Hypoxia upregulates the adenosinergic axis in CLL cells. (A) RT-PCR data or (B) mean fluorescence intensity (MFI) and protein levels of CD39 (ENTPD1) or CD73 (NT5E) expression in CLL cells cultured for 48 hours under normoxic or hypoxic conditions. For CD73 analysis, only the patients that were >30% CD73⁺ at the baseline (17/40) were analyzed. (C) HPLC chromatogram showing adenosine monophosphate (AMP) catabolism in CLL lymphocytes under normoxia (black line) or hypoxia (blue line) and in the presence of the adenosine deaminase inhibitor EHNA (red line). The gray line profile represents the medium alone. (D) Boxplots showing nanomoles of adenosine produced by 10⁶ purified CLL cells after 30 minutes of normoxic or hypoxic culture in the presence of 200 µM AMP (n = 8). The adenosine deaminase inhibitor EHNA (10 µM, 30 min, 37°C) was added where indicated (n = 8). (E) Comparative expression of CD26 (DDP4) mRNA (n = 10) and surface protein levels (n = 10) in circulating CLL lymphocytes under normoxia or hypoxia. (F) Boxplots representing nanomoles of inosine (INO) accumulated by 10⁶ purified CLL cells cultured for 30 minutes in the presence of 200 µM AMP under normoxia or hypoxia. The adenosine deaminase inhibitor EHNA was added where indicated (n = 8). (G-H) RT-PCR data of ENT1 (SLC29A1, n = 10) and ENT2 (SLC29A2, n = 10) nucleoside transporters and A2A receptor (ADORA2A, n = 20) expression under normoxic or hypoxic conditions. (I) RT-PCR data comparing expression of CD39 (ENTPD1), CD73 (NT5E), CD26 (DPP4), ENT1 (SLC29A1), and A2A receptor (ADORA2A) mRNA in the PB or LN of samples obtained concurrently from 9 different patients. Expression was significantly higher in CLL cells obtained from the LN than in paired PB samples. Statistical analyses were performed with the Wilcoxon signed rank test followed by Tukey test. a.u., arbitrary units; ADO, adenosine; INO, inosine; H, hypoxia (gray boxes); N, normoxia (open boxes).