ANGPTL2-containing small extracellular vesicles from vascular endothelial cells accelerate leukemia progression

Abstract

Small extracellular vesicles (SEVs) are functional messengers of certain cellular niches that permit noncontact cell communications. Whether niche-specific SEVs fulfill this role in cancer is unclear. Here, we used 7 cell type-specific mouse Cre lines to conditionally knock out Vps33b in Cdh5+ or Tie2+ endothelial cells (ECs), Lepr+ BM perivascular cells, Osx+ osteoprogenitor cells, Pf4+ megakaryocytes, and Tcf21+ spleen stromal cells. We then examined the effects of reduced SEV secretion on progression of MLL-AF9-induced acute myeloid leukemia (AML), as well as normal hematopoiesis. Blocking SEV secretion from ECs, but not perivascular cells, megakaryocytes, or spleen stromal cells, markedly delayed the leukemia progression. Notably, reducing SEV production from ECs had no effect on normal hematopoiesis. Protein analysis showed that EC-derived SEVs contained a high level of ANGPTL2, which accelerated leukemia progression via binding to the LILRB2 receptor. Moreover, ANGPTL2-SEVs released from ECs were governed by VPS33B. Importantly, ANGPTL2-SEVs were also required for primary human AML cell maintenance. These findings demonstrate a role of niche-specific SEVs in cancer development and suggest targeting of ANGPTL2-SEVs from ECs as a potential strategy to interfere with certain types of AML.

Keywords: Hematology; Leukemias; Stem cells.

Figure 1. BM fluid SEVs bind to LSCs.

(A) The experimental procedure. BM cells were flushed out, and BM fluid SEVs were isolated by ultracentrifugation. The SEVs (100 μg) were labeled with CFSE and cocultured with 2 × 10⁵ L-GMP cells or Mac-1⁺c-Kit⁺ AML cells. (B) Western blot analysis of GM130, TSG101, FLOT1, and VPS33B protein levels in BM cells and BM fluid SEVs. GM130 is a cis-Golgi marker. TSG101 and FLOT1 are SEV markers. (C) Left: Images of LSCs (L-GMP cells) after coculture with CFSE-labeled SEVs. Scale bars: 5 μm. Right: The statistics of membrane-bound and internalized SEVs. In total, 145 AML cells were counted after staining. (D) Flow cytometry (left) and histogram (right) analysis of the CFSE signal of L-GMP cells (n = 3). Ctrl, control. The data represent the means ± SD; ***P < 0.001, Student’s t test. Experiments were conducted 2 times for validation.

Figure 2. Blocking SEV secretion from ECs delays AML progression.

(A) The experimental design. (B–F) Survival curves of AML recipients with different genotypes. Cdh5-Cre;Vps33b^fl/fl (B), Lepr-Cre;Vps33b^fl/fl (C), Osx-CreER;Vps33b^fl/fl (D), Pf4-Cre;Vps33b^fl/fl (E), and Tcf21-CreER;Vps33b^fl/fl (F) are shown (n = 5–8; **P < 0.01, ***P < 0.001, log-rank test). Experiments were conducted 3 times for validation.

Figure 3. EC-SEVs support leukemia development.

(A) Flow cytometry (left) and histogram (right) analysis of the percentage of YFP⁺ leukemia cells in the peripheral blood (PB) of recipients 20 days after transplantation (n = 5; the data represent the means ± SD; **P < 0.01, Student’s t test). (B) Recipient spleen and liver size (left) and weight (right) 20 days after transplantation (n = 3; the data represent the means ± SD; **P < 0.01, Student’s t test). (C) Flow cytometry (left) and histogram (right) analysis shows the percentages of L-GMP cells in the BM of recipients (n = 3; the data represent the means ± SD; **P < 0.01, Student’s t test). (D) Survival curves of Cdh5-CreER;Vps33b^fl/fl mice and Vps33b^fl/fl control mice after AML cell injection (n = 4–5; **P < 0.01, log-rank test). (E) The experimental procedure for BM EC isolation and coculture with AML cells. In brief, BM from Cdh5-CreER;Vps33b^fl/fl mice or Vps33b^fl/fl control mice was crushed and digested, and enriched using anti-CD31 beads. The enriched ECs were transduced with AKT lentiviruses and cocultured with AML cells for 3 days. After coculture, the AML cells were injected into C57BL/6J recipients. (F) Left: The morphology of the enriched and cultured ECs. Scale bar: 40 μm. Right: Flow cytometry analysis to determine the purity of cultured ECs. (G) Survival curves of C57BL/6J recipients injected with AML cells cocultured with WT ECs or Vps33b-null ECs (n = 5; **P < 0.01, log-rank test). Experiments were conducted 2–4 times for validation.

