Overexpression Of IL-15 In Human Lung Cancer Cells Synergize With Irradiation To Enhance The Expression Of Chemokines And The Role Of Antigen Presenting Cells

BackgroundLung cancer is the biggest cause of mortality in China.Because its early symptoms are usually not obvious,most patients are found with advanced,accompanied by lymph node or distant organ metastasis,so they have lost the chance of surgery.At present,the treatments of lung cancer are limited,usually including traditional radiotherapy,chemotherapy,targeted therapy and the latest discovery of PD-1 antibody treatment.However,all aspects of the data suggest that the current treatment of lung cancer is not as efficient as imagined,the average five-year survival rate of lung cancer is still lower than other cancer,such as breast cancer,prostate cancer,etc.So it is particularly important to find some new treatments.In the current research,immunotherapy has been a hotspot.However,in the tumor microenvironment,immune escape is common.Tumor antigens can usually escape the immune surveillance,and tumor cells can express some negative immune molecules,such as the presence of PD-L1 can make immune cells ineffective.In the initiation of tumor immunity,the tumor antigens are first recognized by antigen presenting cells(APC),process them intracellularly,form complexes with MHC molecules,transport them to the cell surface,and then activate T cells.The most powerful antigen-presenting cells in the body are called dendritic cells(DC).The immature DCs have strong phagocytic function,but the antigen-presenting ability is weak,If we promotes the proportion of mature DCs in the tumor microenvironment,it can specifically activate more T cells,enhance immunity and exerting anti-tumor effects.IL-15 is a peptide chain consisting of 114 amino acid residues,mainly produced by mononuclear macrophages and epithelial cells.It has been shown that it plays an important role in activating NK cell.Many clinical studies on IL-15 are currently underway.The IL-15 receptor is a trimer composed of three subunits including IL-15R?,IL-2R?/IL-15R? and IL-2yc/IL-15yc,which share ?(CD122)and yc(CD132)with IL-2,so its function is considered to be similar with IL-2 function.However,according to the literature,IL-15 usually plays a more powerful role than IL-12.The reason may be related to their different receptor assignments.IL-2a and IL-15a are distributed in different cells,IL-2a is mainly expressed in activated B cells and T cells,and IL-15a is mainly expressed in activated DCs and monocytes.The receptor IL-15a expressed on DC can be activated by IL-15 and further signals to cells expressing IL-2R??/IL-15R??,mainly by activating NK cells and CD8+ T cells.Because IL-15 can activate immune effector cells,it plays a important role in enhancing tumor immunity,and can kill tumor cells by activating immune effector cells.Chemokines are a class of small molecule proteins that play an important role in the migration of chemotactic cells,some of which interact with antigen-presenting cells.Lung cancer shows moderate sensitivity to radiation therapy.Radiotherapy can play a certain role in the treatment of lung cancer,but there are still many deficiencies in radiotherapy for lung cancer.In this study,we combined radiotherapy with the cytokine IL-15 to establish a research model of irradiation synergistic immunotherapy to enhance the treatment efficacy of lung cancer.Object1.Analyze the expression level of IL-15 about lung cancer patients and normal people in TCGA database,and further analyze the IL-15 expression level in different stages of lung cancer.2.Denote IL-15-expressing H460/A549 cell subset as H460/IL-15 cells and A549/IL-15 cells.H460/NC and A549/NC cells were set as a control.And to find out after adenovirus infection,if infection had impact on the characteristics of cells.3.To investigate whether overexpression of IL-15 in lung cancer cells can enhance the expression of chemokines after combination with irradiation,and further explore whether IL-15 can promote DC maturation.Methods and Materials1.TCGA database analysis:download data from the TCGA database,integrate the expression data with the survival data,use the function of the R language to do the difference analysis,and use the survival R package for survival analysis.2.The establishment of IL-15 overexpressing lung cancer cell line:lung cancer cells were infected with IL-15 adenovirus and negative control adenovirus respectively.After 