Fig. 4
Exosomes transport TRPM2-AS to HUVECs and promote tumor angiogenesis. A, B EDU, transwell, and tube formation assays verifying the angiogenic ability of HUVECs cultured in Nc-CM/Ts-CM/Ts-CM + del-exo/Ts-exo (A), Nc-exo/Ts-exo/Ts-exo + sh-Ts (B). Nc-CM: conditioned medium from NOZ cells overexpressing empty plasmid. Ts-CM: conditioned medium from NOZ cells overexpressing TRPM2-AS. Ts-CM + del-exo: culture medium from NOZ cells overexpressing TRPM2-AS after exclusion of exosomes. Ts-exo: exosomes from NOZ cells overexpressing TRPM2-AS. Nc-exo: exosomes from NOZ cells overexpressing empty plasmid. Ts-exo + sh-Ts: knockdown TRPM2-AS using shRNA after HUVECs were co-cultured with Ts-exo. Red fluorescence: EDU-positive cells (EDU+). Scale bar: 200 μm (EDU/transwell/tube formation). C Representative images of the immunohistochemical staining of CD34 expression in tissues with high/low TRPM2-AS expression in serum exosomes from 32 GBC patients and Pearson’s correlation analysis of the positive correlation between microvascular density of GBC tissues and TRPM2-AS expression in serum exosomes. D, E EDU, transwell, and tube formation assays verifying the angiogenic capacity of HUVECs with overexpression (D) and knockdown (E) of TRPM2-AS. F-H BALB/c nude mice were treated with Nc-exo/Ts-exo every 2 days after subcutaneous injection of 2 × 106 NOZ cells (F). Representative images of surgically resected GBC tumour. Tumor weight and volume were quantified on day 20 (G) (n = 6). Representative images showing the CD34 expression level in surgically removed GBC tumors (H). Scale bar: 10 μm. I-K BALB/c nude mice were intravenously treated with Nc-exo/Ts-exo every 5 days after intravenous injection of 2 × 106 NOZ cells (I). Representative image and H&E staining of surgically resected lung (J) (n = 5). Immunohistochemical staining showing the microvascular density of pulmonary metastatic nodules (K). Scale bar: 50/200 μm. Data were assessed with unpaired Student’s t test, one-way or two-way ANOVA and presented as mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; ns, no significance