Hypoxia induces metabolic adaptation in CLL cells, triggering protection from drug-induced apoptosis and production of IL-10 in an A2A-dependent manner. (A) RT-PCR analyses of GLUT1 (SLC2A1), MCT4 (SLC16A3), LDH (LDHA), and PKM2 (PKM2) in CLL cells under normoxia or hypoxia (n = 17). Where indicated, cells were treated with the A2A antagonist SCH58261 (10 µM). (B) Representative glycolytic profile obtained by dynamic measurement with the Seahorse in CLL cells cultured under normoxic (red triangles and line) or hypoxic (green squares and line) conditions or hypoxic conditions in the presence of the A2A antagonist SCH58261 (black dots and line). (C) Boxplot showing the percentage of apoptotic cells under normoxia or hypoxia. The A2A antagonist was added to block A2A signaling under hypoxia. Where indicated, cells were treated with ibrutinib (10 µM) or fludarabine (5 µM) before assessing apoptosis (n = 16). (D) RT-PCR data showing the IL-10 mRNA expression on CLL cells cultured under normoxic or hypoxic conditions in the presence of CpG/IL-2. Where indicated, leukemic cells were treated with SCH58261 (10 μM). (E-G) Immunohistochemical (E) and immunofluorescence (G) analyses of IL-10 expression in CLL and reactive LNs. Boxplot showing the percentage of IL-10⁺ area quantified in 10 independent fields from 4 CLL LNs or 3 reactive LNs (F). CD2 (red, open arrow) was used as a T-cell marker; CD20 (green) and Ki-67 (blue, full arrows) stained positive CLL lymphocytes (G). Original magnification ×20, ×40, and ×63. Statistical analyses were performed with the Wilcoxon signed rank and Mann-Whitney U tests. Quantification of brown signal in immunohistochemical analysis was performed with the LAS version 3.8 software (Leica Microsystems). Fluda, fludarabine; H, hypoxia (gray boxes); Oligo, oligomycin; Ibr, ibrutinib; N, normoxia (open boxes); SCH, SCH58261; 2-DG, 2-deoxy-d-glucose.