Fig. 7: RCC-secreted circSPIRE1 is transferred to endothelial cells and suppressed vascular permeability and angiogenesis.

A Left, qRT-PCR analysis of circSPIRE1 expression in sera of 14 RCC patients with metastasis and 14 patients without metastasis. Data represent mean ± S.D.; The P values were determined by Student’s t test. Right, correlation of circSPIRE1 expression between in serum and in primary tumor. B Correlation analysis of circSPIRE1 (red) expression in RCC epithelium and stroma (labeled by CK, purple) and their adjacent endothelial cells (labeled by CD34, green). CircSPIRE1 levels were determined by the FISH score, Spearman rank correlation coefficient (rs), and P value. P value is from Spearman’s test. Scale bar, 50 µm. C Cytoplasmic and exosome RNA fractionation experiment showing that circSPIRE1 mainly localized in the exosome. D Transmission electron microscopy of exosomes derived from original PDX and PDX LM. E Exosome marker TSG101 and CD63 analyzed through western blotting. F RT-PCR analysis of circSPIRE1 expression in HUVECs incubated with exosomes derived from p-CTRL and p-circSPIRE1 cells for 3, 6, 12, 24, and 48 h. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. G Effect of p-CTRL, p-circSPIRE1 and p-circSPIRE1 + Annexin V treatments on circSPIRE1 expression in HUVECs. Data represent mean ± S.D.; dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. H Effect of exosomes derived from circSPIRE1 knockdown and control as well as overexpression and control on tube formation ability of HUVECs by tube formation assay. Mean ± S.D. are provided (n = 3/group). Scale bar represents 100 µm. I Effect of exosomes derived from circSPIRE1 knockdown and control as well as overexpression and control on vascular outgrowth of rat aortic rings. Vascular outgrowth was quantified by counting all sprouts from one ring. Left, representative aortic ring images (n = 3/group). Data represent mean ± S.D.; dot plot reflects data points from three independent experiments. Scale bar represents 200 µm. J Permeability of the HUVEC monolayers to rhodamine–dextran (70 kDa) after exposure to exosomes derived from circSPIRE1 knockdown or overexpression cells for 72 h. Mean ± S.D. are shown. Dot plots reflect data points from three independent experiments. The P values were determined by Student’s t test. K Effect of circSPIRE1 knockdown or overexpression on tumor microvessel genesis. Top, representative tumor stained with CD34 anti-body. Bottom, microvessel density for each group. Data represent mean ± S.D.; dot plots reflect data points from six independent experiments. The P values were determined by Student’s t test. L VEGFR2, Occluding and Claudin5 protein expression in circSPIRE1 overexpression or circSPIRE1 overexpression combined GALNT3 knockdown HUVECs by western blot. M Schematic diagram of mechanism of circSPIRE1 during RCC metastasis in tumor microenvironment.