Experimental Study On The Treatment Of NSCLC By Immunotherapy Combined With ¹²⁵I Seeds

Research background and purpose:In view of the current situation of limited treatment and poor prognosis in non-small cell lung cancer,antitumor mechanism of ¹²⁵I radioactive particles brachytherapy and PD-1/PD-L1 inhibitors immunotherapy is different,about the joint application of treatment for NSCLC has not been reported at present.We have no experience whether the combination therapy can bring clinical benefits to patients with non-smal cell lung cancer.Based on the above analysis,we propose a hypothesis that BRT combined with pd-1 antibody can increase the curative effect of human non-small cell lung cancer.By observing the curative effect of ¹²⁵I radioactive particles combination with PD-1 antibodies to evaluate The feasibility of two combination therapies for the treatment of human non-small cell lung cancer.It is expected to evaluate the feasibility and this possible mechanism in the study and ultimately for the benefit of the patientin in non-small cell lung cancer.Objective:In this study,vitro experiments were conducted first to verify whether the combined application of anti-pd-1 antibody Nivolumab and brachytherapy can more effectively inhibit the growth promote the death of NSCLC tumor cells.Then,vivo experiments were conducted to establish a mouse model of lung cancer.Combined with the application of anti-pd-1 antibody Nivolumab and BRT,the status of the mice and tumor size was tested to verify whether the curative effect could be increased and the tumor inhibition rate could be improved.At the same time,the immune status of mice was detected and analyzed.Transcriptome molecular experiments were conducted to explore the genes and signaling pathways that play the important role in combined therapy,by comparing the transcriptome differences in NSCLC tissues treated with¹²⁵I brachytherapy combined with Nivolumab.It provides a theoretical basis for whether the combined application of the two can bring clinical benefits.Methods:1.Study on the biological behavior of ¹²⁵I combined with immunosuppressant in human lung cancer cellsProtein levels in PD-L1 in three lung cancer cell lines were first detected.Three lungs cancer cell lines,H1299,spc-a1 and A549,were used in this study.Nivolumab is mainly effective for the increased expression of pd-l1,so the cell lines with the highest relative expression of pd-l1 protein which detected by western-blotting was used in the experiment as the lung cancer cell lines.Pd-l1 cell lines with relatively high expression were screened out for further experiments,which were divided into control group,¹²⁵I group,Nivolumab group,¹²⁵I combined with Nivolumab group.Nivolumab was given to selected lung cancer cell lines at a working concentration of 100nM.The initial dose rate of cell culture plane irradiated by ¹²⁵I particle sustained low dose rate?CLDR?was 2.59cgy/h,and cells in exponential growth phase were irradiated by 10Gy.The MTT assay was used to compare the growth inhibition rates of tumor cells in each group,followed by apoptosis assay and Transwell invasion assay.2.Study on the efficacy of combined therapy in mouse transplanted tumor modelA mouse model of transplanted tumor was established.After screening,lung cancer cell lines with high pd-l1 expression was transfected with fluorescent plasmid from and implanted in the forechest of mice.The growth of the transplanted tumor was observed and the size of the transplanted tumor was measured by fluorescence photography.About a week later,grouping experiments could be started when the volume of transplanted tumor reached more than 100mm3.The transplanted tumor mice were randomly divided into three groups,¹²⁵I group,Nivolumab group,¹²⁵I combined with Nivolumab group,starting the drug treatment and ¹²⁵I seed implantation.The ¹²⁵I particles were type 6711 with initial activity of 1mCi.Under aseptic conditions,radioactive ¹²⁵I particles were implanted into the tumor center.Thermoluminescence dosimeter was inserted into the implant needle,and the needle core was gently pushed into the predetermined position,the position of the instrument is parallel to the particle,and the distance is fixed at 0.5cm by vernier caliper.Nivolumab was given 1mg/kg by tail vein injection.Mice were kept for 3weeks to observe the state of the mice and tumor volume changes were measured twice a week to calculate the tumor inhibition rate.HE staining,immunohistochemical and other histopathological identification were performed in sections of tumor tissue samples.The expression levels of pd-l1 protein and pd-l1 mRNA in mouse transplanted tumors were detected.3.Study