Tumor Associated Neutrophils Promote The Migration Of Human Osteosarcoma Cells And Its Mechanism

Background:Osteosarcoma is a primary malignant tumor that often occurs in adolescents under 20 years old,which seriously threatens the lives of children and adolescents.In particular,for patients with metastatic or recurrent osteosarcoma,existing treatments are often difficult to work.However,the study of the tumor microenvironment(TME)that is mainly composed of tumor cells,blood vessels,infiltrating inflammatory cells and so on has undoubtedly opened up a new direction for the treatment of osteosarcoma.In recent years,it has been found that many infiltrating immune cells in TME are significantly correlated with the poor prognosis of tumor patients.Neutrophils infiltrated in tumor tissue are called tumor-associated neutrophils(TANs).Many studies have shown that neutrophils are infiltrated in various types of tumor tissues,and the presence of neutrophils in tumor tissues is significantly correlated with poor prognosis.Objective:1.To investigate the infiltration of tumor-associated neutrophils in osteosarcoma and explore the relationship between tumor-associated neutrophils and the staging and metastasis of osteosarcoma.2.To explore the interaction of tumor-associated neutrophils and osteosarcoma cells in co culture system and the changes of osteosarcoma cells in biological characteristics.3.To study the reason of changes of osteosarcoma cells in biological characteristics and to detect the changes of cytokines after co culture of osteosarcoma cells and neutrophils using cytokine microarray.4.To test the influence of the cytokine on the biological characteristics of osteosarcoma cells.Methods:1.The infiltration of neutrophils in clinical specimens of osteosarcoma was studied by HE and immunohistochemical staining,and the relationship between tumor-associated neutrophils and the stages of osteosarcoma was analyzed.2.The xCELLigence RTCA DP analyzer was used to monitor the effect of different proportions of tumor-associated neutrophils on the migration ability of osteosarcoma SOSP-9607 cells in co culture.3.Cytokine microarray was used to detect the changes of cytokines after co culture of neutrophils and osteosarcoma SOSP-9607 cells.And the expression of cytokines was verified by Western blot.4.The effect of the cytokines on the migration and invasion of osteosarcoma SOSP-9607 cells was confirmed by Transwell migration assay and Transwell invasion assay.Results:1.HE and immunohistochemistry showed that neutrophil infiltration was found in osteosarcoma,and the infiltration of neutrophils was significantly correlated with the stage and clinical metastasis of osteosarcoma.2.The migration ability of osteosarcoma SOSP-9607 cells was significantly enhanced after co culture with neutrophils.3.A variety of cytokines such as IL-6,IL-1 beta,Angiogenin and IGFBP-1 were changed after co culture of osteosarcoma SOSP-9607 cells and neutrophils.Western blot confirmed that a part of the elevated IL-6 was produced by the secretion of neutrophils stimulated by osteosarcoma SOSP-9607 cells.4.IL-6 could enhance the migration and invasion of osteosarcoma cells.And after reducing the expression of IL-6 in osteosarcoma cells,the migration and invasion of osteosarcoma SOSP-9607 decreased.Conclusion:1.Neutrophil infiltration was found in osteosarcoma,and the infiltration of neutrophils is significantly related to the staging and clinical metastasis of osteosarcoma.2.Tumor-associated neutrophils could enhance the migration ability of osteosarcoma SOSP-9607 cells.3.The change of IL-6?IL-1??Angiogenin?IGFBP-1 and other cytokines may be the reason of the migration enhancement of osteosarcoma SOSP-9607 cells.4.The interaction between tumor associated neutrophils and osteosarcoma SOSP-9607 cells is mutual.Tumor cells or their metabolites also could stimulate neutrophils and alter neutrophils protein expression.5.IL-6 can enhance the migration and invasion of osteosarcoma SOSP-9607 cells.Tumor-associated neutrophils can promote the metastasis and invasion of osteosarcoma SOSP-9607 cells by secreting a variety of cytokines,such as IL-6.