| Objective:Oral squamous cell carcinoma(OSCC)is one of the most common malignancies of the head and neck,and its incidence has increased dramatically in recent years.Tongue squamous cell carcinoma is the most common oral cancer with high malignant degree,often with lymph node metastasis and high recurrence rate after treatment.Surgical treatment has a great impact on the facial and oral functions of patients.Radiotherapy and chemotherapy can significantly reduce the scope of tumor,but they have great side effects and limited maintenance time.The five-year survival rate and quality of life of oral squamous cell carcinoma patients are still not optimistic.Therefore,it is urgent to find reliable drug therapeutic targets and therapeutic methods to achieve safer and more effective targeted therapy.Recent advances in molecular targeted therapy and immunotherapy have shown promising prospects for the treatment of tongue squamous cell carcinoma.Oncolytic therapy has been shown to have a good anti-tumor effect in a variety of tumor therapies,and Oncolytic virus(OV)uses malregulated signaling pathways or metabolism to selectively target cancer cells.It kills cancer cells directly and triggers anti-tumor immunity.Vesicular stomatitis virus(VSV),a negative-stranded RNA virus of the genus Vesicular stomatitis,has become a particularly attractive OV because of its extensive cytophagy,rapid replication kinetics,ease of genetic manipulation,and lack of integration into the genome.In addition,VSV-induced tumor lysis can trigger a robust anti-tumor cytotoxic T cell response to viral proteins and tumor-associated antigens,resulting in long-lasting anti-tumor effects.Because of this multifaceted immunomodulatoryproperty,VSV has been extensively studied as an immunoviral therapy alone or in combination with other anti-tumor modalities such as immune checkpoint blockade.The G glycoprotein on the surface of VSV membrane is an important structure of the target cells infected by VSV.The receptor for this protein,the Low-Density Lipoprotein(LDL)Receptor(LDLR),is widely present in human tissue cells,making VSV broadly cytophilic,resulting in low target cell infection efficiency and potentially more adverse effects.Genetic modification of the VSV-G envelope for specific antigen binding has been reported as a means of increasing viral infection of target cells.Meanwhile,several studies have shown that the N-terminal of VSV-G can tolerate multiple amino acid insertions without affecting its function,so finding a suitable tongue squamous cell carcinoma-associated or specific membrane antigen is the key to developing novel recombinant VSV with tongue squamous cell carcinoma targeting.Therefore,we mined the GEO database for tongue squamous carcinomaassociated membrane antigen based on a bioinformatics approach and constructed a recombinant VSV virus with targeting this antigen.The aim is to develop better tumor-targeted gene-viral therapies that combine viral therapy with immunotherapy to improve the efficacy and targeting of tongue squamous cell carcinoma treatment while reducing its side effects.Methods:1.Bioinformatics-based screening of tumor-associated membrane antigens in tongue squamous cell carcinomaGene Expression data and probe annotation file GSE31056 were downloaded from the Gene Expression Omnibus(GEO)database for research.Tongue samples were extracted from the data set according to their anatomical definition.The background correction,log2 transform and quantile normalization of the raw microarray data in CEL format were performed in R using the Robust Multi Array(RMA)averaging algorithm in the Affy package.Differentially Expressed Genes(DEGs)in OTSCC tissues and normal tongue tissues were subsequently identified using the limma package linear model(screening conditions were P<0.05 and |log2FC | >1),and the adjusted P<0.05 was used as the statistically significant cutoff value to screen for differentially significant transmembrane proteins.Thedifferentially significant transmembrane proteins screened from the GEO data were validated by the TCGA database as well as the GEPIA database to select the most appropriate drug targets.2.Construction of a recombinant vesicular stomatitis virus targeting tongue squamous cell carcinomaMutation of amino acid 51 of the M protein of wild-type VSV virus using reverse genetic and targeted mutagenesis techniques.Rescue of blistering stomatitis pseudovirus by single-stranded negative-sense strand RNA virus reverse manipulation technique.The targeting peptide of tongue squamous cell carcinoma-associated membrane antigen screened by bioinformation analysis was fused to the N-terminal of vesicular stomatitis virus G glycoprotein