title: Adenosine-generating ovarian cancer cells attract myeloid cells which differentiate into adenosine-generating tumor associated macrophages - a self-amplifying, CD39- and CD73-dependent mechanism for tumor immune escape.
abstract: BACKGROUND
  Ovarian cancer (OvCA) tissues show abundant expression of the ectonucleotidases CD39 and CD73 which generate immunomodulatory adenosine, thereby inhibiting cytotoxic lymphocytes. Little, however, is known about the effect of adenosine on myeloid cells. Considering that tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) constitute up to 20 % of OvCA tissue, we investigated the effect of adenosine on myeloid cells and explored a possible contribution of myeloid cells to adenosine generation in vitro and ex vivo.
  
  
  METHODS
  Monocytes were used as human blood-derived myeloid cells. After co-incubation with SK-OV-3 or OAW-42 OvCA cells, monocyte migration was determined in transwell assays. For conversion into M2-polarized "TAM-like" macrophages, monocytes were co-incubated with OAW-42 cells. Ex vivo TAMs were obtained from OvCA ascites. Macrophage phenotypes were investigated by intracellular staining for IL-10 and IL-12. CD39 and CD73 expression were assessed by FACS analysis both on in vitro-induced TAM-like macrophages and on ascites-derived ex situ-TAMs. Myeloid cells in solid tumor tissue were analyzed by immunohistochemistry. Generation of biologically active adenosine by TAM-like macrophages was measured in luciferase-based reporter assays. Functional effects of adenosine were investigated in proliferation-experiments with CD4(+) T cells and specific inhibitors.
  
  
  RESULTS
  When CD39 or CD73 activity on OvCA cells were blocked, the migration of monocytes towards OvCA cells was significantly decreased. In vivo, myeloid cells in solid ovarian cancer tissue were found to express CD39 whereas CD73 was mainly detected on stromal fibroblasts. Ex situ-TAMs and in vitro differentiated TAM-like cells, however, upregulated the expression of CD39 and CD73 compared to monocytes or M1 macrophages. Expression of ectonucleotidases also translated into increased levels of biologically active adenosine. Accordingly, co-incubation with these TAMs suppressed CD4(+) T cell proliferation which could be rescued via blockade of CD39 or CD73.
  
  
  CONCLUSIONS
  Adenosine generated by OvCA cells likely contributes to the recruitment of TAMs which further amplify adenosine-dependent immunosuppression via additional ectonucleotidase activity. In solid ovarian cancer tissue, TAMs express CD39 while CD73 is found on stromal fibroblasts. Accordingly, small molecule inhibitors of CD39 or CD73 could improve immune responses in ovarian cancer.
authors.affiliation: Department of Obstetrics and Gynaecology, University of Würzburg, School of Medicine, Josef-Schneider-Strasse 4, 97080 Würzburg, Germany.|Interdisciplinary Centre for Clinical Research, University of Würzburg, School of Medicine, Würzburg, Germany.
authors.forename: Itsaso
authors.initials: I
authors.lastname: Montalbán Del Barrio
authors.affiliation: German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.|Edinger Institute (Neurological Institute), Goethe University, Frankfurt, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Penski
authors.affiliation: Department of Obstetrics and Gynaecology, University of Würzburg, School of Medicine, Josef-Schneider-Strasse 4, 97080 Würzburg, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Schlahsa
authors.affiliation: Department of Obstetrics and Gynaecology, University of Würzburg, School of Medicine, Josef-Schneider-Strasse 4, 97080 Würzburg, Germany.
authors.forename: Roland G
authors.initials: RG
authors.lastname: Stein
authors.affiliation: Department of Obstetrics and Gynaecology, University of Würzburg, School of Medicine, Josef-Schneider-Strasse 4, 97080 Würzburg, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Diessner
authors.affiliation: Department of Obstetrics and Gynaecology, University of Würzburg, School of Medicine, Josef-Schneider-Strasse 4, 97080 Würzburg, Germany.
authors.forename: Achim
authors.initials: A
authors.lastname: Wöckel
authors.affiliation: Department of Obstetrics and Gynaecology, University of Würzburg, School of Medicine, Josef-Schneider-Strasse 4, 97080 Würzburg, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Dietl
authors.affiliation: Institute of Virology and Immunobiology, University of Würzburg, Versbacherstrasse 7, 97078 Würzburg, Germany.
authors.forename: Manfred B
authors.initials: MB
authors.lastname: Lutz
authors.affiliation: German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany.|Edinger Institute (Neurological Institute), Goethe University, Frankfurt, Germany.
authors.forename: Michel
authors.initials: M
authors.lastname: Mittelbronn
authors.affiliation: Department of Obstetrics and Gynaecology, University of Würzburg, School of Medicine, Josef-Schneider-Strasse 4, 97080 Würzburg, Germany.
authors.forename: Jörg
authors.identifier: 0000-0002-0390-4297
authors.initials: J
authors.lastname: Wischhusen
authors.affiliation: Department of Obstetrics and Gynaecology, University of Würzburg, School of Medicine, Josef-Schneider-Strasse 4, 97080 Würzburg, Germany.
authors.forename: Sebastian F M
authors.initials: SFM
authors.lastname: Häusler
keywords: Adenosine; CD39; CD73; Immune escape; Ovarian cancer; Tumor associated macrophages