Potential Antitumor Effect And Mechanisms Of Granulocyte-macrophage Colony-stimulating Factor In Esophageal Cancer

ObjectiveGranulocyte-macrophage colony-stimulating factor(GM-CSF)is one of the most significant hematological growth factors which play well-known roles in hematopoietic regulation.GM-CSF binds to specific receptors,thus stimulate and promote the maturation of bone marrow stem cells,which would proliferate and differentiate into mature granulocytes.In addition,as an immunomodulator,GM-CSF is capable to stimulate dendritic cells(DCs)maturation and enhance its ability to recognize and present tumor antigens.It has been found that GM-CSF can inhibit tumor growth and progression by inducing the formation of tumor microenvironment.GM-CSF has been discovered to be upregulated in a variety of malignant cancers including lung cancer,breast cancer,colorectal cancer,and head and neck cancer,et al.It has been recognized that GM-CSF modulates tumor growth,invasion and metastasis through both immune-dependent and-independent biological pathways.Although many studies have described the potential antitumor effect of GM-CSF,its research in esophageal cancer is still limited and the exact underlying mechanism remains unclear.Thus,this study aims to analyze the biological functions of GM-CSF in human esophageal cancer cell lines and to explore the potential underlying antitumor mechanism.Material and method1.In vivo experiment1.1 The E-cadherin and vimentin expression level,which are respectively considered typical hallmarks of epithelial and mesenchymal phenotypes,were detected by Western blotting(WB)and real-time quantitative polynucleotide chain reaction(qRT-PCR)in both GM-CSF stimulated and non-stumulated Eca109 and 9706 esophageal cancer cells.1.2 Cell Counting Kit-8(CCK-8)was used to detect the proliferation ability of Eca109 and 9706 esophageal cancer cells,both in GM-CSF group and control group at 24h,48h,72h and 96h after stimulation.1.3 The apoptotic capacity of Eca109 and 9706 esophageal cancer cells in both GM-CSF group and control group were detected by flow cytometry.At least 100,000 cells per sample were counted.1.4 To investigate the migration and invasion effect of GM-CSF on Ecal09 and 9706 esophageal cancer cells,transwell assay was applied in 24-well plates according to the manufacturer's instructions.Images of the migrated cells(magnification,x40)and counted(magnification,x100)were captured under an inverted fluorescence microscope.2.In vitro experiment1.1 Immunohistochemistry(IHC)was performed by the streptavidin-perosidase method.The expressions of GM-CSF and E-cadherin were analyzed in paraffin-embedded human esophageal tumor tissues.Staining was scored according to the intensity and staining area as follows:0,no staining;1,weak staining in<50%cells;2,weak staining in>50%cells;3,strong staining in<50%cells;and 4,strong staining in>50%cells.1.2 Expression levels of GM-CSF in tumor tissues of 128 cancer patients were measured and performed survival analyses.HR and 95%Cl for OS were calculated.Results1.Effect of GM-CSF on EMT in esophageal cancer cells1.1 Compared to the controlled cells,the expression of E-cadherin in epithelial phenotype was significantly upregulated while the expression of vimentin was downregulatedin both GM-CSF stimulated Eca109 and 9706 esophageal cancer cells(all p<0.05).Thus,the study demonstrated a significant inhibition of EMT phenomenon in EC cells after GM-CSF stimulation;1.2 Results demonstrated that a significant positive correlation between GM-CSF levels and E-cadherin expression was discovered in 107 esophageal cancer patients(correlation coefficient=0.877,p<0.001).2.Effect of GM-CSF on biological behaviorsin esophageal cancer cells2.1 Compared to the controlled cells,the proliferation rates were significantly downregulated in GM-CSF stimulated Eca109 cells at 72 h(p=0.011)and 96 h(p=0.003)after stimulation.The proliferation rates for 9706 cells at 24 h(p=0.004),72 h(p=0.040),and 96 h(p=0.003)were also significantly decreased in GM-CSF group compared to controlled group.However,the proliferation rates of Eca109 cells incubated for 24 h and 48 h,as well as 9706 cells incubated for 48 h showed no significant difference between GM-CSF and control groups(p>0.05);2.2 Flow cytometry showed that compared to the control group,the apoptotic rates of Eca109 cells(p=0.019)and 9706 cells(p=0.023)were significantly higher in GM-CSF group.The results suggested that GM-CSF couldenhance apoptosis ability of esophageal cancer cells;2.3 The transwell migration assay was used to evaluate the migration and invasion abilities of Eca109 and 9706 cells after GM-CSFstimulation.Results showed that invading cell numbers of Eca109 cells and 9706 cells though the artificial matrix membrane in Transwel chamber was significantly lower in GM-CSF group than that in the control group(all p<0.05),indicating that GM-CSF could inhibit the migration and invasion abilities of esophageal cancer cells.3.Kaplan-Meier survival analysis showed that the different expression of GM-CSF was significantly correlated with OS(p=0.023)and PFS(p=0.012).Cox regression analysis showed that not ony different expression of GM-CSF(HR:0.563,95%CI:0.343-0.924;p=0.023)is an independent prognostic factor for EC patients,but also TNM staging(HR:0.165,95%CI:0.089-0.530;p<0.001)and distant metastasis(HR:3.126,95%CI:2.081-3.573;p=0.001).DiscussionGM-CSF is capable to inhibit the proliferation,migration and invasion of esophageal cancer cells and promote apoptosis capability.In addition,GM-CSF can inhibit EMT in esophageal cancer cells and thus exhibit antitumor effect.The expression of GM-CSF was an independent prognostic factor in esophageal cancer patients.However,the effects of GM-CSF on human EC cells and other cancer cell types require further investigation.Our data may further broaden the potential application of GM-CSF,and suggest GM-CSF as a promising therapeutic agent for antitumor therapy.