Tumor-associated Macrophages Intervene In The Radiosensitivity Of Esophageal Cancer Cells Through VEGF And Its Mechanis

Objective: To study the intervention of tumor-associated macrophages in the radiosensitivity of esophageal cancer cells by regulating VEGF and preliminarily explore the mechanism.Method: Human monocytes THP-1 were induced into tumor-associated macrophages(TAMs)by stimulation of phorbol-12-myristate-13-acetate(PMA)and interleukin(IL-4).The CD68 and CD163 expression were detected using immunofluorescence.Human esophageal squamous cell carcinoma KYSE-150 and TE-1 cells and successfully induced TAMs were co-cultured through the Transwell chamber in a contactless mode,and subsequent experiments were carried out.CCK-8,Transwell invasion assay,cell scratch assay,TUNEL and clonogenesis assay were used to detect cell proliferation,invasion,migration,apoptosis,and radiosensitivity.The mixture of TAMs and KYSE-150 cells was inoculated into BALB/c nude mice in a ratio of 3:1,and the tumor-bearing mice were irradiated locally.Tumor formation rate,time to tumor formation,and changes in tumor volume of each group were observed before and after irradiation.HE staining and immunohistochemistry were used to detect the pathological changes and Ki-67 protein expression level of tumor tissues before and after irradiation.The stable cell lines of KYSE-150 and TE-1 were constructed using LV-VEGFA-RNAi lentivirus as vector.The expression of VEGFA m RNA was detected by q RT-PCR.Western blotting was used to detect the expression levels of the following proteins: VEGFA,MAPK,p-MAPK,BCL-2,Snail,CD68 and CD163.Rusult: Compared with M0 macrophages obtained by PMA and IL-4,the expression of CD68 decreased(P < 0.01),whereas expression of CD163 was significantly increased(P < 0.0001).After co-culture,the proliferation rate of KYSE-150 and TE-1 cells was significantly increased(P < 0.05).There was a significantly higher number of oesophageal carcinoma cells penetrating the Transwell basement membrane in the co-culture group compared to the control group(P <0.01).The scratch-healing width in the co-culture group was narrower than in the control group(P< 0.05),and the rate of apoptosis was reduced(P < 0.01).When the radiation dose was increased,the rate of clone formation for both groups gradually decreased,but the coculture group was significantly stronger than the control group.Compared with the control group,D0,Dq and SF2 in the co-culture group were increased(P < 0.05),the SERDo and SERDq of KYSE-150 cells were 0.94 and 0.92,and the SERDo and SERDq of TE-1 cells were 0.90 and 0.88,respectively.The m RNA and protein expressions of VEGFA in co-culture group were increased(P < 0.01).The tumor volume and weight in co-culture group were significantly higher than those in other groups before and after irradiation(P < 0.01).The morphology and size of transplanted tumor cells were different among the groups.More necrotic tumor tissues appeared in the irradiation group,but to a lesser extent in the co-culture group.The expression of Ki-67 protein in co-culture group was higher than that in other groups before and after irradiation.After down-regulated the VEGFA of KYSE-150 and TE-1 cells,TAMs were co-cultured with VEGFA-nc cells and VEGFA-si cells and irradiated.The cell proliferation rate of the VEGFA-si group after radiation treatment was significantly reduced compared to that of the VEGFA-nc group(P < 0.01).The number of cells penetrating Transwell compartment basement membrane in VEGFA-si group was significantly decreased(P <0.001),the width of scratch healing was increased(P < 0.05),and the rate of apoptosis was significantly enhanced(P < 0.01).With the increase of radiation dose,the clonogenesis ability of VEGFA-si group was also significantly decreased.After VEGFA knockout,D0,Dq and SF2 were decreased(P < 0.05).SERDo and SERDq of KYSE-150 co-cultured cells were 1.18 and 1.18,respectively.SERDo and SERDq of TE-1 co-culture cells were 1.21 and 1.22,respectively.In comparison to the control group,the co-culture group promoted the expression of VEGFA,p-MAPK,BCL-2 and Snail,whereas expression of p-MAPK,BCL-2 and Snail decreased significantly after inhibition of VEGFA expression(P < 0.05).Conclusion:1.TAMs can promote the proliferation,invasion,migration,radiation resistance and inhibit the apoptosis of esophageal cancer cells.2.TAMs may promote the expression of VEGFA in oesophageal cancer cells.3.TAMs affect the radiation sensitivity of esophageal cancer cells by regulating VEGF pathway.