Tumor Associated Vascular Endothelial Cells Promote Monocyte Chemotaxis And Enhance M2 Polarization Through CCL2/CCR2

Objective:To understand the effect of tumor associated vascular endothelial cells on macrophages,and to clarify the source and function of macrophages in tumor microenvironment is conducive to blocking the progress of tumor and enhancing the therapeutic effect of tumor.Therefore,this study aims to investigate the effect of tumor associated endothelial cells on macrophages in oral squamous cell carcinoma and provide a theoretical basis for tumor therapy targeting macrophages.Methods:1.Human tongue squamous cell carcinoma Cal-27 cells and pharyngeal squamous cell carcinoma Fa Du cells were used to induce vascular endothelial cells into tumor associated endothelial cells.The morphological characteristics of normal vascular endothelial cells and tumor related endothelial cells were observed under microscope.The difference of monocyte chemotaxis between normal vascular endothelial cells and tumor related vascular endothelial cells was detected by Trans-well chemotaxis assay.2.RT-q PCR was used to detect the expression of chemokines(CCL2,CCL3,CCL5,CCL8,CXCL12,CXCL16,CSF1);The secretion of CCL2 chemokines was detected by Western blot;The expression of CCR2 on THP-1 was detected by PCR,and the chemotaxis of THP-1 was further detected by adding related inhibitors.3.Human monocytes(THP-1)were induced into adherent macrophages by phorbol ester(PMA)in the conditioned medium of Cal-27 and Fa Du related endothelial cells.Macrophages were cultured in the conditioned media for 24 hours.The expression of macrophage polarization related genes was detected by RT-q PCR.The morphological changes of macrophages were observed under microscope Results:1.The Oral squamous cell carcinoma related endothelial cells was polygonal,the intercellular junctions were loose and arranged disorderly.Compared with normal vascular endothelial cells,Cal-27 and Fa Du related endothelial cells could significantly chemotactic to THP-1.2.RT-q PCR showed that CCL2 m RNA expression was significantly increased in CCL2,CCL3,CCL5,CCl8,CXCL12,CXCL16 and CSF1.Western Blot showed that CCL2 protein was highly expressed in oral squamous cell carcinoma related endothelial cells.PCR results showed that CCR2 receptor existed on THP-1 surface of monocytes.After inhibiting the binding of CCL2 to CCR2,the number of chemotactic cells decreased significantly.3.The morphological changes of macrophages induced by oral squamous cell carcinoma related endothelial cells were round or oval after PMA treatment.Macrophages cultured with tumor related endothelial cells CM elongated and formed many processes similar to astrocytes.There were a lot of granular substances in the cytoplasm.RT-q PCR showed that the m RNA expressions of M2 macrophage related genes TGF-β,IL-10,arg1 and CD163 were significantly increased,while the m RNA expressions of M1 macrophage related genes TNF-α,IL-1 β,i NOS and CD86 were not significantly changed or slightly decreased.Conclusions:Oral squamous cell carcinoma associated vascular endothelial cells can recruit monocyte through CCL2/CCR2 pathway and polarize them into M2 tumor associated macrophages.