| Background:In recent years,immunotherapy has seen successful applications in many solid tumors,but tumor patients who receive immune monotherapy have low overall response rate(ORR)and are prone to acquired drug resistance,so immunization combined with other therapies has become a hot topic in clinical research.Previous studies have shown that immunotherapy combined with the antiangiogenic drug anlotinib can activate the immunosuppressed tumor microenvironment,thereby improving the efficacy of immunotherapy compared with immunomonotherapy,but the specific molecular biology mechanism remains unclear.Objective:To investigate the specific mechanism of action of the immune checkpoint inhibitor tislelizumab in combination with anlotinib in the treatment of lung adenocarcinoma from the perspectives of immune cells,cytokines,intestinal flora,metabolomics,transcriptomics,etc.,so as to provide a basis for better individualized and comprehensive treatment.Methods:(1)Lewis cell line was used to construct a humanized subcutaneous tumor mouse model of programmed death receptor-1(PD-1),and the mice were randomly divided into control group,tislelizumab monotherapy group,anlotinib monotherapy group and two-drug combination treatment group,and the differences in tumor volume between the mice were compared.Murine antibody protein microarray maps were used to detect cytokines,and enzyme-linked immunosorbent assay(ELISA)was used to verify the differences between different groups of cytokines,namely IFN-y,IL-2,IL-23.The expression levels of CD4+T cells,CD8+T cells,CD31,CD34,MVD,and VEGF in tumor tissues were detected by immunohistochemistry.(2)MiSeq sequencing technology was used to sequence and analyze the V3 and V4 regions of the 16S rRNA gene in mouse duodenal feces,and explore the effects of combination therapy on microbial diversity and key bacterial species.(3)Liquid chromatograph mass spectrometer(LC-MS)was used to detect differential metabolites between groups;Principal component analysis(PCA),partial least squares discrimination analysis(PLS-DA),orthogonal partial least-squares discrimination analysis,OPLS-DA)combined with MetPA database and MetaboAnalyst 5.0 for further analysis of differential metabolites and metabolic pathways;The Spearman correlation coefficient was used to construct the correlation coefficient map of different metabolites and intestinal microbiota changes,and the effects of combination therapy on metabolites and metabolic pathways in mice were discussed.(4)RNA sequencing was performed on mouse tumor tissues in each group.In this study,we used weighted correlation network analysis(WGCNA),gene set enrichment analysis(GSEA),and functional and pathway enrichment analysis to screen 10 key genes in the combination treatment group and further explore the expression of Plaur in different treatment groups.Results:(1)The tumor growth was the slowest in the combination treatment group;The serum IL-2 and IFN-γ levels of mice in the combination treatment group were increased,and the number of CD4+T and CD8+T cells in tumor tissues was significantly higher than that in the anlotinib monotherapy group and the tislelizumab monotherapy group,and the expression levels of VEGF,CD31,CD34 and MVD were lower than those in the tislelizumab monotherapy group.(2)Compared with the control group and monotherapy group,the observed index(P=0.01460),CHAO index(P=0.00116)and ACE index(P=0.00863)of mouse feces in the combination treatment group decreased significantly,indicating that the microbial microbiota richness of mice in the combination treatment group decreased.In addition,the content of specific flora such as Rikenellaceae,Verrucomicobia,Verrucomicrobiaceae,and Akkermansia increased in the combination therapy group,suggesting changes in the intestinal microbial microenvironment,which will lay the foundation for the future combination of intestinal microbiota and immune and antiangiogenic drugs.(3)A total of 399 metabolites were expressed differently in the intestinal tissues of mice in the combination group,of which 249 metabolites decreased and 150 metabolites increased.Further analysis of differentially expressed metabolites revealed a total of 6 key metabolic biomarkers.Metabolites:carnosine,4-hydroxy-L-proline content increased in the combination therapy group compared with the single-agent anlotinib group;The content of methoxyisoflavones and docosahexaenoic acid decreased;Compared with the monotherapy tislelizumab group,the content of metabolites:carnosine,4-hydroxy-L-proline,choline was increased in the combination therapy group.Glucose metabolism,vitamin absorption and mTOR signaling pathways were all involved in the anti-tumor process of the combination group.Correlation analysis showed that at the family level,7 altered flora in the combination treatment group were closely related to 10 differential metabolites.(4)Compared with the single-drug group,the combination group has a stronger anti-tumor effect,and its mechanism is related to the interferon-γ response and RNA metabolism of RNA pathway.On this basis,we used 8 algorithms in cytoHubba to screen out 10 key genes,including Mmp9,Plaur,etc.Further studies found that the higher the expression level of Plaur,the weaker its tumor suppressive effect.This study elucidates the molecular mechanism of enhanced antitumor effect after tislelizumab in combination with anlotinib,providing potential therapeutic targets for subsequent treatment.Conclusion:The results of this study show that tislelizumab combined with anlotinib treatment can effectively activate immune cells,and has a certain inhibitory effect on the neovascularization of lung adenocarcinoma,and can cause changes in intestinal flora and metabolites in vivo.In addition,we also found genes and related signaling pathways closely related to the occurrence and development of lung adenocarcinoma such as Mmp9 and Plaur,which provide a basis for clinical individualized treatment and comprehensive treatment. |
PDF Full Text Request