To the editor:
Cancer cells produce unique heterogeneous vesicles1 capable of transferring oncogenic material2,3 to other cells,4,5 with the potential of modulating a tumor-supportive environment.6-8 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 rafts11 was used to identify the cell of origin of EVs in clinical samples. We directly compared plasma samples from CD19+ primary BCP-ALL bone marrow aspirates at diagnosis containing >95% malignant blasts with matched remission samples obtained after 28 days of therapy (Figure 1A, left panel). The diagnostic sample, which predominantly contained BCP-ALL cells, was significantly enriched in CD19+ vesicles, suggesting that the CD19+ LEVs identified were of leukemic origin. By contrast, CD61+ EVs were increased in remission marrow samples as expected in a regenerating marrow (Figure 1A, right panel). NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice are extensively used as a patient-derived xenograft model of BCP-ALL, by us10,12 and others. PKH26-labeled13 BCP cell line SD1 and LEVs derived from SD1 cells were separately transplanted intrafemorally into NSG mice. Bone marrow flushes taken 17 days later showed transplanted LEVs within a proportion of murine bone marrow stromal cells (BMSCs) (Figure 1B, upper). When labeled SD1 cells were introduced (Figure 1B, lower), cells and LEVs were detected within the extracellular space. Confocal microscopy and 3-dimensional modeling confirmed LEV internalization of prelabeled SD1 LEVs by BMSCs after 24 hours in vitro coincubation (Figure 1C). All transplanted mice showed evidence of PKH26+, human CD19+, or dual+ LEVs in peripheral blood plasma (Figure 1D), and femoral flushes (day 14) showed engrafted PKH26+ ALL cells and murine stromal cells with internalized PKH26+ LEVs (Figure 1E).
The effect of LEV internalization by BMSCs was investigated in the human mesenchymal stem cell line HS514 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 energy-producing pathways of the cell and parameters of metabolism were assessed. At 24 hours, HS5 + 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 + 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 + LEVs had comparable extracellular acidification rates (ECARs) (Figure 2E). In the presence of glucose, HS5 + LEVs initiated a sharp increase in ECAR compared with control (∼fivefold), suggesting a higher glycolytic rate. Inhibiting ATP synthase increased ECAR in both HS5 + 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 showed an increase in ECAR, which is suggestive of glycolysis. This was corroborated by the demonstration of significantly increased extracellular lactate production, the end product of aerobic glycolysis, by HS5 + LEV (Figure 2F). To investigate this further, OCR and ECARs were evaluated in the same cultures simultaneously. A shift toward a glycolytic phenotype was observed in HS5 cells treated with either NALM6 or SD1 LEVs (Figure 2G). Overall, these results suggest that uptake of LEVs significantly reduced mitochondrial respiration in recipient stromal cells and their ability to respond oxidatively to stress or metabolic challenge. Because ATP levels and proliferation rates were similar in HS5 and HS5 + LEV, LEVs induced a metabolic switch from oxidative phosphorylation to aerobic glycolysis in stromal cells to meet energy requirements.
Our observations show that both in vitro and in vivo, BCP-ALL cells release a variety of LEVs into the extracellular fluid and circulation that are taken up by BMSCs. LEV-exposed BMSCs undergo a metabolic switch. A similar reprogramming of stromal cells to glycolytic cancer-associated fibroblasts15,16 has been attributed to exosomes released by chronic lymphocytic leukemia cells.4 The altered tumor microenvironment promotes leukemic cell survival17,18 and protects against cytotoxic effects of chemotherapy. A mechanism of this protective effect of the microenvironment appears to be mediated via an adaptation to oxidative stress with decreased mitochondrial electron transfer12 and a switch to glycolysis.19 In this study, we show that the reprogrammed stromal cells generate an excess of lactate, which is released into the extracellular fluid. We speculate that this lactate is used preferentially by tumor cells as a source of energy, a process we have previously termed the reverse Warburg effect.20 Targeting glycolysis21 as well as the redox adaptation12 has been shown to overcome drug resistance in ALL. Modulating the tumor-stromal metabolic interactions offers the development of novel therapeutic strategies to enhance the therapeutic response in ALL and other cancers.
The HS5 cell line was obtained from ATCC and the SD1 and NALM6 from DSMZ.
Animal procedures were approved by Cancer Research UK, Manchester Institute's Animal Welfare and Ethical Review Body, and performed under a project license issued by the United Kingdom Home Office. Six- to 12-week-old NSG mice were transplanted intrafemorally with either 1 × 106 PKH26-labeled ALL cells, SD1 cells, or vesicles from 1 × 107 PKH26-stained ALL cells. Bone marrow flushes from transplanted mice were seeded onto fibronectin and either imaged live or fixed with 3.7% paraformaldehyde and counterstained with Cell Mask green (Life Technologies) and 4′,6-diamidino-2-phenylindole.
To assess BMSC metabolism, HS5 cells were seeded at 1 × 104 cells/well, whereas SD1 or NALM6 were cultured in serum-free RPMI. Dulbecco’s modified Eagle medium diluted 1:1 with either serum-free RPMI or LEV-containing conditioned media was added and incubated for 24 hours. HS5 ± LEVs were washed and replated into XFe96 FluxPaks before equilibration in basal media. The response to both glycolytic and mitochondrial stress was analyzed on using a Seahorse XFe96 extracellular flux analyzer (Seahorse Bioscience, Bothell, MA).
Acknowledgments: This research received funding from the European Union’s Seventh Framework Programme for research, technological development, and demonstration (Grant agreement no. 278514 – IntReALL); a program grant from Cancer Research UK; and an Leukaemia and Lymphoma Research project grant. V.S. is the recipient of an India Alliance Margdarshi Fellowship.
Contribution: S.M.J., C.D., and V.S. designed the research; S.M.J., C.D., A.C., S.H., and M.F. performed research; J.L., Y.D., and O.J.M. contributed to data interpretation; F.S. and M.P.L. contributed vital new reagents and analytical tools; S.M.J., C.D., M.P., S.K., and V.S. analyzed and interpreted data; and S.M.J. and V.S. wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Vaskar Saha, Children’s Cancer Group, Institute of Cancer Sciences, Manchester Academic Health Sciences Centre, Paterson Building, University of Manchester, 550 Wilmslow Rd, Manchester M20 4BX, United Kingdom; e-mail: email@example.com.