The cancer cell-derived culture supernatant and the exosome-enriched fraction significantly enhance the transendothelial migration of breast cancer cells. (A) A scheme for the preparation of the cell culture supernatant, the exosome-enriched fraction and non-exosome fraction. (B) A schematic diagram of the transendothelial migration assay. (C) The transendothelial migration of breast cancer cell MDA-MB-231 after treating human umbilical vein endothelial cells (HUVECs) with the culture supernatant, exosome-enriched precipitate or non-exosome supernatant fractions. Scale bar, 100 μm. (D) Quantitative analyses of the migrated MDA-MB-231. Three independent experiments were performed, in which cell numbers in eight photo images were counted. ** p < 0.01, *** p < 0.001 by unpaired Student’s t-test.

Exosomes enhance the transendothelial migration of breast cancer cells and inhibit the HUVEC tube formation. (A) Expression of exosome markers in MDA-MB-231 exosomes revealed by Western blot analysis. (B) An electron microscopic image of exosomes (red arrows) derived from MDA-MB-231. Scale bar, 100 nm. (C) Granularity and uniformity of exosomes determined by nanoparticle tracking analysis. (D) Images of transendothelial MDA-MB-231 cells after treating HUVECs with different concentrations of exosomes. Scale bar, 100 μm. (E) Migration of MDA-MB-231 through HUVECs layers after treatment with MDA-MB-231-derived exosomes. (F) Image of tube formation in exosome-treated HUVECs. Scale bar, 1 mm. (G) The tube formation in HUVECs treated with different concentrations of exosomes. (H) Time course of the tube formation in HUVECs treated with 100 μg/mL exosomes. Data are shown as means ± standard deviation (SD) and results were from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, no significance by unpaired Student’s t-test.

Exosomes derived from MDA-MB-231 cells regulate the expression of intercellular junction molecules in HUVECs. (A,B) Confocal microscopy images of vascular endothelial cadherin (VE-cadherin) (red) (A) and zona occluden-1 (ZO-1) (red) (B) in exosome-treated HUVECs stained with corresponding antibodies. Exosomes were labeled with Dio (green). Scale bar, 20 μm. (C) Mean fluorescence intensity for VE-cadherin and ZO-1 expression in exosome-treated and control HUVECs. (D) mRNA expression of intercellular junction molecules in exosome-treated and control HUVECs by qRT-PCR analysis. Data are shown as mean ± SD and representative of three independent experiments. ** p < 0.01, *** p < 0.001 by unpaired Student’s t-test.

Exosomal thrombospondin-1 (TSP1) enhances the transendothelial migration of breast cancer cells. (A) The biological processes revealed by Gene Ontology (GO) analysis of exosomal proteome identified from mass spectral data. The numbers after each column indicated the hit number in each subcategory and p-value in parenthesis. (B) Overlapping of GO enriched biological processes for exosomal proteome related to cell–cell junction. The number of genes contained in each cluster is indicated. (C) TSP1 expression in exosomes derived from parent and gene-transfected breast cancer cells by Western blot analyses. (D) Transendothelial migration of MDA-MB-231 after treating HUVECs with exosomes derived from breast cancer cells differentially expressing TSP1. Scale bar, 100 μm. (E) Quantitative analyses of the migrated MDA-MB-231 cells in transwell assay. Data are shown as mean ± SD and representative of three independent experiments. ** p < 0.01, *** p < 0.001 by unpaired Student’s t-test.

Exosomal TSP1 derived from breast cancer cells suppresses the expression of HUVEC intracellular junction proteins. (A) mRNA transcription level of occludin, ZO-1, VE-cadherin, in HUVECs treated with different amount of LSKL. (B) mRNA transcription level of occludin, ZO-1, VE-cadherin, in HUVECs treated with MDA-MB-231-derived exosomes and different concentrations of LSKL. (C) mRNA transcription level of ZO-1 and VE-cadherin in HUVECs treated with exosomes derived from breast cancer cells differentially expressing TSP1. Data are shown as mean ± SD and representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, no significance by unpaired Student’s t-test.

Carcinoma-derived exosomal TSP1 promotes the transendothelial migration of tumor cells in zebrafish. (A) Effect of exosomes derived from breast cancer cells on the expression of VE-cadherin, ZO-1 and CD146 mRNAs. Exosomes derived from MDA-MB-231 cells were injected into 48 h post fertilization (hpf) zebrafish embryos. At 48 h after the exosome injection, 30 embryos injected with exosomes and 30 control embryos were collected for mRNA level analysis by qRT-PCR. (B) Fluorescence imaging of the breast cancer cell migration in zebrafish. Dio-labeled breast cancer cells were injected into the yolk sac of zebrafish. Arrowheads indicate disseminated tumor foci (single tumor cells or cell aggregates) in the tail regions. Scale bar, 500 μm. (C) Quantitative analyses of the breast cancer cells located within the zebrafish tail region. (D) Proportion of zebrafish with the tumor cells migrated to the tail (%). Data are shown as mean ± SD and representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; ns, no significance by unpaired Student’s t-test.

Schematic diagram of transendothelial migration of breast cancer cells with high TSP1 expression via cancer cell-derived exosomes.