no significant differences between the control and tumors. In normal postmenopausal ovary, HtrA3 protein was localized to lutenizing stromal cells and corpus albicans. In serous cystadenocarcinoma, HtrA3 protein was absent in the papillae but detected in the mesenchymal cyst wall.Conclusion. HtrA3 is more extensively downregulated than HtrA1-2 in cancer cell lines. HtrA3, but not HtrA1 or HtrA2, was decreased in primary ovarian serous cystadenocarcinoma and granulosa cell tumors. This study provides evidence that HtrA3 may be the most relevant HtrA associated with ovarian malignancy.

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization.

The chemokine CXCL10 is a potent angiostatic and chemoattractive agent, whose primary role is to recruit activated T-cells into sites of inflammation. We have investigated the expression of CXCL10 in serous epithelial ovarian cancers (SOC), with a focus on specific post-translational modifications that control its function. The study group was comprised of a small cohort of patients diagnosed with high grade (HGSOC) or low grade (LGSOC) ovarian tumors, benign SOC, non-ovarian benign gynecological disease or no pathology. Patients were post-menopausal and had not undergone any prior treatment. CXCL10 expression in tumor tissue was assessed by quantitative PCR and western blotting. Immunoassay was used to analyse plasma CA125, and plasma and urine CXCL10 levels in matched patient samples. Localization and correlation with leukocyte recruitment was determined using immunohiostochemical staining of tumor tissue for CXCL10 and the leukocyte cell surface antigen CD45. Proteolytic processing of CXCL10 was analyzed using MALDI imaging mass spectrometry (IMS), and de novo sequence information obtained by immunoprecipitation and MS/MS analysis. We identified significantly elevated expression of both CXCL10 mRNA and protein in HGSOC, with the highest levels evident in early stage (FIGO stage Ib-c) disease. CXCL10 expression was localized to the cytoplasm of tumor epithelial cells. Despite high levels of CXCL10, however, significant leukocyte infiltration was only observed in low grade tumors or within benign cysts; HGSOC contained significantly fewer CD45+ infiltrating cells in tumor tissue. The proteolytic removal of 2 N-terminal amino acids converts CXCL10 into a potent antagonist of chemotaxis and T-cell recruitment. We therefore investigated the presence of proteolytic modifications to CXCL10 using mass spectrometry. MALDI IMS revealed masses suggesting a 2-amino acid and a 9-amino acid truncation at the N- or C-termini of CXCL10, respectively. These masses also displayed strikingly different localization; N-terminally truncated CXCL10 was present only in tumor epithelium, whilst C-terminally truncated CXCL10 was located only in the intervening regions of stromal tissue. Immunoprecipitation and MS/MS analysis confirmed the presence of N-terminally truncated CXCL10 in HGSOC. Our data identify, for the first time, the presence of the “antagonistic” form of CXCL10 in HGSOC. Antagonistic CXCL10 is present at a clinically early stage of tumor progression and correlates with poor leukocyte infiltration into tumors, suggestive of a direct influence on patient outcomes. We hypothesize that tumor-specific conversion of CXCL10 from agonist to antagonist represents a novel mechanism by which HGSOC may partially evade the early immune response. Our data also suggest inhibition of CXCL10 cleavage may represent a new avenue for the treatment of patients diagnosed with HGSOCs. Citation Format: Andrew N. Stephens, Jyothsna R. Rao, Santanu Deb-Choudhury, Adam Rainczuk. Evidence for the antagonistic form of CXCL10 in high-grade serous epithelial ovarian tumors. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B28.