LEV uptake by BMSCs alters cell metabolism. (A) LEV uptake by BMSCs did not affect the survival or proliferation of BMSCs. HS5 cells were plated into 96-well plates at 4 × 10⁴ cells per well. Unselected LEVs were isolated from serum-free cultures of SD1 and NALM6 cells. LEV-containing conditioned media (24 hours) was diluted 1:1 with Dulbecco’s modified Eagle medium (DMEM)/10% fetal bovine serum (FBS) and added to the HS5 cells. A 3-(4,5 dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium assay was used to assess proliferation and viability of HS5 cells; HS5 + NALM6 LEVs and HS5 + SD1 LEVs over 96 hours. At least 5 wells were assessed at each time point and the experiment repeated 3 times. No significant differences were apparent. (B) Coincubation of BMSCs with ALL LEVs results in activation of AKT. HS5 cells were seeded into large 10-cm cell culture plates at 1.5 × 10⁵ cells and allowed to adhere. LEVs were isolated from 24-hour serum-free RPMI cultures of SD1 cells. HS5 media were replaced with LEV containing conditioned media diluted 1:1 with DMEM/10% FBS and incubated at 37°C 5% CO₂. Cells were washed and lysed on ice at 1, 3, 5, and 24 hours. A 30-µg protein was resolved by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. Blots were probed for pAKT, and then reprobed for AKT and β-Actin. The figure shows increased pAKT in HS5 + LEV compared with control across all time points and is representative of 3 independent experiments. (C) ATP production by BMSC + LEV is reduced compared with control BMSCs. ATP production was measured using the Cell Titer Glo assay (Promega) in HS5 cells exposed to LEVs from either NALM6 or SD1 cells for 24 hours. Data are mean and standard error of the mean (SEM) of 4 independent experiments. (D) BMSC + LEVs have lower resting energetics and are less able to respond to metabolic challenge. HS5 cells were seeded into Seahorse XFe96 cell plates at 1 × 10⁴ cells per well and allowed to attach. LEV-containing conditioned media were diluted 1:1 with DMEM/10% FBS and added to the HS5 cells for 24 hours. Cells were washed and equilibrated in basal media. An XF cell mitochondrial stress test was performed and OCR monitored following the sequential addition of oligomycin an inhibitor of ATP synthase, FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone), which uncouples electron transport from ATP generation in the mitochondria and rotenone/antimycin A, which in turn disrupts the electron transport chain using the XFe96 extracellular flux analyzer. Individual wells were normalized using sulforhodamine B. LEV uptake resulted in a lower basal respiration rate and diminished ability to respond to energetic crisis in the BMSCs. ATP-linked respiration, maximal respiration and spare respiratory capacity (difference in the basal respiratory rate and the maximal respiration) were all significantly diminished in LEV-exposed BMSCs, indicating oxidative stress. Figures are representative of 3 independent experiments with 6 replicates per plate; error bars represent SEM of a representative experiment. ATP-linked respiration: basal respiration minus proton leak reveals ATP production. Maximal respiration occurs when the uncoupler FCCP stimulates the respiratory chain to operate at its maximum speed and mimics a physiological “energetic crisis” and is measured after oligomycin through to before rotenone and antimycin A injection. Spare respiratory capacity refers to the difference between the maximal FCCP-stimulated rate of respiration minus basal respiration and reflects the cells ability to deal with an energetic crisis. (E) BMSC + LEVs use glucose more readily and have a greater glycolytic capacity. HS5 cells, HS5 + NALM6 LEVs, and HS5 + SD1 LEVs were washed and equilibrated in assay media before basal readings. Glycolysis stress test was performed and the ECAR was monitored following the sequential addition of glucose, oligomycin, and 2-deoxy-d-glucose (2DG, a competitive inhibitor of glycolysis using an XFe96 extracellular flux analyzer). Individual wells were normalized using sulforhodamine B. Glycolysis and glycolytic capacity were significantly increased in the LEV-exposed HS5 cells, whereas the glycolytic reserve was comparable to control cells. Figures are representative of 3 independent experiments with 6 replicates per plate; error bars represent SEM of a representative experiment. Glycolysis as indicated by the first measurement of ECAR (attributed to the breakdown of glucose to pyruvate) taken after glucose addition minus the ECAR before glucose addition. Glycolytic capacity refers to increased rate of glycolysis to meet the metabolic demands of the cell following the addition of oligomycin, which blocks mitochondrial ATP synthase minus ECAR before glucose addition. Glycolytic reserve indicates the increase in glycolysis needed to meet cellular energetic requirements without mitochondrial ATP production as calculated by the glycolytic capacity minus glycolysis measurements. (F) BMSC + LEVs produce more lactate compared with control. HS5 cells were seeded as described and the media harvested after 24 hours. The media from BMSC ± LEV from either SD1 cells or NALM6 cells were screened for lactate 24 hours later and compared with control. An increase in lactate was evident from BMSCs + LEVs from either leukemic cell line. Data are expressed as concentration (nmole/cell) as determined from a standard curve and normalized to individual cell counts. Figures are representative of 3 independent experiments with multiple replicates per plate; error bars represent SEM of a representative experiment. (G) LEV-exposed BMSCs switch to a glycolytic phenotype. HS5 cells were exposed to LEVs from either NALM6 or SD1 cells for 24 hours, then washed and equilibrated in mitochondrial stress test media. Extracellular acidification and oxygen consumption were measured in the same well. Although control HS5 cells maintain aerobic respiration, following exposure to LEVs for 24 hours, they switch to a more glycolytic phenotype and become less dependent on mitochondrial respiration. The figure is representative of 3 independent experiments with 6 replicates per plate; error bars represent SEM of a representative experiment. *P < .05; **P < .01; ***P < .005.