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. 2022 Apr 12;13(4):337.
doi: 10.1038/s41419-022-04761-5.

Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells

Aleksandra Georgievski  1   2 , Anaïs Michel  1 , Charles Thomas  1   2 , Zandile Mlamla  1   3 , Jean-Paul Pais de Barros  1   2   3 , Stéphanie Lemaire-Ewing  1   4 , Carmen Garrido  1   2   5 , Ronan Quéré  6   7
Affiliations

Acute lymphoblastic leukemia-derived extracellular vesicles affect quiescence of hematopoietic stem and progenitor cells

Aleksandra Georgievski et al. Cell Death Dis. .

Abstract

Patient-derived xenografted (PDX) models were generated through the transplantation of primary acute lymphoblastic leukemia (ALL) cells into immunodeficient NSG mice. We observed that ALL cells from mouse bone marrow (BM) produced extracellular vesicles (EVs) with specific expression of inducible heat shock protein HSP70, which is commonly activated in cancer cells. Taking advantage of this specific expression, we designed a strategy to generate fluorescent HSP70-labeled ALL EVs and monitor the impact of these EVs on endogenous murine BM cells ex vivo and in vivo. We discovered that hematopoietic stem and progenitor cells (HSPC) were mainly targeted by ALL EVs, affecting their quiescence and maintenance in the murine BM environment. Investigations revealed that ALL EVs were enriched in cholesterol and other metabolites that contribute to promote the mitochondrial function in targeted HSPC. Furthermore, using CD34+ cells isolated from cord blood, we confirmed that ALL EVs can modify quiescence of human HSPC. In conclusion, we have discovered a new oncogenic mechanism illustrating how EVs produced by proliferative ALL cells can target and compromise a healthy hematopoiesis system during leukemia development.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. T-ALL and B-ALL PDX mice produced EVs with membrane-anchored HSP70 expression.
A Flow cytometry showing that few ALL cells (<1%) were expressing high level of HSP70. Immunostaining for HSP70 expression performed without permeabilization on BM cells from T-ALL and B-ALL PDX models. Flow cytometry is shown on hCD45+ cells for T-ALL and B-ALL viable cells (FVS450 negative). B Immunostaining on cells without permeabilization. Cells expressing HSP70 (HSP70 high) or negative for expression (HSP70 neg) were purified by FACS and observed by microscopy. EVs with membrane-anchored HSP70 expression were observed among the population of cells expressing HSP70. Example of microscopy for B-ALL cells. Microscopy with magnification ×63, scale bar represents 10 µm. C Procedure followed to isolate EVs in BM of PDX mice developing ALL. D Quantification and size of EVs isolated from BM with the NTA. Data are shown as means ± SD; n = 9 mice. P value measured by one-way Anova with Tukey’s multiple comparison test; ****P < 0.0001; ns, non-significant. E Procedure followed to isolate stain EVs prior to pull-down and analysis. F Flow cytometry confirming expression of HSP70 by EVs produced in T-ALL and B-ALL PDX mice, compared with control EVs produced by an NSG mouse. Data are representative of two independent experiments. G Microscopy on EVs purified from control NSG and ALL PDX models showing fluorescent EVs stained with HSP70-ATTO488, microscopy with magnification ×63, scale bar represents 5 µm.
Fig. 2
Fig. 2. Murine HSPC intake of T-ALL and B-ALL EVs.
A Procedure followed to identify populations of murine cells that take in fluorescent ALL EVs. B Flow cytometry on total BM cells demonstrating that primitive hematopoietic cells (Sca1+ mCD45+) take in fluorescent T-ALL and B-ALL EVs (ATTO488 EVs). C Flow cytometry gating strategy used to analyze further subpopulations among the primitive Lin− cells and their capacity to take in ALL EVs. D Mean fluorescence intensity (MFI) on Lin− cells showing that HSPC (LSK; Lin− Sca1+ c-Kit+) and HSC (SLAM; LSK CD150+ CD48−) can take in ALL EVs (ATTO488 EVs), while LK (Lin− c-Kit+ Sca1−) cells and the c-Kit negative (c-Kit−) cells cannot. The intake of SLAM cells was higher than LSK cells. Data are compared to c-Kit− cells and are shown as means ± SD; n = 3 mice. P value measured by one-way Anova with Tukey’s multiple comparison test; $P < 0.0001. E Among Lin− cells, cells showing intake of ALL EVs (ATTO488 EVs positive