Effects of CD68 + CD163 + M2 macrophages on tumor angiogenesis in hepatocellular carcinoma / 中华微生物学和免疫学杂志

ABSTRACT

Objective:To study the effects of CD68 + CD163 + M2 macrophages on promoting tumor angiogenesis in hepatocellular carcinoma. Methods:CD68 + M0 macrophages in human spleen tissues were separated by mechanical grinding and magnetic bead separation and used to induce CD68 + CD163 + M2 macrophages in vitro with the presence of IL-4 and IL-13. CD68 + M0 and CD68 + CD163 + M2 macrophages were respectively co-cultured with human umbilical vein endothelial cells (HUVEC) in vitro. CCK-8 assay and scratch test were performed to detect the viability and migration ability of HUVEC. Tube formation assay was used to analyze in vitro angiogenesis. Expression of vascular endothelial growth factor receptor 2 (VEGFR2), Notch1 and Dll4 in HUVEC was detected by Western blot. Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of vascular endothelial growth factor (VEGF) and IL-8 in media. BALB/c nude mice were used to construct HepG2 hepatoma model. CD68 + M0 and CD68 + CD163 + M2 macrophages were respectively injected into the mice. Expression of CD105 in tumor tissues was detected by immunohistochemistry. Expression of VEGF, VEGFR2, Notch1 and Dll4 in tumor tissues was detected by Western blot. Results:(1) IL-4 and IL-13 could induce CD68 + M0 macrophages to differentiate into CD68 + CD163 + M2 macrophages. (2) In the cell experiments, CD68 + M0 macrophages did not significantly promote the angiogenesis of HUVEC, and there was no significant change in the level of VEGF, VEGFR2, Notch1, Dll4 or IL-8. CD68 + CD163 + M2 macrophages could promote the in vitro tube formation of HUVEC, which was related to the activation of VEGF-VEGFR2-Notch1/Dll4 signaling pathway. (3) In the animal experiments, CD68 + CD163 + M2 macrophages could promote the expression of CD105, the formation of tumor vessels and the expression of VEGF-VEGFR2-Notch1/Dll4 signals. Conclusions:CD68 + CD163 + M2 macrophages could promote the formation of tumor vessels in hepatocellular carcinoma by secreting angiogenic factors including VEGF and activating VEGF-VEGFR2-Notch1/Dll4 signals.