Figure 4. EC-SEVs have no effect on normal hematopoiesis.

(A–C) Cellularity analysis of Cdh5-CreER;Vps33b^fl/fl mice and Vps33b^fl/fl littermate control mice. The total BM cell numbers (A), frequencies of hematopoietic progenitor cells (B), and frequencies of HSCs/multipotent progenitors (MPPs) (C) are shown (n = 4–6; the data represent the means ± SD, Student’s t test). (D) The percentage of CD45.2⁺ donor cells in the PB of recipient mice at the indicated time points after competitive BM transplantation. The donor cells from Cdh5-CreER;Vps33b^fl/fl mice and Vps33b^fl/fl littermate control mice were CD45.2⁺ (n = 10; the data represent the means ± SEM, Student’s t test). (E) The short-term and long-term multilineage reconstitution capacities of donor-derived (CD45.2) PB cells in recipients (n = 6; Student’s t test). (F) The percentage of CD45.2⁺ donor-derived HSCs in the BM of recipients (n = 6; the data represent the means ± SD, Student’s t test). (G) The percentage of CD45.1⁺ donor cells in the PB of recipient mice at the indicated time points after BM transplantation. The donor cells were CD45.1⁺ (n = 10; the data represent the means ± SEM, Student’s t test). (H) The short-term and long-term multilineage reconstitution capacities of donor-derived (CD45.1) PB cells in recipients (n = 6; Student’s t test). (I) The percentage of CD45.1⁺ donor-derived HSCs in the BM of recipients (n = 6; the data represent the means ± SD, Student’s t test). Experiments were conducted 2–3 times for validation. GMP, granulocyte-macrophage progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte-erythrocyte progenitor; CLP, common lymphoid progenitor; MkP, megakaryocyte progenitor.

Figure 5. EC-SEVs contain high levels of ANGPTL2 protein.

(A) The experimental procedure for mass spectra analysis. The SEVs were collected from the culture supernatant of WT ECs, Vps33b-knockdown ECs, and normal BM fluid. The proteins were extracted from the SEVs for mass spectrometry. (B) Mass spectra results. The overlap in the proteins enriched in WT EC-SEVs versus BM-SEVs and WT EC-SEVs versus Vps33b-knockdown (KD) EC-SEVs is shown. (C) Western blot analysis of ANGPTL2 (A2), β-actin, and CD63 protein levels in BM ECs and EC-SEVs. (D) qRT-PCR detection of Angptl2 mRNA in the indicated cells (n = 3; the data represent the means ± SD). β-Actin was used as an internal control. Experiments were conducted 2–3 times for validation.

Figure 6. ANGPTL2-SEVs from ECs support leukemia development.

(A and B) Flow cytometry (A) and histogram (B) analysis of the percentages of GFP⁺ leukemia cells in the PB of recipients 16 days after transplantation (n = 5; the data represent the means ± SD; *P < 0.05, **P < 0.01, 1-way ANOVA followed by Dunnett’s test). Ctrl, control. (C) Survival analysis of recipients injected with AML cells cocultured with the indicated SEVs (n = 5; *P < 0.05, **P < 0.01, log-rank test). (D) The colony-forming ability of AML cells cocultured with the indicated SEVs (n = 3; the data represent the means ± SD; *P < 0.05, **P < 0.01, 1-way ANOVA followed by Dunnett’s test). (E) The in vivo administration of EC-SEVs. Equal volumes of PBS or EC-SEVs (40 μg) were intravenously injected every 5 days for 40 days. (F) The percentages of GFP⁺ leukemia cells in the PB of the recipients at indicated time points (n = 4–6; **P < 0.01, ***P < 0.001, Student’s t test). (G) Survival curves of AML recipients injected with PBS or EC-SEVs (n = 4–6; **P < 0.01, log-rank test). Experiments were conducted 2–3 times for validation.

Figure 7. VPS33B regulates ANGPTL2-SEV release.

