Metabolic reprogramming of bone marrow stromal cells by leukemic extracellular vesicles in acute lymphoblastic leukemia

Abstract

Cancer cells produce unique heterogeneous vesicles 1 capable of transferring oncogenic material 2,3 to other cells, 4,5 with the potential of modulating a tumor-supportive environment. [6][7][8] We have previously reported the presence of lipid-enriched, membrane-bound subcellular vesicles at the periphery of acute lymphoblastic leukemia (ALL) cell lines. 9,10 We now extend these findings to describe heterogeneous anucleate vesicles released into extracellular fluids in vitro and in vivo by primary B-cell precursor (BCP) ALL blasts and cell lines. Leukemic extracellular vesicles (LEVs) were internalized by stromal cells, and induced a metabolic switch.Extracellular vesicles (EVs) are enclosed in lipid bilayers originating from the cell of origin, released by both normal and cancer cells.1 Here, the BCP cell-specific membrane protein CD19 present within membrane lipid rafts 11 was used to identify the cell of origin of EVs in clinical samples. We directly compared plasma samples from CD19 1 primary BCP-ALL bone marrow aspirates at diagnosis containing .95% malignant blasts with matched remission samples obtained after 28 days of therapy ( Figure 1A LEVs ( Figure 1E). The effect of LEV internalization by BMSCs was investigated in the human mesenchymal stem cell line HS5 14 exposed to LEVs released by the BCP-ALL cell lines SD1 and NALM6. Proliferation and viability assays revealed no significant differences from control ( Figure 2A). Despite a sustained increase in AKT phosphorylation over 24 hours ( Figure 2B), nonsignificant reductions in adenosine triphosphate (ATP) concentrations were observed ( Figure 2C). Next, the 2 major energyproducing pathways of the cell and parameters of metabolism were assessed. At 24 hours, HS5 1 LEVs showed a reduced oxygen consumption rate (OCR) compared with control, were less sensitive to the inhibition of ATP by oligomycin, and did not change OCR when electron transport from ATP generation in the mitochondria was uncoupled ( Figure 2D). Disrupting the electron transport chain (rotenone/antimycin A) reduced OCR to a comparable level in all cells, suggesting that the rate of oxygen consumption from nonmitochondrial sources was comparable. HS5 1 LEV have a significantly reduced spare respiratory capacity, an indicator of a decreased ability to respond to stress or metabolic challenge ( Figure 2D). Overall, these results suggest that uptake of LEVs significantly reduced mitochondrial respiration in recipient stromal cells.In the absence of glucose, HS5 and HS5 1 LEVs had comparable extracellular acidification rates (ECARs) ( Figure 2E). In the presence of glucose, HS5 1 LEVs initiated a sharp increase in ECAR compared with control (;fivefold), suggesting a higher glycolytic rate. Inhibiting ATP synthase increased ECAR in both HS5 1 LEVs and controls, but more sharply in the latter. Following the addition of 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, ECARs returned to base levels in both control and LEV-exposed cells. Thus, in the presence of glucose, LEV-exposed HS5 s...

Investigating chemoresistance to improve sensitivity of childhood T-cell acute lymphoblastic leukemia to parthenolide

“…The switch to glycolysis led to an increased release of the metabolite lactate, which might be used as an additional source of energy by leukemia cells and confer chemoresistance. 34 …”

Large extracellular vesicles can be characterised by multiplex labelling using imaging flow cytometry

“…We previously reported a population of large EVs released by leukaemic cells which were actin-rich and contained intact organelles (11). These large EVs could be internalised by normal stromal cells and induced a switch in the preferred metabolic pathway of the recipient cells (9). Additionally, we found that leukaemia-derived EVs expressed a surface marker indicative of their parent cell (CD19) and could be detected in the peripheral blood of murine models and patient bone marrow plasma (9).…”

“…Large EVs, defined as >200 nm by recent guidelines set out by the International Society of Extracellular Vesicles (1), include cancer cell-derived oncosomes, dead cell-derived apoptotic bodies and platelets, and are visible by light microscopy (9). In published literature, EVs larger than 1 µm have historically been assumed to be apoptotic bodies (10).…”

“…These large EVs could be internalised by normal stromal cells and induced a switch in the preferred metabolic pathway of the recipient cells (9). Additionally, we found that leukaemia-derived EVs expressed a surface marker indicative of their parent cell (CD19) and could be detected in the peripheral blood of murine models and patient bone marrow plasma (9). Taken together, our previous work and existing large EV literature suggest that large EVs, often discarded in techniques to isolate smaller EVs and exosomes, could be considered as extensive reservoirs of biomolecules useful to study EV biogenesis and function, and to identify clinically relevant biomarkers for disease detection and treatment monitoring.…”

Extracellular Vesicles in Hematological Malignancies: From Biology to Therapy

“…GAL3was internalized by ALL cells, where it stimulated transcription of endogenous GAL3 mRNA and was a protection toward drug treatment [109]. Also, ALL Exo could be captured from stromal cells, inducing a metabolic switch from oxidative phosphorylation to aerobic glycolysis [110]. …”