Exploration Of Anti-angiogenic Drugs Sensitizing Radiation-based Combination Therapy And Ameliorating Radiation-induced Pulmonary Fibrosis

PART 1 Exploration of Anti-angiogenic Drugs Sensitizing RadiotherapyChapter 1 Rh-Endostatin Improves Efficacy of Concurrent Chemoradiotherapy for Locally Advanced Non-Small Cell Lung Cancer:A Systematic Review and Meta-AnalysisBackground We aimed to clarify the benefits of the addition of rh-endostatin into concurrent chemoradiotherapy(CCRT)versus CCRT alone for locally advanced non-small cell lung cancer(NSCLC)by a meta-analysis.Methods PubMed,Embase,Cochrane Central Register of Controlled Trials,Wanfang and Chinese National Knowledge Infrastructure(CNKI)were systematically screened from inception to November 2020 using the prespecified terms.Prospective trials(evaluating or)comparing the efficacy of rh-endostatin combined with CCRT and CCRT for locally advanced NSCLC were included.The primary endpoints were risk ratios(RR)for objective response rate(ORR)and disease control rate(DCR).The secondary endpoints were RRs for overall survival(OS)and adverse events(AE).Results Ten studies with 716 patients were included in this meta-analysis.Rh-endostatin combined with CCRT significantly improved ORR and DCR compared with CCRT.The RRs of ORR and DCR for rh-endostatin combined with CCRT versus CCRT were 1.263(95%CI:1.137-1.403,p<0.001)and 1.274(95%CI:1.124-1.444,p<0.001),respectively.Rh-endostatin combined with CCRT significantly improved one-year survival rate compared with CCRT with pooled RR=1.113(95%CI:1.006-1.231,p=0.038).Rh-endostatin combination treatments had similar incidences of main adverse events compared with CCRT(p>0.05).Conclusion Rh-endostatin combined with CCRT is associated with significantly higher ORR,DCR and survival rate than CCRT with similar incidences of main adverse events in NSCLC.Chapter 2 Anlotinib Enhances the Antitumor Activity of High-Dose Irradiation Combined with Anti-PD-L1 by Potentiating the Tumor Immune Microenvironment in Murine Lung CancerBackground Radioimmunotherapy has become one of the most promising strategies for cancer treatment.Preclinical and clinical studies have demonstrated that antiangiogenic therapy can improve the efficacy of immunotherapy and sensitize radiotherapy through a variety of mechanisms.However,it is undefined whether angiogenesis inhibitors can enhance the effect of radioimmunotherapy.In this study,we aim to explore the role of anlotinib(AL3818)on the combination of radiotherapy and immune checkpoint inhibitors in Lewis lung carcinoma mouse.Methods C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control.Immune response and immunophenotyping including the quantification and activation were determined by flow cytometry,multiplex immunofluorescence,and multiplex immunoassay.Results Triple therapy(radiotherapy combined with anti-PD-L1 and anlotinib)increased tumor-infiltrating lymphocytes and reversed the immunosuppressive effect of radiation on the tumor microenvironment in mouse model.Compared with radioimmunotherapy,the addition of anlotinib also boosted the infiltration of CD8+T cells and M1 cells and caused a decrease in the number of MDSCs and M2 cells in mice.The levels of IFN-y and IL-18 were the highest in the triple therapy group,while the levels of IL-23,IL-13,IL-1β,IL-2,IL-6,IL-10,and Arg-1 were significantly reduced.NF-κB,MAPK,and AKT pathways were downregulated in triple therapy compared with radioimmunotherapy.Thus,the tumor immune microenvironment was significantly improved.As a consequence,triple therapy displayed greater benefit in antitumor efficacy.Conclusion Our findings indicate that anlotinib might be a potential synergistic treatment for radioimmunotherapy to achieve better antitumor efficacy in NSCLC patients by potentiating the tumor immune microenvironment.Chapter 3 Famitinib Enhances the Antitumor Effect of Radioimmunotherapy in Murine Lung CancerObjective Combining antiangiogenesis therapy with radioimmunotherapy is believed to further improve anti-tumor