Study On TAMs Promoting Epithelial-to-mesenchymal Transition The Ability Of Invasion And Metastasis In Oral Squamous Cell Carcinoma

Background: Oral squamous cell carcinoma(OSCC) is the most common malignant oral cancer. Cancer cell invasion and metastasis are closely related to the death in OSCC. A growing number of studies confirm that tumor-associated macrophages(TAMs) is a prominent component of inflammatory microenviroment in tumor which can induce epithelial-mesenchymal transition(EMT) and promote the invasion and metastasis in many tumors. However, the impact on OSCC and the mechanism underline still unclear. Therefore, we studied the effects of TAMs on invasion and metastasis of oral cancer cells and its related mechanism.Objective: To investigate whether TAMs would promoted EMT and promoting tumor cell invasion and metastasis in OSCC and explore the molecular biological mechanism of TAMs-induced EMT in OSCC cell lines;Methods: First, we used phorbol 12-myristate 13-acetate( PMA) and macrophage colony-stimulating factor(M-CSF)to make THP-1 monocytes differentiated into TAMs, and then we collected the culture media of TAMs as condition medium(CM) to stimulate oral squamous cancer cells(HN4,HN6 and SCC9) and send TAMs conditioned medium to MS protein spectrum analysis.Secondly, we use the digital gene expression profiling analysis of gene expression difference of SCC9 initial evaluation under TAMs, then real-time RT-PCR, Western blot andimmunofluorescence microscopy was applied to detect the changes of the exprssion of epithelial and mesenchymal markers. The ability of invasion and metastasis of oral squamous cells was measured by wound-healing assay, flow cytometry cycle and transwell invasion assay. Finally, to further determine the mechanism of TAMs in oral cancer cell EMT and invasion, RT-PCR were used to assess the expression of transcription inhibitory factors in three kinds of oral cancer cell and Western blotting were conducted to assess the activation of the NF-κB pathwayResults: First,when treated with PMA and M-CSF, THP-1 cells were differentiated to TAMs. we detected the macrophage marker CD68 and the tumor associated macrophage marker CD163 were expression significantly and a variety of inflammatory cytokines and chemokines in the TAMs CM.Secondly,TAMs induced three OSCC cells(HN4 、 HN6 and SCC9) to undergone EMT with the down-regulation of E-cadherin andβ-catenin and the up-regulation of vimentin and fibronectin, and their morphology changed into a fibroblast-like phenotype. Compared to control group, the migratory and invasive activities of three OSCC cells were increased after TAMs treatment. Finally, TAMs may be through regulation of transcription inhibition factor Snail, ZEB and Twist and activation of NF- kappa B pathways to promote the incidence of oral cancer EMT.Conclusions: 1, The successful construction of TAMs model in vitro. 2, TAMs promoted EMT in HN4,HN6 and SCC9 cells and enhanced their invasive and metastatic properties. 3,TAMs promoted the transcription inhibitory factor Snail,Twist, ZEB expression and activation of NF- kappa B pathways in HN4,HN6 and SCC9 cells.