TGF-β1 And IL-17A Comediate The Protumorigenic Phenotype Of Neutrophils To Regulate The Epithelial-mesenchymal Transition In Oral Squamous Cell Carcinoma

Background and Objective: The role of neutrophils in cancer has been the subject of intense research in recent years.One major theme that has emerged is that not all neutrophils are equal in the field of cancer.However,it remains unclear what induces the protumorigenic or antitumorigenic phenotype predominate in tumor.Here,we found that infiltrated neutrophils and elevated TGF-β1 and IL-17 A in OSCC tissues.Therefore,this study aimed to investigate the effect of TGF-β1 and IL-17 A on neutrophils in OSCC and examined the underlying role of stimulation of neutrophils in OSCC cells.Methods: Immunofluorescence staining was used to observe infiltrated neutrophils in OSCC tissues.TGF-β1 and IL-17 A expression in OSCC tissues was determined by immunohistochemistry staining.Quantitative PCR was used to examine neutrophil-associated markers level after treatment with recombinant human TGF-β1 and IL-17 A.The source of TGF-β1 and IL-17 A in OSCC tissues were used to examine by immunofluorescence staining.OSCC cell migration,proliferation,invasion,stemness and EMT were analyzed after treatment with conditioned medium from TGF-β1/IL-17A-activated neutrophils.The levels of neutrophil-associated markers in OSCC tissues were examined by immunofluorescence staining,and quantitative PCR was performed to detect peripheral blood neutrophil-associated markers.Result:(1)Immunofluorescence staining showed that enriched of neutrophils infiltration in OSCC tissues;(2)The publicly available cancer gene database Oncomine and immunofluorescence staining indicated that the expression of TGF-β1 and IL-17 A were elevated in OSCC tissues versus normal mucosa;(3)The log-rank test indicated that high TGF-β1 expression was associated with worse overall survival rates than low TGF-β1 expression,but elevated IL-17 A expression was not significantly correlated with the overall survival rate;(4)Stimulation of neutrophils with TGF-β1 or IL-17 A alone did not dramatically influence the level of N2-associated markers,but their synergistic administration up-regulated the expression of MMP9,VEGF-A and BV8 and decreased the levels of CCL3,ICAM-1 and TRAIL;(5)The sources of TGF-β1 and IL-17 A include but are not limited to multiple immune cell types in the OSCC microenvironment;(6)TGF-β1/IL-17A-modulated neutrophils significantly promote OSCC cell migration,proliferation,invasion and stemness;(7)TGF-β1/IL-17A-activated neutrophils induce the EMT process in OSCC cells via the NF-κB signaling pathway;(8)Immunofluorescence staining showed that expression of MMP9 in neutrophils was significantly higher in OSCC tissues than in normal mucosa,and the level of CCL3 in neutrophils was lower in OSCC tissues than in the controls.(9)We noted that the expression of MMP9 in peripheral blood neutrophils was significantly higher in OSCC patients(n=83)than in control volunteers(n=40),and the level of CCL3 in peripheral blood neutrophils was lower in OSCC patients(n=83)than in control volunteers(n=40).The receiver operating characteristic(ROC)curve analysis revealed that the area under the curve(AUC)of the combined measurement of MMP9 and CCL3 in peripheral blood neutrophils was 0.912,the sensitivity and specificity of this combination assay for detecting OSCC in patients were 79.5% and 92.5%,respectively.Conclusions: TGF-β1 and IL-17 A cooperated to augment the protumor functions of neutrophils,thereby promoting EMT in OSCC cells via the NF-κB signaling pathway.In addition,the combination of neutrophil-associated markers may serve as a diagnostic and predictive method to screen for patients with OSCC.