The Effects Of Tumor Derived Vegf On Endothelial Cells To Disrupt Immune Functions

BACKGROUNDWith the increasing of the incidence of cancer, tumor has become one of the main threats to human health and lifespan. According to the American cancer statistics, in 2015, the United States will have 1,658,370 new cancer cases, and 589,430 cases of patients will die of it. The incidence of breast cancer in American women cancer patients accounted for the first place, and mortality rate is accounted for the second place. In recent years, the incidence of breast cancer in china has been increasing year by year. The incidence and metastasis of cancer is a complicated process influenced by a lot of factors such as the tumor cells and host immune environment. It was found that the host immune environment plays an important role in the process of cancer incidence and metastasis. Tumor cells can secret immune suppressive products such as VEGF, PGE2 and TGF-β. Furthermore, they can also recruit host cells to aid in indirectly suppressing immune function, so patients with tumors always have defects in immune effect cell function. In these normal host cells, vascular endothelial cells is not only the physical barrier between the blood vessels and surrounding tissues, but also can secrete immune factors and regulate immune functions. While endothelial cells have been examined their role in tumor-induced immune suppression, the mechanisms remain unclear. This study aims to explore the mechanism by which tumors induce endothelial cells and the means by which endothelial cells disrupt immune functions both in mouse and human cells.METHODSBreast cancer cells were cultured for 24 hours, then the cultural supernatants were collected for backup. Human umbilical vein endothelial cells (HUVEC) were divided into four groups and respectively treated by 1640 medium, VEGF, MCF-7 cultural supernatants and SU5416+MCF-7 cultural supernatants for 24h. Then all the groups were switched to 1640 medium and cultured to 24h. The levels of VEGF, PGE2, IL-6 and IL-12 in 24h conditioned cultural supernatants were assayed by ELISA. Experiments of CCK-8, ELISPOT and flow cytometry were processed on peripheral blood T cells after treatment with the 24h conditioned cultural supernatants.30 female BALB/c mice of 6-8 weeks were randomly divided into 3 groups:(1) the PBS group: injection of PBS on the fourth breast; (2) the 4T1 groups:mammary gland injection of 4T1 cells (3) the TC group:mammary gland injection of 4T1 cells+ intragastric infusion of celecoxib. All groups of mice were collected blood samples by eyeball extirpating. Serum of PBS,4T1 and TC group was assayed of IL-6, IL-10 and IL-12 levels by ELISA. COX-2 expression of PBS and 4T1 groups in lungs were determined by Western blot and (q)RT-PCR. Another parts of lungs in PBS and 4T1 groups were separated into the mouse pulmonary vascular endothelial cells (MPVEC). The levels of PGE2 in the MPVEC 24h cultural supernatants were assayed by ELISA.RESULTS1.Breast cancer cells induced HUVEC to increase the immune suppressant factors.After treatment with the culture supernatants of MCF-7, the level of VEGF, PGE2 and IL-6 were increased in the T-sup group compared with those in control.2.Breast cancer cells induced HUVEC to increase the immune enhancement factors.After treatment with the culture supernatants of MCF-7, the level of IL-12 were decreased in the T-sup group compared with those in control.3.T cells proliferation is restrained after exposure to the HUVEC conditioned media.CCK-8 indicated that there is statistically significant differences between T cells treated with conditioned media for 48h and media alone.4.Treatment with the HUVEC conditioned media reduced the immune function of T cells.ELISPOT incline that T cells suppressed less IFN-γ production in response to the treatment of conditioned media than those in control.5.Treatment with the HUVEC conditioned media reduced CD8+ T cells function. Flow cytometric analysis demonstrated that with the treatment of conditioned media, there was a significant reduction in the production of IFN-γ in CD8+ T cells compared to control group.6.Immune suppressant factors in the blood of tumor-bearing mice was increase. ELISA showed that the levels of IL-6 and IL-10 were clearly elevated in tumor-bearing mice compared with normal group.7.Immune enhancement factors in the blood of tumor-bearing mice was decrease.ELISA showed that the levels of IL-12 was clearly elevated in tumor-bearing mice compared with normal group.8. The production of VEGF secreted by breast cancer cells induced the function of HUVEC.The culture supernatants of MCF-7 is rich in VEGF. After treatment, the level of VEGF, PGE2 and IL-6 were increased and the the level of IL-12 were decreased in the VEGF group compared with those in control. Then they had no statistically significant differences after the treatment of SU5416, VEGF receptor antagonist.9.COX2/PGE2 is up-regulated in the lung of tumor-bearing mice. (q)RT-PCR and Western blot analysis of COX2 expression in lung tissues confirmed that increased PGE2 level was measured in the 4T1 group. The level of PGE2 was increased in MPVEC 24h cultural supernatants of the 4T1 group.10.Celecoxib treatment could antagonize the immune suppression caused by the vascular endothelial cells which were induced by breast cancer cells.After the intragastric infusion of celecoxib, the levels of IL-6, IL-10 and IL-12 in the TC group were recovered compared with the PBS group and 4T1 group.CONCLUSION1.Breast tumor cells skews endothelial cells to disrupt the immune function.2.The influence on immune function is concerned with the high expression of PGE2 which is induced by VEGF..