1.The Landscape Of RNA Splicing Alterations And Its Biological And Clinical Implications In Lung Adenocarcinoma 2.Single-cell Transcriptomics Of Peripheral Blood Reveals Anti-tumor Systemic Immunity Induced By Oncolytic Virotherapy

Background:Alternative RNA splicing is one of the most important mechanisms of posttranscriptional gene regulation,which contributes to protein diversity in eukaryotes.It is well known that RNA splicing dysregulation is a critical mechanism in tumor pathogenesis and the rationale for the promising splice-switching therapeutics for cancer treatment.Although we have a comprehensive understanding of DNA mutations,abnormal gene expression profiles,epigenomics,and proteomics in lung adenocarcinoma(LUAD),little is known about its aberrant alternative splicing profiles.Objective:To systematically study the RNA splicing alterations in LUAD and reveal their biological and clinical implications.To find critical splicing events that play key roles in LUAD and serve as candidate drug targets.To lay the foundation for the development of splice-switching therapies for LUAD.Methods:RNA splicing alterations in LUAD were revealed based on the multi-omics data generated from over 1000 samples.These sequencing data were collected from Cancer Hospital,Chinese Academy of Medical Sciences,The Cancer Genome Atlas(TCGA),and Gene Expression Omnibus(GEO).Key results were derived from comprehensively analysis of these multi-omics sequencing data and were validated by wet experiments,such as western blot,immunohistochemistry,colony formation assay,and so on.Results:We identified 3688 aberrant alternative splicing events(AASE)in LUAD,most of which were alternative promoter and exon skip.The specific regulatory roles of RNA binding proteins,somatic mutations,and DNA methylations on AASE were comprehensively interrogated.We dissected the functional implications of AASE and concluded that AASE mainly affected biological processes related to tumor proliferation and metastasis,such as "Ras signaling pathways" and "cell-matrix adhesion".These are previously unknown mechanisms for tumorigenesis.We also noticed that one subtype of LUAD with a particular AASE pattern was immunogenic and have a better prognosis and response rate to immunotherapy,which may provide meaningful knowledge for precision immunotherapy for LUAD patients.Conclusions:To our knowledge,this study is the first one focusing on revealing the AASE in LUAD by using multi-omics data generated from large-scale samples.These findings revealed novel events related to tumorigenesis and tumor immune microenvironment,represented a sufficiently striking advance in the discovery of aberrant molecular events and their biological meanings in LUAD,laid the foundation for the development of splice-switching therapies for LUAD,and promotes the application of this promising therapies in clinical settings.Background:Oncolytic viruses(OV)that improve immune status are favorable candidates for optimizing immunotherapy strategies.Existing studies have focused on characterizing the disturbance of the tumor microenvironment(TME)by OV.However,the changes in systemic immunity induced by OV were largely ignored,which would prevent the further understanding and optimization of OV.Objective:To comprehensively reveal the impact and significance of OV on systemic immunity,deeply explore the tumor killing ability and mechanism of OV,and search for molecular markers for companion diagnostics of OV.Methods:The HSV-2-based oncolytic virus OH2 was used to treat tumor-bearing mouse models.The peripheral blood samples were then collected for single-cell RNA sequencing(scRNA-seq).The scRNA-seq data were analyzed using Cell Ranger,Seurat,and other bioinformatics tools.Key findings were further validated by ELISA,immunohistochemistry,flow cytometry,in vivo experiments,and clinical samples.Results:Our data showed that OH2 therapy effectively activated systemic immunity and induced a sustained anti-tumor immune response.One major impact of OH2 on systemic immunity was to boost CCL5 production,which correlated with clinical response.Besides,the cytotoxic ability of peripheral cytotoxic Cd8+T cells and mature NK cells was elevated by OH2.Further analysis revealed that the interaction of monocytes with T cells and NK cells was critical for systemic immune remodeling and activation.We also found that systemic immune responses induced by OH2 could effectively reshape the microenvironment of distant tumor lesions and inhibit their progression.Conclusions:This study is the first to comprehensively characterize the effects of OH2 therapy on systemic immunity,which not only sheds new light on the anti-tumor mechanisms of OH2,but also contributes to the establishment of companion diagnostics for OH2 treatment and the improvement of oncolytic therapy strategies.