Hypoxic exosomes facilitate angiogenesis and metastasis in esophageal squamous cell carcinoma through altering the phenotype and transcriptome of endothelial cells

Abstract

Background: In cancer progression, hypoxia, or low oxygen tension, is a major regulator of tumor aggressiveness and metastasis. However, how cancer cells adapt to the hypoxia and communicate with other mesenchymal cells in microenvironment during tumor development remains to be elucidated. Here, we investigated the involvement of exosomes in modulating angiogenesis and enhancing metastasis in esophageal squamous cell carcinoma (ESCC).

Methods: Differential centrifugation, transmission electron microscopy and nanoparticle tracking analysis were used to isolate and characterize exosomes. Colony formation and transwell assay were performed to assess the proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs). The tube formation assay and matrigel plug assay were used to evaluate the vascular formation ability of HUVECs in vitro and in vivo respectively. An in vivo nude mice model was established to detect the regulatory role of exosomes in ESCC progression. Microarray analysis was performed to analyze the transcriptome profiles in HUVECs.

Results: Exosomes derived from ESCC cells cultured under hypoxia played a better role in promoting proliferation, migration, invasion and tube formation of HUVECs in vitro and in vivo than exosomes from ESCC cells cultured under normoxia. Moreover, hypoxic exosomes significantly enhanced the tumor growth and lung metastasis compared with normoxic exosomes in nude mice models. Interestingly, endothelial cells were programmed by hypoxic and normoxic exosomes from ESCC cells which altered the transcriptome profile of HUVECs.

Conclusions: Taken together, our data identified an angiogenic role of exosomes from ESCC cells which shed light on the further application of exosomes as valuable therapeutic target for ESCC.

Keywords: Angiogensis; ESCC; Exosomes; Metastasis.

Fig. 1

Identification of the purified extracellular vesicles. a Transmission electron micrographs of extracellular vesicles derived from ECA109 and KYSE410. b The nanoparticle concentration and size distribution of the extracellular vesicles derived from ECA109 and KYSE410. c The expression level of CD9 and TSG101 (exosome specific markers) in extracellular vesicles

Fig. 2

Uptake of exosomes derived from ECA109 and KYSE410 by HUVECs at 15 min, 60 min, 2 h and 4 h. HUVECs were cultured with exosomes (25 μg /mL) from ECA109, or exosomes (25 μg /mL) from KYSE410, or in the absence of exosomes (Exosome (−)). Fluorescence microscopy images showing the internalization of exosomes by HUVECs. Blue: Nucleus stained with DAPI. Red: PKH26-labeled exosomes. Green: Phalloidin-iFluor 488 Reagent. Scale bar, 50 μm

Fig. 3

The regulatory role of normoxic and hypoxic exosomes in the proliferation, cell cycle distribution, migration and invasion of HUVECs. HUVECs were cultured with exosomes (25 μg /mL) from ECA109 that cultured in normoxic environment (norm-Exo (ECA109)) or hypoxic environment (hypo-Exo (ECA109)), or exosomes (25 μg /mL) from KYSE410 that cultured in normoxic environment (norm-Exo (KYSE410)) or hypoxic environment (hypo-Exo (KYSE410)), or in the absence of exosomes (Exosome (−)). The proliferation of HUVECs was detected by colony formation assay (a). The graph summarizes the results of three independent experiments (b). The cell cycle of HUVECs were analyzed by flow cytometry. Representative pictures of the cell cycle distributions in HUVECs (c). The graph summarizes the results of three independent experiments (d). Transwell assays were used to investigate the migratory (e) and invasive (g) abilities of HUVECs. The graph summarizes the results of three independent experiments of migration (f) and invasion assay (h). Data was presented as mean ± standard deviation (SD).*P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Hypoxic exosomes promoted angiogenesis in vitro and increased the vessel density in vivo. HUVECs were plated with matrigel and cultured with exosomes (25 μg /mL) or not. Representative pictures of tube formation were taken after stained with Calcein-AM (a). The tube formation ability was quantified by measuring the total branching length (b). Matrigel containing exosomes, or not, were injected subcutaneously into the nude mice. Representative images of the general observation of matrigel plugs (c). In vivo neovascularization induced by exosomes was measured by H&E staining. Representative pictures of neovascularization were shown in (d) and quantified for blood vessel density (e). Data was presented as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Hypoxic exosomes promoted tumor proliferation and angiogenesis in situ and facilitated lung metastasis. Xenograft transplanted tumor models were established through subcutaneously injecting ECA109 (a) or KYSE410 (b) into the nude mice. Then animals were randomly divided into five groups and injected with PBS, 10 μg exosomes from normal squamous esophageal epithelial cell line (HET-1A), exosome release inhibitor (GW4869, 1 mg/kg), 10 μg exosomes from normoxic ECA109 or KYSE410 cells (norm-Exo), or 10 μg exosomes from hypoxic ECA109 or KYSE410 cells (hypo-Exo) every 2 days (a and b). ECA109 or KYSE410 tumor weigh was measured and shown in c and e. Tumor growth curves for ECA109 or KYSE410 tumor models were shown in d and f. Then tumors were analyzed by immunofluorescence microscopy for Ki67 and CD31. Representative pictures of Ki67 were shown in g and h, and quantified for cell proliferation (i and j). Representative pictures of CD31 were shown in k and l, and quantified for vascular density (m and n). Representative images of the general observation the lungs with metastasis nodules and the corresponding H&E images of the tumor edges in lungs (o and p). The metastasis lung nodules were quantified (q and r). Data was presented as mean ± standard deviation (SD). *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Microarray analysis revealed differentially expressed RNAs between different groups. a Scatter-Plot of differentially expressed RNAs variations between HUVECs in the control group and norm-Exo group. Dots above the top line (red) and below the bottom line (green) indicated the fold change of the RNAs is more than 1.5 between the two groups. Heat map of the dysregulated mRNA, lncRNA and circular RNA expression in control group and norm-Exo group. b Scatter-Plot of differentially expressed RNAs variations between HUVECs in the control group and hypo-Exo group. Heat map of the dysregulated mRNA, lncRNA and circular RNA expression in control group and hypo-Exo group. Eight hundred and thirty nine down-regulated mRNAs (c), 113 up-regulated mRNAs (d), 232 down-regulated lncRNAs (e), 99 up-regulated lncRNAs (f), 692 down-regulated circular RNAs (g) and 86 up-regulated circular RNAs (h) were identified according to the intersection of transcriptome between norm-Exo group and hypo-Exo group

Fig. 7

Function annotation of dysregulated genes and hub genes identification. The results of GO biological process enrichment (a) and KEGG signaling pathways analysis (b) were presented as bubble chart. The size of bubble indicate the number of genes enriched in corresponding annotation and the color indicate the –log10 value of false discovery rate (FDR). The PPI network for dysregulated genes that enrichment in cell cycle (c) and cell migration (d) were plotted