48h,use inverted fluorescence microscope to observe the infection efficiency,and the mRNA expression of IL-15 was detected by qRT-PCR,and cell supernatants and cell lysates were collected for protein expression levels by ELISA.3.Detection of cell characteristics after adenovirus infection:EDU,plate cloning experiments were used to detect the proliferative capacity of lung cancer cells in the positive virus group,the negative virus group,and the untransfected virus group,and to investigate whether adeno-virus transfection affects tumor cells..4.Chemokine expression detection:use qRT-PCR to detect if IL-15 can promote the expression of chemokines,and detect whether IL-15 can synergistic irradiation to further increase the expression of chemokines.5.Co-culture of tumor cells and DC cells:Peripheral blood of normal donors was used,and human lymphocyte separation liquid was used to isolate mononuclear cells,then differentiated into immature DCs by IL-4 and GM-CSF.On the sixth day,the DC cells were collected,and divided into four groups including positive virus group,negative virus group,untransfected virus group and not co-cltured group.The DC cell were collected 24 hours later,and DC maturation ratio was measured by flow cytometry.Results1.The results of TCGA data analysis indicated that IL-15 was differentially expressed between lung cancer patients and normal people.The downloaded TCGA data were analyzed using the R language's own function,and there was a significant difference in expression between normal and lung cancer patients(p<0.0001),IL-15 expression was higher in normal people than in lung cancer patients.The data were further classified according to the stage of lung cancer.It can be seen that the later the stage,the expression level of IL-15 gradually decreased.2.Successfully constructed a lung cancer cell line that overexpressed IL-15.After IL-15 overexpressing adenovirus and negative control virus were transfected into lung cancer cells,they were observed under inverted fluorescence microscope at 48h,the H460/IL-15?H460/NC?A549/IL-15?A549/NC has obvious green fluorescence,indicating that the virus transfection efficiency was high.The results of qRT-PCR and ELISA confirmed that IL-15 expression was up-regulated both at the RNA level and protein level after adenovirus transfection.3.Adenovirus transfection did not affect the nature of tumor cells.In the transfection positive virus,negative virus and non-transfected virus cells,the proportion of EDU positive cells was not statistically different,and the colony formation rate was also not statistically different.4.IL-15 promotes the expression of chemokines,and synergize with irradiation to promote the expression of chemokines.After overexpressing IL-15 in lung cancer cells,the upregulation of chemokines CCL2,CCL5,CXCL9,and CXCL10 can be detected.The expression level of chemokines also changed after 1-5 days after irrasiation.However,after irradiation was taked to lung cancer cells overexpressing IL-15,the results showed that the expression of chemokines was further increased,which was more obvious than the increase of irradiation alone,and can last for a longer time.5.Tumor cells overexpressing IL-15 can promote DC maturation.The H460/IL-15.?H460/NC?H460/blank?A549/IL-15?A549/NC?A549/blank were co-cultured with DC,and the DC maturity ratio was detected by flow cytometry.The results indicated that the transfected positive virus group DC cells matured most.6.Lung cancer cells overexpressing IL-15 promote more DC cell maturation after receiving radiotherapy.Cell culture medium,untreated group of lung cancer cells,lung cancer cells overexpressing IL-15,and the lung cancer cell transfected with IL-15 after irradiation co-cultured with DC cells,and detect the DC maturation ratio by flow cytometry.The results suggest that lung cancer cells transfected with IL-15 and exposed to irradiation at the same time can promote more DC cell maturation.Conclusions1.The expression level of IL-15 in normal people is higher than that in lung cancer patients,at the same time,the later the stage,the lower the expression of IL-15.2.IL-15 transfection of adenovirus did not affect the nature of tumor cells.3.IL-15 can increase the expression of chemokines CCL2,CCL5,CXCL9 and CXCL10.4.IL-15 can promote DC cell maturation.5.IL-15 can further increase the expression of chemokines promoted by radiotherapy.6.IL-15 can synergistic radiotherapy to further increase DC cell maturation.