cells; pos) and no intake (ATTO488 EVs negative cells; neg). To examine the cell intake of EVs, Lin− cells are treated with HSP70-ATTO488 EVs, for 30 min to 2 h. Data showing that ALL EVs have the potential to rapidly bind, within 30 min, ~4% of the Lin− cells. Data are shown as means ± SD; n = 4 biological replicates. P value measured by one-way Anova with Tukey’s multiple comparison test; ****P < 0.0001. F CFU assay is assessed for 30,000 pos and neg cells purified by FACS, after 7 days on semi-solid media. Data showing that pos cells are more primitive, while they produce more CFU-GM colonies when compared with neg cells. Quantity of CFU (left panel) and distribution (right panel). Data are shown as means ± SD; n = 3 mice. P value measured by two-tailed unpaired Student’s t test; ****P < 0.0001; ns, non-significant.
Fig. 3
Fig. 3. Intake of T-ALL and B-ALL EVs by lipid raft-enriched HSPC.
A Procedure followed to identify populations of murine Lin− cells that take in fluorescent ALL EVs. Murine Sca1+ cells were purified with magnetic beads and stained with CTB prior to incubation with fluorescent ALL EVs (HSP70-ATTO488). B Flow cytometry on different populations showing that LSK and HSC (SLAM cells) express high levels of lipid rafts (CTB-AF555) as well as intake of ALL EVs (ATTO488 EVs). Sca1+ cells show intermediate levels in expression of lipid rafts and ALL EV intake, while Sca1− cells are not positive for lipid rafts expression or for intake of ALL EVs. Representative of two independent experiments. C Flow cytometry on HSC showing that when Sca1+ cells are pretreated for 2 h with Methyl-β-cyclodextrine (Mβc), this affects the lipid rafts staining (CTB-AF555) as well as the intake of ALL EVs (ATTO488 EVs). D Microscopy on murine Sca1+ cells showing that the population of primitive hematopoietic cells which take in fluorescent EVs (ATTO488 EVs) correspond to cells also displaying lipid raft staining with the cholera toxin subunit B (CTB-AF555). Magnification ×40, scale bar represents 5 µm. Representative of two independent experiments. E Single cell sorting on positive cells showing colocalization score (R) between EV intake and lipid rafts. Data shows box-and-whisker plots (n = 30 cells for each conditions), presented as medians (central line), first and third quartiles (bottom and top of boxes, respectively), and whiskers (extreme values). Fluorescent optical sections of cells, magnification ×63, scale bar represents 5 µm.
Fig. 4
Fig. 4. Injection of ALL EVs in vivo affects maintenance of murine HSC.
A NSG mice were injected with fluorescent EVs (ATTO488 EVs) by i.v. injections at 1010 particles/mouse. Mice were sacrificed 24 h after the injection, and florescence was determined by flow cytometry on Lin−, LSK (c-Kit+ Sca1+) and SLAM (LSK CD150+ CD48−) cells. B Procedure followed to treat NSG mice with ALL EVs or control EVs, through three consecutive i.v. injections, at 1010 particles/mouse/every 10 days. Ten days after the third injection, mice were sacrificed to perform experiments on Sca1+ cells. C The absolute number of Sca1+ cells detected in BM. Data are shown as means ± SD; n = 4 mice. D The number of hematopoietic progenitors (CD45+ Sca1+ c-Kit+ cells) and HSC (SLAM; LSK CD150+ CD48− cells) were determined by flow cytometry. Data are shown as means ± SD; n = 4 mice. E Sca1+ cells (105 cells) isolated from mice were analyzed on semi-solid methylcellulose media for growth of murine hematopoietic CFU. Data show the percentage of total CFU and CFU-GM per Sca1+ cells, as well as the distribution among colonies. Data are shown as means ± SD; n = 4 mice. F Sca1+ cells (3 × 105 cells) were injected in C57BL/6.SJL (Ly.1) mice and the hematopoietic reconstitution was examined by flow cytometry, 4 weeks after the transplantation. Absolute number of CD45.2 total and Lin− BM cells. Data are shown as means ± SD; n = 3 transplanted mice. G Hematopoietic reconstitution for progenitors (LSK cells) and HSC (SLAM cells). Absolute number of CD45.2 cells in BM. Data are shown as means ± SD; n = 3 transplanted mice. On this figure, P value is measured by one-way Anova with Tukey’s multiple comparison test; **P < 0.01; ***P < 0.001; ns, non-significant.
Fig. 5
Fig. 5. Exhaustion of HSPC but not MSC in ALL PDX mice.
