Study On Enhancing The Immunogenicity Of Whole Cell Lung Cancer Vaccine By Combining Bcg Vaccine With Targeted Inhibition Of Oncogene Myc

Background:Lung cancer is a common malignant tumor globally,with Non-Small Cell Lung Cancer(NSCLC)being the primary type.Lung Adenocarcinoma(LUAD)is the most common subtype.Patients often present with covert symptoms,leading to late-stage diagnosis.Researchers are seeking innovative treatment methods such as lung cancer vaccines.The Myc gene family is a key regulatory gene,encoding transcription factors involved in various biological processes such as cell proliferation,growth,apoptosis,differentiation,and metabolism.Among the members of the Myc family,c-MYC and MYCN have the greatest impact on cancer.Myc can induce tumor cell proliferation and inhibit immune cell infiltration.Early unmodified whole-cell vaccines have poor immunogenicity,limiting their anti-tumor effects.Research indicates that targeting Myc can increase the immunogenicity of tumor cells.Adding Bacille Calmette-Guérin(BCG)as an adjuvant to tumor vaccines can enhance the activity of antigen-specific T lymphocytes and strengthen the immune effect of the vaccine.Objective:In this study,we used the TCGA-LUAD database to analyze the role of Myc in the LUAD Tumor Microenvironment(TME).Based on these findings,we further evaluated the effect of BCG combined with a Myc-targeted mouse Lewis Lung Carcinoma(LLC)whole-cell vaccine on the treatment of mouse LLC tumors in experiments,to determine the effect of BCG adjuvant combined with targeted Myc inhibition on the immunogenicity of the LLC whole-cell vaccine.Methods:We analyzed the TCGA database to study the features of immune cell infiltration and differential gene expression in LUAD tissues in the context of c-MYC and MYCN amplification.We used KEGG enrichment analysis to identify significantly different cancer-related pathways and conducted protein interaction network analysis.We used the Kaplan-Meier survival analysis method to screen for genes significantly associated with patient survival rates.LLC cells were co-cultured with Myc-targeted inhibitors(+)-JQ1 and I-BET151(0.4μM)for 4 days,and Myc and key immune-related gene expression differences were detected by Quantitative Real-Time Polymerase Chain Reaction(RT-qPCR).We studied the conditions for preparing a whole-cell vaccine from LLC irradiated by ultraviolet light,and divided C57BL/6N mice into six groups:(1)PBS group,(2)BCG group,(3)Irradiated LLC group without Myc-targeted inhibition,(4)Irradiated LLC group with Myc-targeted inhibition,(5)Irradiated LLC+BCG group without Myc-targeted inhibition,(6)Irradiated LLC+BCG group with Myc-targeted inhibition.In the mouse model,various irradiated LLC vaccines were administered on days 4,11,and 18 after injection of live LLC cells.The anti-tumor effects of different LLC vaccines were evaluated by measuring tumor volume,survival rate,the proportion of CD3~+CD8~+T cells in the spleen and tumor,and the expression levels of serum Tumor Necrosis Factor-alpha(TNF-α)and Interferon-gamma(IFN-γ).Results:(1)Database analysis results showed that c-MYC and MYCN amplification was related to a decrease in the proportion of Cytotoxic T Lymphocytes(CTLs)and an increase in Tregs cells.In the high c-MYC group,the expression of CD274(PD-L1),CTLA4,PDCD1(PD-1),ANXA1,NT5E,AXL,APOE,VEGFA,VEGFC,TAP(including TAP1,TAP2,TAPBP)increased(W=30491.5～43793.5,P<0.05).In the high MYCN expression group,the expression of CD274,CD3D,CD4,CD8A,MHC-I(including HLA-A,HLA-B,HLA-C),MHC-II(including HLA-DP,HLA-DR),PSMB,THBS1 decreased(W=53368680.5,P<0.05).Furthermore,low Myc expression could activate immune-related pathways in LUAD tissue samples.(2)RT-qPCR results showed that after 4 days of co-cultivation with(+)-JQ1 and I-BET151,the expression levels of c-MYC,MYCN,PD-L1,PSMB(including PSMB8 and PSMB9),DHX37,CD73,THBS1 in LLC cells significantly decreased(t=3.99～13.51,P<0.05).These genes are related to immune suppression and tumor angiogenesis.This finding confirmed the similar trend of differential expression of homologous genes in human LUAD cells under low c-MYC and MYCN expression in the TCGA-LUAD database.(3)Animal experiments showed that compared to the irradiated LLC group without Myc-targeted inhibition,the proportion of CD3~+CD8~+T cells in the spleen and tumor and the expression levels of serum TNF-αand IFN-γin the irradiated LLC group with Myc-targeted inhibition were all increased,tumor growth slowed,survival rates increased,and the anti-tumor effect of the irradiated LLC+BCG group with Myc-targeted inhibition was better than the irradiated LLC group with Myc-targeted inhibition.Conclusion:This study indicates that Myc is an appropriate gene target for enhancing cell immunogenicity in LUAD,and BCG as an adjuvant can effectively enhance the anti-tumor effect of the LUAD whole-cell vaccine.A LUAD whole-cell vaccine produced by targeting Myc inhibition combined with BCG shows promise as an effective anti-tumor immunotherapy.