Exosomes from Plasmodium-infected hosts inhibit tumor angiogenesis in a murine Lewis lung cancer model

Y Yang, Q Liu, J Lu, D Adah, S Yu, S Zhao, Y Yao, L Qin, L Qin, X Chen, Y Yang, Q Liu, J Lu, D Adah, S Yu, S Zhao, Y Yao, L Qin, L Qin, X Chen

Abstract

Previous research to investigate the interaction between malaria infection and tumor progression has revealed that malaria infection can potentiate host immune response against tumor in tumor-bearing mice. Exosomes may play key roles in disseminating pathogenic host-derived molecules during infection because several studies have shown the involvement and roles of extracellular vesicles in cell-cell communication. However, the role of exosomes generated during Plasmodium infection in tumor growth, progression and angiogenesis has not been studied either in animals or in the clinics. To test this hypothesis, we designed an animal model to generate and isolate exosomes from mice which were subsequently used to treat the tumor. Intra-tumor injection of exosomes derived from the plasma of Plasmodium-infected mice provided significantly reduced Lewis lung cancer growth in mice. We further co-cultured the isolated exosomes with endothelial cells and observed significantly reduced expression of VEGFR2 and migration in the endothelial cells. Interestingly, high level of micro-RNA (miRNA) 16/322/497/17 was detected in the exosomes derived from the plasma of mice infected with Plasmodium compared with those from control mice. We observed that overexpression of the miRNA 16/322/497/17 in endothelial cell corresponded with decreased expression of VEGFR2, inhibition of angiogenesis and inhibition of the miRNA 16/322/497/17 significantly alleviated these effects. These data provide novel scientific evidence of the interaction between Plasmodium infection and lung cancer growth and angiogenesis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Identification of exosomes characteristics. (a) Using Nanosight to detect particle numbers of exosomes isolated from plasma of mice in different groups (*P<0.05, compared with naïve ex and LLC ex groups). (b) Using Nanosight to detect size of exosome isolated from plasma of mice in different groups. (c) A representative electron microscopic image of exosomes from mice plasma; scale bar 200 nm. (d) Confirmed by western blot for CD63, CD9, CD81 and Hsp70 exosomes markers from plasma of mice.
Figure 2
Figure 2
Exosomes suppressed tumor growth. (a) Longitudinal exosomes intra-tumor injection (each groups n=6). (b) C57BL/6 mice were inoculated with 5 × 105 lewis lung cells subcutaneously. Exosomes were injected intra-tumor from day 7 when tumor reached 3 × 3 mm2 in size. Tumor growth was monitored (V=ab2/2). Bars correspond to mean±s.d. (*P<0.05 Naïve ex versus Py ex; LLC ex versus Py+LLC ex).
Figure 3
Figure 3
Exosomes inhibited angiogenesis. (a) At 19 days, tumors were collected and presented as shown. (b) Tumors displayed varying degrees of feeding tumor vessels along the undersurface of the surrounding dermis at 19 days post inoculation. Quantification of blood vessels feeding to tumors treated with exosomes of different groups, or PBS. n=5. (*P<0.05). (c) Immunohistochemical analysis of CD31 expression in tumor tissues. Scale bar 20 μm (***P<0.001 Py ex and Py+LLC ex groups compared with PBS, Naïve ex and LLC ex groups).
Figure 4
Figure 4
Exosomes uptake by MS1 cells in vitro. (a) Confocal images of cultures exposed to exosomes at 6 h. The red channel is representative of Dir emission, the blue channel is Hoechst nuclear stain. Fluorescent signals are merged with transmission images. Scale bar is 20 μm. (b) MS1 co-culture with different exosomes at 24 h and uptake confirmed by western blot for red blood cell special marker CD235a.
Figure 5
Figure 5
The effect of exosomes on tube formation and migration in endothelial cell. (a) Endothelial cell tube formation assay showed interference of network assembly of MS1 cells on pre-solidified Matrigel in medium containing exosomes. Scale bar 500 μm (*P<0.05). (b) The invasion activity of MS1 cell incubated with different exosomes using transwell method. Scale bar 100 μm (NC: Negative control, PC: Positive control, ***P<0.001). (c) Endothelial cell migration was measured after co-culture with different groups exosomes for 24 h. Lines indicated the edge of the ‘wound’ directly after making the scratch. Statistical analysis of the width of the scratches is shown on the right (*P<0.05).
Figure 6
Figure 6
Exosomes inhibited vegfR2 expression in MS1 cells. (a) MS1 cells were cultured in vitro and exosomes were added in the culture medium for 24 h. VEGFR2 mRNA expression was detected using qPCR (*P<0.05). (b) Western blotting analysis of phospho-VEGFR2 and VEGFR2 expression in MS1 cells co-culture with exosomes; Quantitative results of western blotting assay showed thatVEGFR2 and phospho-VEGFR2 expression (*P<0.05).
Figure 7
Figure 7
miRNAs is overexpressed in plasmodium-infected mice plasma exosomes, downregulated VEGFR-2 and inhibited tube formation. (a) qPCR detected the level of miRNAs expression in exosomes of four groups. (b) VEGFR2 is a target gene of miR(16-5p/322-5p/497-5p/17-5p). (c) Luciferase reporter assay was performed using 293T cells as described in the Materials and methods section (*P<0.05,compared with miRNA negative control; # P<0.05, compared with miRNAs add inhibitors). (d) The relative expression of VEGFR2 mRNA in MS1 cells transfected with different combination of miRNAs based on qPCR (*P<0.05, compared with miRNA negative control). (e) The protein level of VEGFR2 in MS1 cells transfected with two combination of miRNAs based on western blotting. (f) Endothelial cell tube formation assay showed interference of network assembly of MS1 cells on pre-solidified Matrigel. Scale bar 500 μm (miRNA NC: Transfection of miRNA negative control; miRNAs: Transfection of miR (16-5p/322-5p/497-5p/); miRNAs+inhibitors: Transfection of miR (16-5p/322-5p/497-5p/17-5p) and inhibitors. *P<0.05).
Figure 8
Figure 8
miRNAs inhibitors rescued the effect of exosomes inhibition on VEGFR2 expression and tube formation in endothelial cells. (a) The relative expression of VEGFR2 mRNA in MS1 cells co-cultured with different groups exosomes and transfected with miRNAs inhibitors based on qPCR (***P<0.01). (b) The protein level of VEGFR2 in MS1 cells co-culture with Py or Py+LLC exosomes and miRNAs inhibitors based on western blotting. (c) Endothelial cell tube formation assay showed interference of network assembly of MS1 cells on presolidified Matrigel. Scale bar 500 μm (miRIn: Transfection of miR (16-5p/322-5p/497-5p/17-5p) inhibitors. ***P<0.01).
Figure 9
Figure 9
Schematic of plasma exosomes from plasmodium-infected mice inhibition of angiogenesis through miR(16/322/497/17) to target VEGFR2.

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