Study On Tumor Microenvironment And Extracellular Neutrophils Traps In Peripheral Blood In Patients With Gastric Cancer As Molecular Markers For Diagnosis And Efficacy Prediction Of Chemotherapy

Background&Aim Gastric Cancer(GC)is one of the most common malignant tumors in China,which lacks of effective biomarkers for diagnosis and efficacy prediction.Studies have shown that Neutrophil infiltration in gastric cancer group is associated with prognosis,and the Neutrophil lymphocyte ratio(NLR)in peripheral blood is associated with prognosis.On the one hand,Neutrophil Extracellular Traps are an important way for neutrophils to play their roles.In recent years,the role of NETs in malignant tumors has received extensive attention.On the other hand,tumor microenvironment is the main cause of multidrug resistance.Hence,this topic aims to explore the value of tumor microenvironment and peripheral blood NETs in gastric cancer patients as molecular markers for the diagnosis and chemotherapy efficacy of gastric cancer.Methods Firstly,the co-expression of nets-related proteins,Neutrophil elastase(NE)and Citrullinated histone H3(H3Cit)in 30 samples of gastric cancer and adjacent tissues was detected by immunofluorescence staining to verify the presence of NETs in the microenvironment of gastric cancer tissues.Then,the absorbance(Optical density,OD)values of NE-DNA complexes in peripheral blood of 122 patients with advanced gastric cancer,83 preoperative patients with gastric cancer,85 postoperative patients with gastric cancer,70 patients with benign gastric disease and 85 healthy patients were measured by enzyme-linked immunosorbent assay(ELISA)to analyze the differences in peripheral blood NETs expression in these five populations.The short-term efficacy of the first-line therapy was evaluated in 62 patients with advanced gastric cancer.Results 1.The colocalization of NE and H3 Cit in gastric cancer tissues was observed by immunofluorescence staining in 30 patients with gastric cancer,and the existence of NETs was confirmed.Neutrophils were found to accumulate in the tumor tissues by ne-immunohistochemisty,and this aggregation was reduced in the adjacent tissue samples from the same patient(P<0.001).Compared with tumor tissue samples,the number of NETs formation per field in paracancer tissue samples was significantly lower(P<0.001).2:NETs levels in plasma and serum in peripheral blood gradually decreased in the following groups: patients with advanced gastric cancer,patients before gastric cancer,patients after gastric cancer,patients with benign gastric disease and healthy people;3:Pearson correlation coefficient or Spearman non-parametric analysis was used to determine the statistical significance of NETs levels in gastric cancer tissues and peripheral blood samples of the same patient(plasma: Spearman r=0.456,P=0.011;Serum: Spearman r=0.580,P=0.001).4:As a serum marker,NETs value to the diagnosis of gastric cancer(AUC = 0.909)is better than that of Carcinoembryonic antigen(CEA)(AUC = 0.805)and Carbohydrate antigen 19-9(CA19-9)(AUC = 0.634).5:Correlation between NETs and clinicopathological features.(1)the presence of lymph node metastasis indicates a higher level of NETs in the serum;(2)Certain hematologic indicators in peripheral blood,such as CEA CA19-9 Neutrophil count and Neutrophil to lymphocyte ratio(NLR),were also correlated with NETs levels(3)the level of NETs in peripheral blood samples is affected by whether the patient has received surgery or not.6:There was no significant difference in PFS between patients with low peripheral blood NETs level and those with high NETs level(plasma: 5.0 months and 5.5 months,respectively,P=0.686;Serum: 4.6 and 6.0 months,respectively,P=0.160).In addition,the Proportional risk regression model(Cox)shows that the level of NETs in peripheral blood is not an independent prognostic factor for PFS in patients with advanced gastric cancer receiving first-line treatment.Conclusion 1.NETs were found in both tumor tissues and PB in patients with gastric cancer,and the level of NETs was correlated between gastric cancer tissues and PB,as well as between PB serum and plasma.2.The level of PB NETs in gastric cancer,benign gastric lesions and healthy people showed a gradually decreasing trend.3.The level of PB NETs was higher than CEA and CA19-9 in the diagnosis of gastric cancer.4.PB NETs level was correlated with lymph node metastasis,tumor load,WBC count,NLR,and chemotherapy efficacy in patients with gastric cancer,but was not correlated with PFS.