Dextran Sulfate Inhibits Gastric Cancer Angiogenesis By Interfering The Polarization Of M2-type Macrophages

Objective To explore the effect of dextran sulfate on the polarization and recruitment of M2-type macrophages,and the indirect effect of dextran sulfate on the angiogenesis,invasion and migration of gastric cancer cells through M2-type macrophages.Methods Part Ⅰ Nude mice metastasis models were established by the abdominal cavity inject method,the intraperitoneal metastasis tumor of nude mice was observed 14 days later.Immunohistochemistry and Western blot were used to assess the expression level of M2-type macrophage molecular marker CD163,CD206 and angiogenesis related protein CD34,VEGF and VEGFR2 in metastatic tumors.Immunohistochemistry and immunofluorescence were used to measured the infiltration degree of M2-type macrophages and microvessel density in gastric cancer and paracancerous tissues,and the expression and tissue localization of CD163 and CD34 were analyzed.Part Ⅱ THP-1 monocytic cells were differentiated into M0,and M2 macrophages,and identified by cell morphology,the surface markers CD68 and CD163/CD206 of M0 and M2 macrophages were verified by immunofluorescence and Western blot,and the results suggested that the induction was successful.DS was added during macrophage induction and polarization,and divided into M0 group,M0 DS group,M2 group and M2 DS group.The expression of CD163 and CD206 and the key factors of the polarization pathway IL-6,STAT3 and p-STAT3 of each group were measured by Western blot.M2-type macrophage specific cytokines Arg-1 and IL-10 in the supernatant of macrophages were measured by ELISA.The chemotactic recruitment ability of macrophages in the four groups was detected by Transwell migration assay.Established co-culture model of macrophages and gastric cancer cells and divided into GC group,M0+GC group,M0DS+GC group,M2+GC group and M2DS+GC group.The supernatant of co-culture system was collected for the tube formation experiment,and the expression level of VEGFR2 in vascular endothelial cells was detected by Immunocytofluorescence.The ability of cell invasion and migration in gastric cancer was evaluated by Transwell invasion and Wound healing assay separately,the expression of angiogenesis and invasion and migration related proteins VEGF,MMP-2 N-cadherin,Vimentin in co-cultured gastric cancer cells were measured by Western blot.Results Part Ⅰ The diffuse distribution of grayish-white tumor nodules was observed in the control group 14 days later,and the number of tumor nodules was significantly higher than DS group(P=0.014);Immunohistochemistry results indicated that the infiltration degree of M2-type macrophages and MVD in DS group was significantly lower than that in control group(P<0.001);Western blot indicated that the expressions of CD163,VEGF and VEGFR2 in the DS group were declined than control group(P=0.037,P=0.002,P=0.001).Immunohistochemistry results showed that the M2-type macrophages and MVD in human gastric cancer tissues were significantly increased(P=0.004,P=0.001),and the number of M2-type macrophages was positively correlated with MVD(r=0.7013,P<0.001);Immunofluorescence double staining showed that M2-type macrophages were mostly infiltrated around microvessels,and located in tumor stroma.Part Ⅱ Under inverted microscope,M0 macrophages were adherent round,and M2-type macrophages were protuberant cell.Immune cell fluorescence and Western blot were used to verify the surface markers CD68 and CD163/CD206 of M0 and M2 macrophages,and the results indicated that the induction was successful.CCK8 assay showed that DS concentration of 0-0.5% had no cytotoxicity to M0 and M2 type macrophages.After the treatment of macrophage polarization with DS,Western blot indicated that DS could reduce the expression of CD206,CD163,IL-6 and p-STAT3(P<0.001,P<0.01).ELISA results indicated that DS down-regulated the secretion levels of IL-10 and Arg-1 in the supernatant of macrophages(P<0.05).Transwell assay result indicated that DS could significantly inhibit the chemotactic recruitment of M0 and M2-type macrophages(P<0.001).After co-culture with M2-type macrophages,the angiogenic ability of gastric cancer cells was enhanced(P<0.001),Immunocytofluorescence showed that the expression of VEGFR2 on HUVECs was increased(P<0.001),Transwell and Wound healing showed that invasion and migration ability was significantly enhanced(P<0.001),Western blot results indicated that the expressions of angiogenesis,invasion and migration related proteins VEGF,MMP-2,N-cadherin and Vimentin in gastric cancer cells were significantly upregulated(P<0.001).After co-culture with M2-type macrophages treated with DS,the angiogenic ability of gastric cancer cells was decreased,the expression of VEGFR2 was weakened,the ability of invasion and migration were decreased(P<0.01,P<0.001),the expression of VEGF,MMP-2,N-cadherin and Vimentin down-regulated(P<0.05,P<0.01,P<0.001).Conclusion DS can reduce the infiltration of M2 type macrophages and the density of microvessels in peritoneal cavity metastases of gastric cancer in nude mice.Both M2-type macrophages and microvessel density were highly expressed in human gastric cancer tissue,and there was a positive correlation between them.DS inhibits the angiogenesis,invasion and migration of gastric cancer cells by affecting the polarization of M2-type macrophages.