Transcriptome Analysis Of Lung Adenocarcinoma And In Vivo Toxicity And Immune Regulation Of Anti-PD-L1 Antibodies

Objective: To analyse the prognosis of survival and expression regulation of transcription factors in lung adenocarcinoma patients based on RNA sequencing data from lung adenocarcinoma patients in the TCGA database;to preliminarily explore the adverse effects that multiple intravenous injections of anti-PD-L1 antibodies can have on normal mice and the effects on tumour-infiltrating immune cells,using PD-L1 inhibitors as a representative.Methods: Analysis of public data in TCGA database by Survival,edge R,Seurat and NMF packages in R program,classification of lung adenocarcinoma patients by NMF clustering,analysis of differential genes by edge R,analysis of survival by Survival package,screening of signature genes by Random Forest,analysis of immune microenvironment by The immune microenvironment was analyzed by ESTIMATE algorithm;PD-L1 concentration in mouse tissues was measured by protein immunoblotting(Western Blot)and quantitative real-time fluorescence PCR(q RT-PCR);anti-PD-L1 antibody was administered to mice by tail vein injection at a dose of 200 ?g per mouse,once a week,to observe the status and The mice were treated with the anti-PD-L1 antibody by tail vein injection at a dose of 200 ?g/each,and the status and survival time of the mice were observed.The ejection fraction,short-axis shortening rate and end-diastolic volume of the left ventricle were measured by echocardiography;the electrocardiographic characteristics and body temperature of the mice were measured by a small animal vital signs monitor;the concentration of thyroxine in the serum of the mice was measured by enzyme-linked immunolabeling;and the changes in the immune microenvironment of the mice after treatment with the anti-PD-L1 antibody were measured by data from the GEO database.Results: We screened 240 differentially expressed transcription factors by analysing gene expression profiles and comparing them with the list of transcription factors in the transcription factor database;the 240 differentially expressed transcription factors were used to classify lung adenocarcinoma patients into three subgroups,with significant differences in gene expression paradigms between groups and significant differences in survival prognosis between the N1 and N2&N3 groups(p=0.00089).A random forest screen identified 30 representative genes that could be used to predict patient staging and prognosis;in the anti-PD-L1 antibody assay,mice receiving four anti-PD-L1 antibody injections had a 60% mortality rate.Further examination of these mice by ultrasound and electrocardiography revealed that the mice had severely impaired cardiac ejection and compensatory myocardial hyperplasia;electrocardiograms suggested myocardial ischaemia and significant impairment of cardiac conduction or autonomic rhythm;HE staining showed compensatory hyperplasia in the heart tissue of mice injected with anti-PD-L1 antibody.In addition,the body temperature of mice injected with antiPD-L1 antibody decreased abnormally: the average body temperature of mice was 38.2?,while 10 minutes after the injection of anti-PD-L1 antibody,the average body temperature of mice dropped to 37.23?,and they showed a significant depressed state,and the level of thyroxine T4 in the body dropped to one-third of that in the control group;and the supplementation of L-thyroxine could significantly alleviate these symptoms,and at the same time could significantly reduce mice.Anti-PD-L1 antibody-treated mice showed a decrease in infiltrating T cells and an increase in both NKT cells and macrophages in the tumour microenvironment;PD-1 expression was increased in all types of cells in the tumour microenvironment.Conclusion: After grouping patients with lung adenocarcinoma by differentially expressed transcription factors,there were significant differences in gene expression between groups of patients,from which the characteristically expressed differential genes or transcription factors were extracted to predict the survival of lung adenocarcinoma patients as a Biomarker for bioassay;anti-PD-L1 antibodies caused reduced cardiac ejection and abnormal cardiac conduction in mice;inhibition of PD-1/PD-L1 Immunoregulation is a likely cause of primary hypothyroidism;anti-PD-L1 antibodies also have a significant effect on immune cell fractionation in mice.While anti-PD-L1 antibodies are used clinically to treat tumour patients,it is important to be alert to the possible cardiac damage and negative neuroimmune regulation caused by anti-PD-L1 antibodies in humans.