efficacy,but there is still a lack of evidence.This study aims to investigate the role of tumor vascular-targeted agent famitinib in the combination of radiotherapy and immune checkpoint inhibitor in murine lung cancer.Methods Effect of VEGFA and HIF1A on clinical prognosis and tumor immune microenvironment was analyzed using public data.Enrichment analyses of post-irradiation gene expression and mRNAs related to immunotherapy efficacy were carried out based on GEO datasets.C57BL/6 mouse subcutaneous tumor model was used to evaluate the antitumor effects of different treatment schemes.The tumor immunophenotyping was identified by flow cytometry.Results We demonstrated that high level of VEGFA and HIF1A expression in lung cancer was related to poor prognosis and immunosuppressive tumor microenvironment.In mouse model,the triple therapy of famitinib,radiotherapy and immunotherapy had the most dramatic antitumor activity.It significantly increased tumor infiltrating lymphocytes and reversed the immunosuppressive state of tumor microenvironment in mice.Compared with radioimmunotherapy,the addition of famitinib further promoted the infiltration of CD8+T cells and M1 type tumor associated macrophages,and reduced the number of myeloid suppressor cells.Therefore,triple therapy converted the immunosuppressive tumor microenvironment into an immunostimulatory one.Conclusion Famitinib can synergize with radioimmunotherapy by regulating tumor immune microenvironment in murine lung cancer.PART 2 Mapping of mRNA Alterations in Radiation-Induced Pulmonary Fibrosis and an Exploratory Study on Alleviating Radiation-Induced Pulmonary FibrosisBackground There is no definite conclusion of the etiology of radiation-induced pulmonary fibrosis,and the effective interventions are still lacking.In this study,based on radiation-induced pulmonary fibrosis mouse model,we aim to identify genes responsive to irradiation,compare the difference in genome expression between normal lung tissues and the irradiated ones,provide the mRNA alteration mapping,and to explore the potential interventions of radiation-induced pulmonary fibrosis.Methods C57BL/6 mice were exposed to a single 16Gy or 20Gy thoracic irradiation to establish the radiation-induced pulmonary fibrosis mouse model.Lung tissues were harvested at 3 and 6 months after irradiation for histological identification.The global gene expression of lung tissues was assessed by RNA sequencing.Differentially expressed genes were identified and subjected to functional and pathway enrichment analysis.Infiltration of immune cells was evaluated by CIBERSORT.Recombinant human endostatin was administered in radiation-induced pulmonary fibrosis mouse model to explore whether the anti-angiogetic strategy can alleviate radiation-induced pulmonary fibrosis.Results At 3 months after radiation,317 mRNAs were significantly upregulated and 254 downregulated in the 16Gy group.203 mRNAs were upregulated and 149 downrcgulated significantly in the 20Gy group.At 6 months after radiation,651 mRNAs were significantly upregulated and 131 downregulated in the 16Gy group.106 mRNAs were significantly upregulated and 4 downregulated in the 20Gy group.Several functions and pathways including angiogenesis,epithelial cell proliferation,extracellular matrix,complement and coagulation cascades,TNF signaling pathway,NOD-like receptor signaling pathway,HIF-1 signaling pathway,cellular senescence,myeloid leukocyte activation,regulation of lymphocyte activation,mononuclear cell proliferation,immunoglobulin binding were significantly enriched in irradiation groups based on the differentially expressed genes.The process of radiation-induced pulmonary fibrosis was almost the same between thoracic irradiation groups and irradiation combined with recombinant human endostatin groups.Conclusion Irradiation responsive genes were identified.The differentially expressed genes were mainly associated with cellular metabolism,epithelial cell proliferation,cell injury,and immune cell activation and regulation.