Mice were injected with 5 × 105 cells (T-ALL) or 105 cells (B-ALL) and murine Sca1+ cells were recovered when mice were developing ALL disease (day 35). A The number of Sca1+ cells recovered from the BM of control NSG mice and PDX mice developing T-ALL or B-ALL. Data are shown as means ± SD; n = 5 mice. B Analysis of Sca1+ cells by flow cytometry showing loss of hematopoietic progenitors (CD45+ c-Kit+) and HSC (SLAM; CD150+ CD48−) in T-ALL and B-ALL PDX mice. Data are shown as means ± SD; n = 5 mice. C 25% of the Sca1+ cells were cultured in methylcellulose media and hematopoietic CFU were observed at day 7; scale bar represents 5 mm. Data are shown as means ± SD; n = 3 mice. D 75% of the Sca1+ cells were cultured in media for growth of mesenchymal CFU for 10 days, scale bar represents 1 cm. Data are shown as means ± SD; n = 3 mice. On this figure, P value is measured by one-way Anova with Tukey’s multiple comparison test; ***P < 0.001; ****P < 0.0001; ns non-significant.
Fig. 6
Fig. 6. T-ALL and B-ALL EVs affect HSPC maintenance in vitro.
Murine Lin− cells were treated ex vivo with EVs isolated from NSG control mice or ALL PDX models. A After 24 h, as assessed by flow cytometry on LSK gating cells, treatment with ALL EVs affects the numbers of HSC (SLAM). B This is further corroborated by an attrition of the primitive LSK CD34− cells. C Flow cytometry on LSK gating cells showing a loss of quiescent cells (in G0). On this figure, data are shown as means ± SD; n = 4 mice. P value are measured by one-way Anova with Tukey’s multiple comparison test; **P < 0.01; ***P < 0.001; ns, non-significant.
Fig. 7
Fig. 7. T-ALL and B-ALL EVs are enriched with metabolites and cholesterol.
A UPLC-LC-MS/MS data showing the quantity of all lipids (nmol/1010 particles) identified in T-ALL EVs and B-ALL EVs compared with NSG EVs. Triacylglycerides (TG), diacylglycerides (DG), ceramides (Cer), sphingomyelins (SM), phospho-cholines (PC), phospho-ethanolamines (PE), phospho-glycerols (PG), phospho-inositols (PI). B Distribution of each family of lipids in T-ALL EVs, B-ALL EVs and NSG EVs. C Data showing increased quantities of other amino acids measured by UPLC LC-MS/MS in T-ALL EVs and B-ALL EVs when compared with NSG EVs. D Data showing how cholesterol was highly present in T-ALL EVs and B-ALL EVs when compared with NSG EVs. ALL EVs also show elevated levels of ATP and glucose; n = 4 mice. P value measured by one-way Anova with Tukey’s multiple comparison test; **P < 0.01; ****P < 0.0001. E Flow cytometry data showing a relevant loss of quiescent cells (G0) with the lipid fractions isolated from T-ALL or B-ALL EVs when compared with lipids isolated from control NSG mice. Data are shown as means ± SD; n = 4 mice. P value measured by one-way Anova with Tukey’s multiple comparison test; ***P < 0.001; ns, non-significant.
Fig. 8
Fig. 8. T-ALL and B-ALL EVs activate mitochondrial activity in HSPC.
A Extracellular acid production measured with the Seahorse on Lin− cells after 24 h of treatment with T-ALL EVs, B-ALL EVs, or NSG EVs (2 × 109 particles); n = 4 mice, in technical triplicates. B Oxygen consumption rate (OCR) measured with the Seahorse on Lin− cells after 24 h of treatment with T-ALL EVs and B-ALL EVs, compared with NSG EVs. Data showing that the basal and maximal respiration were increased following 24 h of Lin− cell exposure to ALL EVs (2 × 109 particles). Basal respiration corresponds to means of OCR measured for points 1–4 and maximal respiration for points 8–10. Oligomycin (1.5 μM), FCCP (1 μM), antimycin A, rotenone, (AA/Rot, 0.5 μM/0.5 μM); n = 4 mice, in technical triplicates. C Flow cytometry showing increased detection of the mitochondrial membrane potential states following the exposure of Lin− cells to ALL EVs (2 × 109 particles) for 24 h. Cells were treated with TMRM for 30 min before flow cytometry recording. Data performed on Lin− cells and gating on LK, LSK and LSK CD34− cells. Example of plot for TMRM recording with dashed line corresponding to mean fluorescence intensity (MFI) for Mock. On this figure, (A, B) data are shown as means ± SD; n = 4 mice in technical triplicates, (C) data are shown as means ± SD; n = 4 mice. P value measured by one-way Anova with Tukey’s multiple comparison test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, non-significant.
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