on the mechanism of combination therapy through transcriptome molecular experimentPathological specimens of tumor-bearing mice were divided into ¹²⁵I group,combination therapy group.Tissue RNA was extracted,and the fragments were sorted and sequenced on the computer.The sequencing data were filtered after quality control.RSEM software was used to calculate the gene expression in each sample,and the expression differences between samples were compared.Based on the comparison results,the differential expression of genes in each sample was analyzed by using edgeR software,and the enrichment analysis of GO and KEGG pathways was conducted for the differentially expressed genes to find the genes and signal pathways that play an important role after the difference experiment results.Result:1.Screening of lung cancer cell lines and cell biology experimentsThe expression of pd-l1 protein in H1299 was the highest among the three lung cancer cell lines detected by western-blotting.H1299 was selected as the experimental lung cancer cell line,and the lung cancer cell lines of group ¹²⁵I,Nivolumab and ¹²⁵I combined with Nivolumab were treated with drugs and irradiation respectively.The results showed that the tumor inhibition rate of the combined treatment group was higher than that of the other two groups.The flow cell experiment showed that the tumor apoptosis rate of the combination treatment group was significantly higher than the single treatment group.The Transwell experiment showed that the combined treatment group could inhibit tumor cell invasion more effectively.2.In vivo experiments on tumor-bearing miceThrough the observation and measurement of different treatment methods in the tumor-bearing mouse model,curve mapping and tumor inhibition rate calculated.It is found that tumor growth inhibition is more obvious in the combined treatment group compared with the radiation seed implantation group and the immunotherapy group,and the effect was significant.The analysis of the transplanted tumor tissue showed that the pd-l1 protein level and mRNA expression level were the highest in the ¹²⁵I group,followed by the Nivolumab group,and the lowest in the combined treatment group.3.Transcriptome molecular experimentBy comparing the cell transcriptome differences under different treatment conditions,the genes and signaling pathways that play an important role in different habitats were explored.The results showed that 651 mRNAs were significantly up-regulated or down-regulated.GO analysis showed mRNA which have been upregulated involved in cellscellular responses to cytokine stimulus,responses to cy-tokines,immune responses,the cytokine-mediated signaling pathway,responses to chemicals,and cytokine activity.Subsequent pathway analysis revealed that abnormally upregulated mRNA was involved in IL23-mediated signaling events,IFN-?signaling,the NF-?B signaling pathway,chemokine receptor binding of chemokines,GPCR ligand binding,and metabolic disorders of biological oxidation enzymes.Downregulated mRNAs were participanted in Ca²⁺pathway,and ras-independent pathway in NK cell-mediated cytotoxicity.Conclusion:1.In non-small cell lung cancer cell lines with high expression of pd-l1,¹²⁵I combined with Nivolumab can significantly inhibit the growth of tumor cells,increase their apoptosis and inhibit tumor invasion and other biological behaviors.2.In vitro experiments showed that ¹²⁵I combined with Nivolumab could effectively inhibit tumor growth in tumor-bearing mice,and the analysis of transplanted tumor tissue showed that pd-l1 protein level and mRNA expression in the combined treatment group were lower than those in the single-factor treatment group.3.Transcriptome experiments showed that combined therapy could affect abnormal mRNA expression.By comparing the cell transcriptome differences under different treatment conditions,the genes and signaling pathways that play an important role in different habitats were explored.The results showed that 651 mRNAs were significantly up-regulated or down-regulated.GO analysis showed mRNA which have been upregulated involved in cellscellular responses to cytokine stimulus,responses to cy-tokines,immune responses,the cytokine-mediated signaling pathway,responses to chemicals,and cytokine activity.Subsequent pathway analysis revealed that abnormally upregulated mRNA was involved in IL23-mediated signaling events,IFN-?signaling,the NF-?B signaling pathway,chemokine receptor binding of chemokines,GPCR ligand binding,and metabolic disorders of biological oxidation enzymes.Downregulated mRNAs were participanted in Ca²⁺pathway,and ras-independent pathway in NK cell-mediated cytotoxicity.