Fig. 4

CD39 and CD73 activity on OvCA cells promote monocyte migration in transwell chambers. Primary human monocytes were placed in the upper inserts of transwell plates while SK-OV-3 (a) or OAW-42 (b) OvCA cells were seeded in the respective bottom compartments. To explore a potential influence of ectonucleotidases, CD39 activity in tumor cells was inhibited by 100 μM ARL67156 whereas CD73 was inhibited with 100 μM α,β-methyleneadenosine-5′-diphosphate (APCP). Equal amounts of solvent (DMSO) were added to the otherwise untreated controls. To exclude unwanted effects of the inhibitors on migrating monocytes, ovarian cancer cells were pre-incubated with the inhibitors for 30 min before being washed with PBS. To assess the effect of adenosine on monocyte migration directly, positive controls with the metabolically stable adenosine receptor agonist adenosine-5′-N-ethylcarboxamide (NECA) (used at 100 nM) were also included, both in the absence and in the presence of APCP and ARL67156 (n = 3, * indicates p < 0.05, ** denotes p < 0.01, as assessed by unpaired Student’s t-test). As additional control, A2A receptor signalling was blocked during the assay by SCH58261 (in a). Under all conditions, migration of monocytes through the 8 μm wide and 10 μm thick pores was analyzed after 4 h by staining transmigrated cells for CD11c followed by flow cytometric analysis. Tumor cells from the bottom compartment were identified by co-staining for EpCAM expression