NTP pools were extracted from CLL cells after 24 hours (n?=?11) or 48 (n?=?6) h of culture and analyzed with high-performance liquid chromatography. Because OxPhos and energy production feed into nucleotide biosynthesis, we measured intracellular NTP pools. was observed in CLL cells cocultured with M2-10B4 and HS-5 stromal lines. In contrast, heterogeneous changes in the extracellular acidification rate (a measure of glycolysis) were observed in CLL cells cocultured with stromal cells. Ingenuity Pathway Salicylamide Analysis of CLL cells’ metabolomics profile indicated stroma-mediated stimulation of nucleotide synthesis. Quantitation of ribonucleotide pools showed a significant two-fold increase in CLL cells cocultured with stromal cells, indicating that the stroma may induce CLL cellular bioenergy and the RNA building blocks necessary for the transcriptional requirement Salicylamide of a prosurvival phenotype. The stroma did not impact the proliferation Salicylamide index (Ki-67 staining) of CLL cells. Collectively, these data suggest that short-term conversation (24 hours) with stroma increases OxPhos and bioenergy in replicationally quiescent CLL cells. test assuming unequal variances or a paired two-sample test for means where required. Results Stromal Cell Conversation Up-Regulates Mitochondrial OxPhos in CLL Cells We first assessed the effect that stromal cells have on mitochondrial OxPhos in CLL cells. The OCR profiles of NK.Tert cells cultured alone (blue line) Salicylamide and 1 patient’s CLL cells cultured alone in suspension (red line) or cocultured with NK.Tert cells (green line) are shown in Physique 1and and and C). Stromal Cell Conversation Does Not Significantly Affect Substrate Uptake and Mitochondrial Functionalities in CLL Cells Because stromal cells mediated OxPhos augmentation in CLL cells, we investigated whether stromal cells also induce CLL cells to switch their carbon source. We measured substrate uptake in seven patients’ CLL cells cultured alone or cocultured with NK.Tert cells (Physique 4A). Compared with CLL cells cultured alone, CLL cells cocultured with stromal cells generally had lower glucose uptake, which indicated these cells’ utilization of an alternative carbon source. Open in a separate window Physique 4 Stromal cells do not significantly affect substrate uptake and mitochondrial functionalities in CLL cells. (A) Effect of stroma on glucose (Glu) and glutamine (Gln) uptake in CLL cells. [3H]2-Deoxy-d-glucose was used to determine the cellular uptake of the substrate in CLL patient samples in suspension versus CLL patient samples in cocultures with NK. Tert cells (P?=?.03; paired t-test; n?=?7). Three technical replicates were used for each suspension and cocultured condition. Disintegrations per minute (DPM)/60 minutes were normalized to 106 cells. [3H]-glutamine was used similarly, and its cellular uptake was measured in CLL patient samples before and after NK.Tert coculture; DPM/15 minutes were normalized to 106 cells (P?=?.2; paired t-test; n?=?7). (B) Mitochondrial functional assays in CLL cells. The geometric means (decided from flow cytometry data) of patients’ CLL cells were analyzed to compare mitochondrial reactive oxygen species (ROS) (around the left y-axis) before and after NK. Tert coculture (P?=?.2). Similarly, three patient samples were analyzed for mitochondrial outer membrane potential (MOMP) and mitochondrial mass (on the right y-axis) before and after coculture (P?=?.47). (C) Immunoblot analysis of whole-cell extracts of 6 patients’ CLL cells cultured alone (control; C) and cocultured with NK. Tert cells (N). Proteins were extracted and analyzed using antibodies against all 5 mitochondrial respiratory chain complexes (I, II, III, IV, and V). (D) Effect of stromal cells on CLL mtDNA copy number. DNA was extracted from four patients’ CLL cells cultured alone or cocultured with NK.Tert cells. qPCR analysis for mtDNA in CLL cells cultured alone (black bars) and CLL cells cocultured with stromal cells (green bars) was performed in triplicate (P?=?.192). Previous studies have shown that many malignant cell types increasingly catabolize glutamine to supplement their increasing metabolic needs [24]; hence, we measured glutamine uptake in CLL cells cultured alone or cocultured with stromal cells. Compared with CLL cells cultured alone, CLL cells cocultured with stromal cells had a heterogeneous increase in glutamine uptake, although this increase was not statistically significant (Physique 4A). We assessed the effect of stromal cells on other CLL mitochondrial functionalities, such as reactive oxygen species, mitochondrial outer membrane potential, and mitochondrial mass. Flow cytometry Salicylamide revealed no significant differences in these parameters in CLL mitochondria (Physique 4B). Because OCR assays revealed that stromal cells up-regulate the mitochondrial electron transport chain activity in CLL cells, we sought to SELL determine whether stromal cells also affect mtDNA copy number and the expression.