CXCL1/EGF-mediated Interaction Between Tumor Cells And Macrophages In The Migration And Invasion Of Oral Squamous Cell Carcinoma

Objective: Oral squamous cell carcinoma(oral squamous cell carcinoma;OSCC)is the most common malignant tumor in oral cancer,accounting for more than 90% of oral cancers.OSCC is an oral malignancy with a poor prognosis,with a five-year survival rate of less than 50%.OSCC cells are highly aggressive,and OSCC often undergoes local invasion and distant lymph node metastasis;Most patients die from recurrent tumors or metastatic cancer.Tumor microenvironment(TME)is a complex environment in which various cells secrete a large number of factors to lay a solid foundation for tumor migration,invasion and metastasis.In TME,tumor-associated macrophages(TAM)are one of the most abundant cells,typically accounting for 30% to 50% of tumor stromal cells.M2 macrophages are closely related to OSCC invasion and metastasis.Chemokine(C-X-C motif)ligand 1(CXCL1)belongs to one of the CXC chemokines and is highly expressed in various tumor cells.Studies have shown that CXCL1 is highly expressed in OSCC and plays an important role in the regulation of cytokines secreted by TAM.The binding of epidermal growth factor(EGF)secreted by TAM and epidermal growth factor receptor(EGFR)also plays an important role in OSCC migration and invasion.The latest research shows that the interaction between tumor cells and TAM plays an important role in tumor invasion and metastasis.Therefore,this experiment aims to explore the role of interaction between tumor cells and TAM in OSCC invasion and metastasis in the oral tumor microenvironment.It also provides a theoretical basis and experimental basis for targeted OSCC therapy.Material and Methods:1、Experimental materials: Cal27 cell line、Hacat cell line、Raw264.7 cell line2、Methods:(1)Hacat and Cal27 cells were divided into two groups to detect the gene and protein secretion level of EGF by Real-time PCR and ELISA.Raw264.7 cells were divided into three groups with tumor supernatant,tumor supernatant and CXCL1.Control group,TCM group and TCM+CXCL1 group intervened in Cal27 cells.The migration and invasion ability of Cal27 cells were detected by scratch,Transwell migration and invasion test.(2)Raw264.7 is divided into Control group,TCM group and TCM+CXCL1 group through different processing;Real-time PCR,flow cytometry and ELISA were used to detect the expression and secretion levels of CD206,CD86,Arg-1 and protein EGF in Raw264.7 cells under different interventions.Raw264.7 cells were divided into Control group,CXCL1 group,CXCL1+SB225002(CXCR2m Ab)group by different treatments;Real-time PCR and ELISA were used to detect the expression level of EGF-related genes and proteins in Raw264.7 cells under various interventions.Knock down CXCL1 in Cal27 cells.First,use PCR to verify the knockdown efficiency;Then the supernatant was used to interfere with Raw264.7 cells,and the level of EGF gene and protein secretion was detected by PCR and ELISA.Raw264.7 is divided into Control group,CXCL1 group,CXCL1+SB225002(CXCR2m Ab)group through different treatments;Real-time PCR,ELISA and Western Bolt were used to detect the expression level of related gene EGF and protein in Raw264.7 cells under different interventions.(3)Cal27 cells were divided into control group,EGF group and EGF+AG1478(EGFR m Ab)group by different stimulation;Real-time PCR,ELISA,Western Bolt and immunofluorescence assay were used to detect the expression level of E-cad,N-cad,Vim related to epithelial-mesenchymal transformation of Cal27 cells under different interventions.CCK-8 cell proliferation test,scratch,Transwell migration and invasion test are used to explore the effect of macrophages on the biological behavior of tumor.(4)Cal27 cells were treated with the same concentration of EGF for different times to detect the expression of phosphorylated EGFR;At the same time,Cal27 was divided into Control group,EGF group and EGF+AG1478 group through different treatments;Western Blot test to detect EGFR and NF-κ B pathway proteins(p-EGFR,p-P65).The expression of CXCL1 was detected by Real-time PCR,ELISA and Western Bolt.Results:1.Real-time PCR and ELISA results showed that the expression level of CXCL1 gene and protein of Cal27 was higher than that of Hacat.The results of scratch,Transwell migration and invasion experiments showed that the TCM group could significantly promote the migration and invasion of Cal27 cells compared with the control group;TCM+CXCL1 group further enhanced the migration and invasion ability of Cal27 cells.2.Real-time PCR results showed that the gene expression levels of CD206,CD86 and Arg-1 in TCM+CXCL1 group and TCM group were significantly higher than those in control group.The results of flow cytometry showed that the expression level of CD206 in TCM+CXCL1 group was significantly higher than that in TCM group,and that in TCM group was significantly higher than that in Control group;The expression of CD86 in TCM group was higher than that in Control group,but there was no statistical difference between TCM+CXCL1 group and TCM group.ELISA results showed that the level of EGF protein secretion in TCM+CXCL1 group was significantly higher than that in Control group and TCM group.Real-time PCR and ELISA showed that the level of EGF gene and protein secretion in CXCL1 group was significantly higher than that in Control group;The CXCL1+SB225002 group inhibited the level of EGF gene and protein secretion.When CXCL1 was knocked down,the level of EGF gene and protein secretion of macrophages interfered by supernatant was significantly decreased.3.The proliferation results of CCK-8 experiment showed that the EGF group had a trend but no statistical difference compared with the control group after 24 hours of intervention;After 48 hours of intervention,the proliferation ability of oral squamous cell Cal27 in EGF group was significantly enhanced compared with that in Control group.The proliferation of Cal27 cells in EGF+AG1478 group was significantly inhibited.The results of cloning experiment showed that EGF group could promote the proliferation level of Cal27 cells,while EGF+AG1478 group inhibited the proliferation level of cancer cells.The results of scratch,Transwell migration and invasion experiments showed that EGF group could significantly promote the growth of Cal27 cells compared with Control group;After treatment with EGF+AG1478,the growth of Cal27 cells was significantly inhibited.Real-time PCR,Western Bolt and immunofluorescence tests showed that the level of E-cad gene,the EMT-related index of EGF group,decreased,and the level of N-cad and Vim gene increased compared with the control group;The results of EGF+AG1478 group were contrary to those of EGF group.E-cad protein level and fluorescence intensity also decreased significantly,while N-cad protein level increased.In EGF+AG1478 group,N-cad protein level was significantly inhibited while E-cad protein level was up-regulated.4.Western Bolt experiment results showed that phosphorylated protein EGFR was continuously activated with time;EGF group level of phosphorylated proteins EGFR and NF-κB increased,EGF+AG148 group inhibited level of EGFR and NF-κB Phosphorylation.Real-time PCR,ELISA and Western Bolt experiments showed that the gene and protein levels of CXCL1 were up-regulated in EGF group;EGF+AG1478group reversed the results.Conclusion: CXCL1 and EGF were highly expressed in Cal27 cells and tumor associated macrophages,respectively.CXCL1 derived from tumor cells can promote the polarization of Raw264.7 cells towards M2 direction and make macrophages secrete EGF.At the same time,EGF secreted by macrophages can promote the proliferation,migration,invasion and epithelial-mesenchymal transformation of Cal27 cells.In addition,EGF derived from macrophages can also promote the secretion of more CXCL1 by tumor cell Cal27,and then form a positive feedback loop of CXCL1/EGF between tumor cells and macrophages.