by molecular cloning technique,and the recombinant protein was used to replace VSV G glycoprotein to construct a novel recombinant VSV.3.Effects of recombinant vesicular stomatitis virus on tongue squamous cell carcinoma in vitro and in vivoTo investigate the effect of recombinant vesicular stomatitis virus on tongue squamous carcinoma cells in vitro using flow cytometry,Western blot,q RT-PCR,etc.C3 H mice were subcutaneously grown with mouse squamous carcinoma cells SCC7 to construct a mouse tongue squamous carcinoma transplantation tumor model.UV-VSV,r VSV,α PD-1 and Ig G treatment were given after grouping.After 3 weeks,the tumors were removed intact,and the tumor volumes and weights of the different treatment groups were compared.Peripheral blood and spleen of mice were collected to analyze the effect of combined treatment with r VSV and PD-1 antibody on immune infiltration in mice.Results:1.Bioinformatics-based screening to obtain the tongue squamous carcinomaassociated membrane antigen ICAM-1Several tumor-associated membrane antigens,such as ICAM-1 and FN1,were obtained by differential analysis of the gene expression dataset of tongue squamous cell carcinoma in the GEO database,and ICAM-1 was selected as the target of drug therapy for tongue squamous cell carcinoma after validation byTCGA and GEPAI databases.2.Construction and screening of recombinant blister-causing stomatitis virus targeting ICAM-1 in tongue squamous carcinomaThe pVSV△M51-△ G-eGFP(+)plasmid was constructed by successfully mutating amino acid 51 of the VSV M protein using reverse genetic and targeted mutagenesis techniques and inserting the fluorescent reporter gene e GFP into the VSV genome.Successful rescue of VSV△M51-△ G-e GFP-G virus by singlestranded negative-sense strand RNA virus reverse manipulation technique.The ICAM-1 targeting peptide was successfully constructed at the N-terminal end of VSV-G,and it was verified that the fusion expression protein IBPG still maintained its targeting ability to ICAM-1.The plasmids expressing the fusion protein IBPG and VSVG were transfected into Vero cells,and VSV△M51-△Ge GFP-G virus was inoculated after 10 h.The VSV△M51-△ G-e GFPIBPG/G(r VSV)virus was successfully encapsulated.3.Targeting ICAM-1 recombinant vesicular stomatitis virus(r VSV)kills tongue squamous carcinoma cells in vitro and in vivo through multiple pathwaysInfection with rVSV enhances the antigen-presenting ability of SCC25 and SAS cells in vitro.Flow cytometry assay revealed increased expression of MHC1,MHC2,and CD1 d in SCC25 and SAS cells after 24 h of infection with r VSV.r VSV infection causes immunogenic cell death in tongue squamous carcinoma cells.The release of ATP was significantly increased in SCC25 and SAS cells after infection with r VSV virus.We found increased mobilization of calreticulin on the surface of SCC25 and SAS cells after 24 h infection with r VSV by flow cytometry assay.r VSV infection induces expression of proinflammatory genes associated with anti-tumor immune response in tongue squamous carcinoma cells.m RNA expression levels of chemokines in many attracting myeloid cells(CCL3,CCL4)and lymphocytes cells(CCL5,CXCL9,CXCL10,CXCL11,CXCL16)were significantly increased in tongue squamous cell SCC25 and SAS cells after infected with r VSV.The increased expression of TNF,IFN-α and IL-6 transcripts in SCC25 and SAS cells infected with rVSValso indicated that r VSV virus has the potential to "immune heat" tongue squamous carcinoma.In a mouse model of squamous carcinoma of the tongue,inoculation with r VSV significantly inhibited the growth of SCC7 tumors compared to negative controls,and the r VSV combined with the immune checkpoint inhibitor PD-1 antibody treatment group showed more sustained immune activation and immune infiltration,and exhibited superior tumor suppression.Conclusions:1.Differential expression gene screening based on tongue squamous cell carcinoma microarray data is a feasible tool to search for therapeutic targets of tongue squamous cell carcinoma.2.The recombinant vesicular stomatitis pseudovirus VSV△M51-△ G-eGFP-IBPG /G(r VSV)with ICAM-1 targeting was successfully rescued and screened.3.Infection with rVSV increased the antigen-presenting capacity of tongue squamous carcinoma cells and caused their immunogenic cell death and increased their expression of inflammation-associated chemokines and cytokines.PD-1antibody inhibition significantly increased T-cell activation and immune infiltration induced by VSV treatment,and recombinant VSV combined with PD-1 antibody treatment significantly inhibited tumor growth in tongue squamous carcinoma-bearing mice... |
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