Bone marrow stromal cells promote aerobic glycolysis in primary CLL cells. (A) The ECAR, which quantifies proton production as a surrogate for the overall glycolytic flux was evaluated in CLL cells (n = 4) following a 6-day culture in the presence or absence of stromal contact under baseline conditions and upon glucose administration using an XFe96 flux analyzer. (B) Glucose uptake in primary CLL cells (n = 6) cultured for 6 days in the presence or absence of HS-5 as quantified by FACS based on the mean fluorescence index (MFI) of the incorporated fluorescent glucose analog 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose). (C) Density of the glucose transporter GLUT3 was assessed on primary CLL cells (n = 6) cultured in the presence or absence of HS-5 by FACS. (D) ECAR was measured in purified primary CLL cells (n = 4) following a 6-day culture in the presence or absence of stromal contact under basal conditions, in response to glucose, and upon blocking the mitochondrial ATP generation by oligomycin. The resulting (compensatory) effects on ECAR following the interference with the mitochondrial energy metabolism represent the maximal glycolytic capacity and are shown as a percentage of the baseline measurement (set as 100%). (E) Changes in the relative gene expression of key glycolytic enzymes including hexokinase-2 (hk2), lactate dehydrogenase A (ldha), pyruvate dehydrogenase kinase-1 (pdk1), enolase-1 (eno1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), and glucose-6-phosphate dehydrogenase (g6pdh) were determined by qPCR in primary CLL cells (n = 5-8) upon stromal contact for 6 days as compared with cells cultured alone (set as 1). (F) ECAR was measured in response to glucose in purified primary CLL cells (n = 4-6) following culture in the presence or absence of contact to primary human bone marrow-derived MSCs from 3 healthy donors or primary HLFs from 1 donor. (G) The oxygen consumption rate (OCR) indicative for mitochondrial respiration was measured by an XFe96 flux analyzer under baseline conditions in purified primary CLL cells (n = 4) following a 6-day culture in the presence or absence of stromal contact. (H) In addition to measurements under basal conditions, OCR was assessed in response to the indicated mitochondrial inhibitors. The resulting effects on OCR are shown as a percentage of the baseline measurement (set as 100%) for each treatment. Alterations after oligomycin and FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) are indicative for respiration linked to ATP production and the maximal respiratory capacity, respectively. (I) The ΔΨM was semiquantified using the potentiometric dye JC-1 (Cayman Chemical) by FACS. CLL cells (n = 3) cultured with/without HS-5 cells were comparatively assessed. (J) ATP levels were measured in lysates from purified CLL cells (n = 5) cultured alone or in the presence of HS-5 cells using a colorimetric assay. (K) The % specific cell death as assessed by FACS is shown for purified CLL cells treated for 24 hours with 2-deoxy-d-glucose (2-DG) (n = 5) and diclofenac (n = 3) in the presence or absence of HS-5 cells. Bars indicate the standard error of the mean. * P < .05; ** P < .005; *** P < .001.