Anales RANF

P.22 INVESTIGATION OF HEPARIN-INDUCED ECTONUCLEOTIDASE-INHIBITION TO COUNTERACT ADENOSINE-MEDIATED IMMUNOSUPPRESSION IN THE TUMOR MICROENVIRONMENT H. J. Maximilian Schuh, Vittoria Lopez, Martin Schlesinger, Christa E. Müller, Gerd Bendas University of Bonn, Department of Pharmaceutical Chemistry, Bonn, Germany Background: The tumor microenvironment is of crucial importance for cancer cell survival and proliferation. A multitude of different host cells, such as granulocytes, macrophages and different lymphocyte subtypes are recruited to the growing tumor nodules or the sites of metastatic niche. To escape or mitigate the attack of the immune surveillance, certain tumor cells express ectonucleotidases that generate high levels of extracellular adenosine by degradation of released ATP in the tumor microenvironment. Since adenosine is known for its potent suppressive activities on immune cells, while ATP shows the opposite effect, the pharmacological inhibition of ectonucleotidase activity of cancer cells appears an intriguing immuno-oncological approach to foster an anti-tumor immune response. Aim: Early reports described an inhibition of crude ecto-nucleotidase preparations by heparin. We have recently studied various heparin preparations on purified ecto- nucleotidase subtypes, confirming potent effects on certain ecto-nucleotidases (manuscript in preparation). To investigate whether these effects have an impact on ATP degradation and on adenosine levels in cancer cells, we established methods i) to analyze ATP degradation and adenosine formation; and ii) to study potential consequences on immune cell activities. Methods: Various human carcinoma cell lines were investigated by qPCR with respect to their mRNA expression of various ectonucleotidases. The supernatant of the glioblastoma cell line U87 was treated with ATP, and the effect of heparin on ATP degradation and the formation of its hydrolysis products, including adenosine, was analyzed by capillary electrophoresis (CE). Moreover, activated CD4 + -T-lymphocytes were treated with adenosine to monitor concentration-dependent effects on their stage of proliferation by flow cytometry. Results: The glioblastoma cell line U87 was selected as a cell model due to its high expression of ectonucleotidases. A suitable CE method was established for detecting nucleotide degradation and adenosine formation, as well as the effect of heparin on extracellular nucleotide metabolism. Proliferation of CD4 + -T-lymphocytes was slightly affected by adenosine in preliminary studies, but further experiments are required to confirm these effects. Conclusions: Heparin is a well-accepted, and guideline-based component of the clinical treatment of cancer patients. Discussions on further heparin effects, going beyond the inhibition of the coagulation system in oncology are still ongoing. Our data provide evidence that heparin is able to reduce the degradation of ATP to adenosine and accordingly impact multiple cellular effects of adenosine. It is tempting to speculate that heparin fosters the immune response towards cancer cells in an immunosuppressive tumor microenvironment.