Fig. 3.

Exosomes released by colorectal cancer-associated fibroblasts enhance endothelial cell angiogenesis and up regulate VEGF in HUVECs. A. CAF-derived exosomes enhanced the proliferation rate of HUVECs in a dose-dependent manner. B–C. Treatment of HUVECs with 100 μg/ml of CAF-derived exosomes resulted in increased cell migration in comparison with cells incubated with PBS or an equivalent of NF-derived exosomes. D. Representative photomicrographs illustrate that HUVECs treated with Exo CAF (100 μg/mL) form more and longer tube-like structures on Matrigel compared to control conditions. E, F. Quantitative assessment of the tube length and branch. Treatment of the CAF-derived exosomes (100 μg/mL) resulted in the increased total tube length (E) and the number of tube branches (F) in HUVECs. G-I. Assessment of VEGFA levels at both mRNA (G) and protein (H) levels in HUVECs treated with 100 μg/mL exosomes derived from either CAF or NFs. The original bots of Fig. 3H are provided in Supplementary Fig. S6. For quantitative analysis, the density of bands was assessed using ImageJ software and represented as relative intensity (I). J. Quantitative measurements of secreted VEGF using ELISA revealed elevated secretion of VEGFA in HUVECs treated with 100 μg/mL CAF-derived exosomes compared to control conditions and those treated with 100 μg/mL NF-derived exosomes. K, L. Western blot analysis showed the increased phosphorylated level of VEGFR2 in HUVECs incubated with 100 μg/mL CAF-derived exosomes. There was no significant difference in VEGFR2 phosphorylation levels between HUVECs incubated with NF-derived exosomes and those treated with PBS. The original bots of Fig. 3K are provided in Supplementary Fig. S7. For quantitative analysis, the density of bands was assessed using ImageJ software and represented as relative intensity (L). The columns represent the means of three independent experiments, with the bars indicating the standard deviation (SD). ns: not significant, * p-value <0.05, ** p-value <0.01, *** p-value <0.001.