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Fig. 6 | Journal for ImmunoTherapy of Cancer

Fig. 6

From: 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

Fig. 6

Macrophages from in vitro co-culture with OvCA cells generate adenosine via CD39 and CD73. Human macrophages were generated and polarized in transwell coculture with OAW-42 OvCA cells. For control purposes, M1 macrophages were induced with LPS (10 μg/ml) and IFN-γ (1 μg/ml). After coculture with ADORA2A-overexpressing, RIP1-CRE transfected sensor cells, a luciferase-based reporter assay was performed to determine production of biologically active adenosine [45]. To confirm that adenosine generation depends on ectonucleotidase activity, CD39 was blocked with ARL67156 (MФ (Co-culture) + ARL) during coculture. Likewise, CD73 was inhibited by APCP (MФ (Co-culture) + APCP) and to inactivate both ectonucleotidases, ARL67156 and APCP were also combined (MФ (Co-culture) + ARL + APCP). To the control groups (MФ (Co-culture) and MФ (IFN-γ + LPS)), only the solvent DMSO was added (n = 3). Significance levels were determined by Student’s t-test. p-values < 0.01 were considered as highly significant (**)

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