Application Of Peripheral Blood Neutrophil Extracellular Traps In The Diagnosis And Progression Of Malignant Tumors

Background and purpose:The morbidity and mortality of malignant tumors remain high and have become a global economic and medical burden,and there is a lack of highly specific diagnostic and progression markers.It has been demonstrated that in the tumor microenvironment,neutrophil extracellular traps(NETs)can promote thrombosis,invasion and metastasis,and inflammatory responses associated with malignancy and that the level of infiltrating NETs in tissues correlates with the level of NETs in peripheral blood.Still,the relationship between the level of NETs in peripheral blood and malignant tumors is currently lacking clinical information for in-depth studies.Methods:The cellular experiments: The release of NETs was detected by fluorescence and immunofluorescence using phorbol myristate acetate(PMA),conditioned medium for glioma cells LN18 co-cultured with neutrophils in vitro,respectively.The clinical study: We collected 263 patients with malignant tumors(55 gliomas,101 ovarian cancers,64 colorectal cancers,and 43 lung cancers)and 75 healthy controls,and measured the levels of NETs markers citrullinated histone H3(citH3)and cell free DNA(cfDNA)in the serum of the subjects using ELISA and fluorescent nucleic acid staining for quantification,respectively.In addition,the levels of systemic inflammation-related parameters,including neutrophils(Ne),lymphocytes(Ly),monocytes(M),and platelets(PLT),were measured in the whole blood of the subjects using the laboratory Sysmex-XN2000 automated hematology analyzer,and calculate the neutrophil-to-lymphocyte ratio(NLR),monocyte-tolymphocyte ratio(MLR),platelet-to-lymphocyte ratio(MLR),platelet-tolymphocyte ratio(PLR),systemic immune inflammation index(SII),and systemic inflammation response index(SIRI)levels.To assess the value of citH3 and cfDNA levels in the peripheral blood of patients with malignancy in the diagnosis of malignancy,the occurrence of combined venous thromboembolism(VTE),clinical staging,and systemic inflammatory response.Results.:1.PMA stimulated neutrophils in vitro for 1-4h,and the fluorescence staining and quantification results showed that the area of PMA-stimulated neutrophils releasing NETs increased significantly higher than the control with time(P < 0.01 or P <0.001);immunofluorescence co-staining of citH3 and DNA further confirmed the generation of NETs.The conditioned medium of glioma cells LN18 was co-cultured with neutrophils in vitro for 4 h.The fluorescence staining and quantification results showed that the area of NETs release from the conditioned medium-stimulated neutrophils was significantly higher than that of the control(P < 0.0001);immunofluorescence co-staining of citH3 and DNA further confirmed the production of NETs.2.Tumor patients were divided into tumor group and each type of tumor group(i.e.,glioma,ovarian cancer,colorectal cancer,and lung cancer groups).The levels of citH3 and cfDNA in the peripheral blood of patients in the tumor group and each type of tumor group were higher than those in the control group(P < 0.0001).3.ROC analysis of citH3,cfDNA,and citH3+cfDNA increased sequentially in the area under the curve(AUC)and the Youden’s index in the tumor group and each type of tumor group.4.The tumor patients were divided into a non-combined VTE group and a combined VTE group according to whether they were combined with VTE within a short period after the clinical intervention.The two groups’ comparison of citH3 and cfDNA levels before clinical intervention was not statistically significant.5.The patients in both the tumor group and each type of tumor group were grouped according to clinical stage.In the tumor group,citH3 and cfDNA levels were higher in patients with advanced(stage Ⅲ-Ⅳ)tumors than in patients with early(Ⅰ-Ⅱ)stages(P < 0.0001);in each type of tumor group,except for citH3 in the lung cancer group,the levels of citH3 and cfDNA in all other types of tumor groups were higher in patients with advanced stage than in patients with early-stage(P < 0.05 or P <0.01).That is,the levels of peripheral blood citH3 and cfDNA in tumor patients increased with increasing clinical stage.6.The correlations between peripheral blood citH3,cfDNA levels,and systemic inflammation-related parameters in the tumor group and each type of tumor group were analyzed by Spearman nonparametric analysis.cfDNA was positively correlated with Ne,M,MLR,and SIRI in the tumor group,in patients with two and more types of tumors(P < 0.05 or P < 0.01),and in patients with two and more types of tumor patients were positively correlated with NLR and SII(P < 0.05 or P < 0.01),and negatively correlated with Ly(P < 0.05).That is,cfDNA levels in the peripheral blood of tumor patients were positively correlated with Ne,M,NLR,MLR,SII,and SIRI,and negatively correlated with Ly.7.The patients in both the tumor group and each type of tumor group were grouped according to clinical stage.cfDNA was positively correlated with M,MLR,and SIRI in the advanced tumor group and patients with two or more types of advanced tumors(P < 0.05 or P < 0.01),and with Ne,NLR,and SII in patients with two or more types of advanced tumors(P < 0.05 or P < 0.01).That is,the correlation between cfDNA and Ne,M,NLR,MLR,SII,and SIRI was more significant in patients with advanced tumors.Comparing the levels of systemic inflammation-related parameters in patients with different stages in the tumor group and each type of tumor group,the levels of NLR,MLR,SII,and SIRI increased with increasing clinical stages in patients in the tumor group and both types of tumor groups(P < 0.05 or P < 0.01),and PLR increased with increasing clinical stage in patients in both types of tumor groups(P < 0.05 or P< 0.01).That is,the levels of systemic inflammation-related parameters NLR,MLR,PLR,SII,and SIRI in tumor patients increased with increasing clinical stage.Conclusion:NETs levels in peripheral blood can distinguish healthy controls from patients with malignancy.NETs may participate in tumor-induced systemic inflammatory responses by interacting with circulating inflammatory cells,thereby promoting tumor progression.NETs can be used as a marker for the diagnosis and progression of malignancy.