The Roles Of Tumor Associated Macrophages On Tumor Progression And Prognosis Of Non-small Cell Lung Cancer

Objective:Tumor microenvironment is composed of tumor cells,immune cells,macrophages,tumor-associated fibroblasts and extracellular matrix,which is necessary for tumor development and invasion and related to effective tumor therapy.Tumor-associated macrophages serve as a vital part of tumor microenvironment,among which M2-type tumor-associated macrophages can promote tumor growth and metastasis by secreting inflammatory cytokines,matrix degrading enzymes and vascular endothelial growth factors.Clinical therapy targeting tumor-associated macrophages has become a new direction in the immunotherapy of non-small cell lung cancer.The aim of this study was to explore the correlation between tumor-associated macrophages and the clinicopathological features as well as EGFR gene mutation and PD-L1 expression through evaluating the expression of CD206 and CD68 in non-small cell lung cancer.The effects of CD206 and CD68 expression on disease-free survival and independent prognostic factors affecting prognosis were determined.Regulatory effects of tumor-associated macrophages on inflammatory cytokines were explored by evaluating the expression of TNF-αand NF-κB on immune cells to provide multiple options for survival benefits and clinical treatment.Methods:1.115 specimens of non-small cell lung cancer were collected.CD206immunohistochemical staining was used to detect M2 type tumor associated macrophages and CD68 immunohistochemical staining was used to detect pan tumor associated macrophages.The number of tumor associated macrophages in tumor nests and stroma was calculated separately and the relevance with clinical pathological features was investigated.Meanwhile the associations between tumor associated macrophages and EGFR gene mutation and PD-L1 expression were examined.2.Kaplan-Meier survival curve was established and Log-Rank analysis was used to compare the effects of high and low expression of CD206 and CD68 in tumor nests and stroma on disease-free survival.Logistic regression model was used to determine independent prognostic factors.3.The expression of TNF-αand NF-κB in immune cells of non-small cell lung cancer cells was detected by immunohistochemical staining.The expression intensity was observed under microscope and the correlation between the expression of NF-κB and TNF-αand tumor-associated macrophages was analyzed.Results:1.The number of CD206~+and CD68~+macrophages in tumor nests and stroma was significantly higher than those in normal tissues(P<0.01).Moreover,the number of CD206~+and CD68~+macrophages in tumor stroma was significantly higher than those in tumor nests(P<0.01).The number of CD206~+macrophages in tumor nests was negatively correlated with tumor size and clinical stage(P=0.024,P=0.046).The number of CD206~+macrophages in stroma was positively correlated with tumor size(P=0.006).The number of CD68~+macrophages in tumor nests was negatively correlated with tumor size and TNM stage(P=0.012,P=0.013).The number of CD68~+macrophages in stroma was negatively correlated with distant metastasis(P=0.039).2.The correlation between the number of CD206~+macrophages and the clinicopathological characteristics had sex-based differences in NSCLC.In female NSCLC patients,the number of CD206~+macrophages in the tumor nests was negatively correlated with clinical stage(P=0.042)and distant metastasis(P=0.014).In male patients,the number of CD206~+macrophages in the tumor nests was correlated with patient age and EGFR mutation(P=0.02,P=0.008).3.The correlation between the number of CD206~+and CD68~+macrophages and clinicopathological features had discrepancy among different histological types of NSCLC.In adenocarcinoma,the number of CD206~+macrophages in the tumor nests was negatively correlated with clinical stage(P=0.001),distant metastasis(P=0.018)and tumor size(P=0.002).The number of CD68~+macrophages was negatively correlated with tumor clinical stage(P=0.025)and size(P=0.002).In tumor stroma,the number of CD206~+macrophages was positively correlated with tumor size and clinical stage(P=0.025,P=0.001)and the number of CD68~+macrophages was negatively correlated with lymph node metastasis(P=0.035).In squamous cell carcinoma,only the number of CD206~+macrophages in the nests was correlated with gender(P=0.036),while no relevance was identified among other factors.4.Kaplan-Meier survival curve analysis showed that patients with high expression of CD206~+and CD68~+macrophages in tumor nests had significantly higher disease-free survival than patients with low expression of CD206~+and CD68~+macrophages(P=0.047,P=0.03).In females,disease-free survival was significantly higher in patients with high expression of CD68~+macrophages in tumor nests than in patients with low expression(P=0.02).In males,CD206~+and CD68~+macrophages were not significantly associated with disease-free survival.The results of Logistic regression model showed that the infiltration of CD68~+macrophages in the tumor nests was an independent prognostic factor for female NSCLC patients,while histological type was an independent prognostic factor for male NSCLC patients.5.The number of CD68~+macrophages in tumor nests was positively correlated with the expression of TNF-αon immune cells.In males with non-small cell lung cancer,the number of CD206~+macrophages in tumor nests was negatively correlated with the expression of NF-κB on immune cells.Conclusions:1.The correlation between M2-type tumor-associated macrophages and tumor progression of NSCLC is influenced by tumor location with positive correlation in tumor stroma while negative correlation in tumor nests.The number of pan tumor-associated macrophages was negatively with tumor progression both in tumor nests and stroma.2.In addition to infiltration location,sex and tumor histologic types are important factors affecting the roles of tumor associated macrophages in non-small cell lung cancer.The infiltration of M2-type tumor associated macrophages have greater effects on tumor progression in female patients compared with those in male patients.The infiltration of M2-type tumor associated macrophages and pan tumor associated macrophages showed predilection in adenocarcinoma compared with squamous carcinoma.3.In male patients with NSCLC,M2-type tumor-associated macrophages in tumor nests are closely related to EGFR gene mutations while no correlation is detected in female patients,suggesting that EGFR pathway plays a more prominent role in regulating the polarization of tumor-associated macrophages in male patients.4.The high expression of both M2-type and pan tumor-associated macrophages in tumor nests favors good prognosis.In female patients,the high expression of pan tumor-associated macrophages in tumor nests is an independent prognostic factor affecting the prognosis.In male patients,histological type is an independent prognostic factor in patients with NSCLC.5.Pan tumor-associated macrophages would affect tumor progression and invasion via inducing the secretion of TNF-αin NSCLC.