(A) Western blot analysis of ANGPTL2, TSG101, FLOT1, and VPS33B protein levels in BM fluid SEVs from Vps33b^fl/fl mice and Cdh5-Cre;Vps33b^fl/fl mice. (B) Colocalization of CD63 and ANGPTL2 in WT and VPS33B-null ECs. (C) Flow cytometry (left) and histogram (right) analysis of the percentages of YFP⁺ AML cells in the PB of the indicated recipients injected with AML cells cocultured with Ctrl-SEVs or ANGPTL2-SEVs (n = 5; the data represent the means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA with Tukey’s multiple-comparison test). (D) Survival analysis of the indicated recipients injected with AML cells cocultured with Ctrl-SEVs or ANGPTL2-SEVs (3 μg per 1 × 10⁵ AML cells) (n = 5; *P < 0.05, **P < 0.01, log-rank test). (E) The in vivo administration of ANGPTL2-SEVs into Cdh5-Cre;Vps33b^fl/fl mice and Vps33b^fl/fl control recipients. Equal volumes of Ctrl- or ANGPTL2-SEVs (20 μg) were intratibially injected every 5 days for 20 days. (F) Survival analysis of AML recipients injected with Ctrl- or ANGPTL2-SEVs (n = 5; *P < 0.05, **P < 0.01, log-rank test). (G) The percentage of L-GMP cells in BM of indicated recipients (n = 5; the data represent the means ± SD; *P < 0.05, **P < 0.01, 1-way ANOVA with Tukey’s multiple-comparison test). Experiments were conducted 2 times for validation.

Figure 8. ANGPTL2-SEVs bind to LILRB2.

(A) Immunoelectron microscopy showing the location of ANGPTL2 protein in SEVs. Scale bars: 100 nm. (B) Flow cytometry (left) and histogram (right) analysis of ANGPTL2-SEVs binding to LILRB2-chimeric reporter cells. In this experiment, LILRB2-chimeric reporter cells will express GFP when ANGPTL2-SEVs bind to LILRB2 on the cell surface (n = 3; the data represent the means ± SD; **P < 0.01, Student’s t test). The stable chimeric receptor reporter cell system has been previously described (49). The extracellular domain of LILRB2 was chimerically fused with the intracellular domain of paired immunoglobulin-like receptor (PILR). Upon binding with ANGPTL2-SEVs, the receptor can further recruit the adaptor DAP-12 to activate the NFAT promoter, followed by the increase in GFP expression in reporter cells. (C) Colony-forming ability of Pirb^+/+ and Pirb^–/– AML cells cocultured with control- or ANGPTL2-SEVs (10 μg per 50,000 AML cells) (n = 3; the data represent the means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA followed by Dunnett’s test). (D) The percentage of AML cells in the PB of recipients injected with Pirb^+/+ and Pirb^–/– AML cells cocultured with control- or ANGPTL2-SEVs (n = 5; the data represent the means ± SD; *P < 0.05, ***P < 0.001, 1-way ANOVA followed by Dunnett’s test). (E) Survival curves of recipients injected with Pirb^+/+ and Pirb^–/– AML cells cocultured with control- or ANGPTL2-SEVs (n = 5; **P < 0.01, ***P < 0.001, log-rank test). Experiments were conducted 2–4 times for validation.

Figure 9. ANGPTL2-SEVs can target human LSCs.

(A) The experimental procedure for studying the effect of ANGPTL2-SEVs on human AML cells. 293T cells were transduced with Ctrl vectors or ANGPTL2 vectors. Ctrl-SEVs or ANGPTL2-SEVs were collected from the supernatant of the transduced 293T cells. Human AML cells (1 × 10⁶) were cocultured with 10 μg Ctrl-SEVs or ANGPTL2-SEVs, and then colony assay and transplantation into NOD/SCID mice were performed. (B) Images of CD34⁺ human AML cells captured after coculture with CFSE-labeled SEVs. Scale bar: 5 μm. (C) Flow cytometry (left) and histogram (right) analysis of the CFSE signals of CD34⁺ human AML cells (n = 3; the data represent the means ± SD; ***P < 0.001, Student’s t test). Experiments were conducted 3 times for validation.

Figure 10. ANGPTL2-SEVs support human AML development.

(A) The colony-forming ability of human AML cells after coculture with Ctrl-SEVs or ANGPTL2-SEVs (n = 3; the data represent the means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test). (B) Flow cytometry (left) and histogram (right) analysis of the percentages of human CD45⁺ AML cells in the PB of recipients injected with AML cells after coculture with Ctrl-SEVs or ANGPTL2-SEVs (n = 3–5; the data represent the means ± SD; *P < 0.05, **P < 0.01, Student’s t test). (C) Survival analysis of recipients injected with AML cells after coculture with Ctrl-SEVs or ANGPTL2-SEVs (n = 5; *P < 0.05, log-rank test). Experiments were conducted 2–3 times for validation.

Figure 11. A model showing how EC-derived SEVs regulate AML development.

VPS33B controls the release of ANGPTL2-SEVs from ECs, which further enhances leukemogenesis via its binding to the receptor LILRB2.