Abstract Background: Ectonucleotidases CD39/CD73 have been reported to play an important role in functional supression of various immune cells via adenosine that is generated locally in the tumor microenvironment. In patients with ovarian cancer (OvCa) exosomes released by tumor cells (TEX) are abundant in body fluids, including the plasma or ascites and may be involved in tumor progression. Based on the observations suggesting that TEX carry proteins that are expressed on tumor cells from which TEX originate, we hypothesized that CD39+ and CD73+ TEX could deliver these enzymes to distant immune cells. Adenosine produced fom ATP in the presence of TEX could suppress functions of these cells or elevate suppressor activity of regulatory T cells (Treg) , The aim of the study was to: (1) investigate the expression and clinical significance of CD39, CD73, adenosine deaminase (ADA) and CD26 in OvCa tissues and in TEX isolated from OvCa body fluids; (2) determine whether OvCa TEX metabolize exogeneous ATP to adenosine. (3) characterize the molecular profile of TEX; and (4) test whether OvCa TEX can suppress activities of NK cells and up-regulate suppressive activity of Treg. Methods: The expression of CD39, CD73, ADA and CD26 in OvCa tissues was determined by immunohistochemistry (IHC). The relationship between expression of these enzymes and clinicopathological characteristics was analyzed. TEX were isolated from supernatant of two OvCa cell lines (A2780, SKOV-3) and from the patients' plasma by exclusion chromatpgraphy followed by ultracentrifugation, as previously described.. Western blots were used for molecular characterization of TEX. NK cells and Treg were separated from the PMBC of normal donors and co-cultured with TEX. The phenotype of NK cells and Treg was evaluated by flow cytometry. ATP hydrolysis was measured using a luciferase detection assay. Results: By IHC in tissue sections, 70% of tumor cells were CD39+, 77% were CD73+ and 100% were CD26+ADA+.Expression levels of the ectonucleotidases varied from strong to moderate, and patients with a more advanced disease stage had tumors showing strongest CD73 expression (p<0,05). Exosomes isolated from plasma of OvCa patients were enriched in TEX which carried LAMP-1, CD63, TGF-β1, MAGE3/6, CD39, CD73, ADA and Ep-CAM. In contrast, exosomes isolated from the plasma of healthy donors carried LAMP-1 and CD63. TEX obtained from OvCa patients hydrolyzed more exogeneous ATP than did TEX from OvCa cell line supernatants (p<0.05) and produced more adenosine (p<0,05). After co-incubation with TEX, normal NK cells downregulated expression of NKG2D, NKp44 and NKp46 (p<0,05) and Treg up-regulated expression of Perforin, FasL, CCR7 (p<0,05). Co-incubation of Treg with TEX resulted by increased suppression of responder cells (p<0,01). Conclusion: Similar to OvCa tumor cells in tissue exosomes isolated from the plasma of OvCa patients were found to carry enzymatically-active ectonucleotidases and to produce extracellular adenosine. These exosomes were capable of down-regulating NK cell functions and up-regulating Treg activity in vitro. The same TEX-mesiated mechanisms could contribute to tumor-induced immune suppression characteristic of OvCa and resulting in tumor immune escape and OvCa progression. Note: This abstract was not presented at the conference. Citation Format: Marta E. Szajnik-Szczepanski, Magdalena Derbis, Michal Lach, Paulina Patalas, Marcin Michalak, Hanna Drzewiecka, Marta Glura, Ewa Nowak-Markwitz, Marek Spaczynski, Theresa L. Whiteside. The adenosine pathway in ovarian carcinoma: Tumor cells and tumor-derived exosomes express CD39 and CD73 ectonucleotidases, produce adenosine and mediate immune suppression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A79.

BACKGROUND:In patients with Ovarian Cancer (OvCa) exosomes released by tumor cells are present in the plasma and could be involved in tumor progression. This study examines the association between the exosome presence/protein content in plasma of OvCa patients and disease outcome, response to standard therapy and/or tumorresistance to therapies in patients studied at diagnosis and also serially during and after therapy.DESIGN AND METHODS:Exosomes were purified from OvCa patients' plasma (n=22), patients with benign tumors (n=10) or (n=10) healthy controls (NC) using ultracentrifugation. Exosomes were visualized by scanning electron microscopy. Their protein content was measured. The presence of MAGE 3/6 and TGF-β1 in exosomes was evaluated in Western blots.RESULTS:The OvCa patients' plasma contained higher levels of exosomal proteins (p<0.05) compared to those isolated from plasma of patients with benign tumors or NC. Exosomes isolated from OvCa patients's plasma carried TGF-β1 and MAGE3/6, which distinguished OvCa patients from those with benign tumors and NC. High protein levels of exosomes were seen in newly diagnosed patients; however in advanced stages of OvCa patients the protein content of isolated exosomes was significantly higher than that of early stages. The exosome levels variably changed during/after chemotherapy, and correlations between the changes in exosomal protein levels and clinical data suggested that the protein content of exosomes might be useful in predicting responses to therapy and prognosis in OvCa patients.CONCLUSION:Analysis of plasma exosomes levels offers a novel approach to diagnosis and monitoring response to therapies in OvCa patients.