Abstract: BACKGROUND: Exosomes can release RNA, proteins and other signaling molecules to the surrounding environment to influence tumor development. Angiopoietin-2 expression is significantly increased in oral squamous carcinoma tissues and angiopoietin-2 overexpression is closely associated with tumor lymphangiogenesis, increased microvessel density, and poor prognosis of the patients. However, it is not clear whether exosomes of oral squamous cell carcinoma origin contain angiopoietin-2 and influence tumor development and angiogenesis. 
OBJECTIVE: To investigate the potential mechanism of exosomes regulating angiogenesis in oral squamous cell carcinoma.
METHODS: (1) The effects of Cal-27 and Scc-25 cell supernatants on the proliferation and migration of human umbilical vein endothelial cells were observed by CCK8 and Transwell assays. (2) The exosomes in the supernatants of Cal-27 and Scc-25 cells were extracted and identified using an exosome extraction kit. (3) Immunofluorescence assay was used to detect whether human umbilical vein endothelial cells could take up Cal-27 and Scc-25 exosomes. (4) Western blot assay was used to detect the expression of angiopoietin-2 in Cal-27 and Scc-25 cells and exosomes. (5) Cal-27 and Scc-25 cell exosomes at different mass concentrations (25 and 50 µg/mL) were used to intervene in human umbilical vein endothelial cells. Western blot assay, CCK-8 assay, Transwell, and tube formation experiments were used to detect the expression of angiopoietin-2 and CD34 in human umbilical vein endothelial cells and their effects on the proliferation, migration, and tube formation of human umbilical vein endothelial cells. (6) Lentivirus transfection was used to construct Cal-27 and Scc-25 cells and exosomes with overexpression and knockdown of angiopoietin-2. qRT-PCR and western blot assay were used to detect the transfection efficiency. Western blot assay, CCK-8 assay, Transwell, and tube formation experiments were used to detect the regulatory effects of Cal-27 and Scc-25 exosomes with overexpression and knockdown of angiopoietin-2 on human umbilical vein endothelial cells. (7) Twenty 5-week-old BALB/cA female mice were randomly divided into four groups: control group, Cal-27 exosome group, Cal-27 exosome group with angiopoietin-2 overexpression, and Cal-27 exosome group with angiopoietin-2 knockdown, with five mice in each group. Mice were subcutaneously implanted with Cal-27 cells. When the tumor volume grew to 50 mm3, exosomes were injected peritumorally on day 10 after tumor implantation according to the group assignment. The control group was injected with an equal amount of PBS every 2 days. On day 30, tumor tissues were collected for hematoxylin-eosin staining, Ki67 and CD31 immunohistochemical staining. 
RESULTS AND CONCLUSION: (1) Compared with the control group, the supernatants of Cal-27 and Scc-25 cells promoted the proliferation and migration of human umbilical vein endothelial cells (P < 0.05). (2) Human umbilical vein endothelial cells could take up Cal-27 and Scc-25 exosomes. (3) Angiopoietin-2 was contained in Cal-27 and Scc-25 cells and exosomes. With the increase of exosome concentration, the expression levels of angiopoietin-2 and CD34 proteins in human umbilical vein endothelial cells increased, and the proliferation, migration, and tube formation of human umbilical vein endothelial cells were enhanced (P < 0.05). (4) Cal-27 and Scc-25 exosomes overexpressing angiopoietin-2 further promoted the proliferation, migration, and tube formation of human umbilical vein endothelial cells (P < 0.05). Cal-27 and Scc-25 exosomes with knockdown of angiopoietin-2 inhibited the proliferation, migration, and tube formation of human umbilical vein endothelial cells (P < 0.05). (5) Animal experiments showed that compared with the control group, the tumor volume of the Cal-27 exosome group did not increase significantly, but the expression levels of Ki67 and CD31 increased. The tumor volume of the Cal-27 exosome group with angiopoietin-2 overexpression increased significantly, and the expression levels of Ki67 and CD31 increased significantly. The tumor volume and the expression levels of Ki67 and CD31 in the Cal-27 exosome group with angiopoietin-2 knockdown decreased (P < 0.05). These results indicate that oral squamous cell carcinoma-derived exosomes participate in tumor angiogenesis by delivering angiopoietin-2 and affect tumor development.  

Key words: oral squamous cell carcinoma, exosome, angiopoietin-2, human umbilical vein endothelial cell, CD34 protein, CD31 protein, tube formation experiment, angiogenesis