Objective To investigate the killing effect of the recombinant oncolytic influenza virus OvFlu-GM-CSF,constructed using reverse genetics(RG)technology,in combination with chemotherapy drug,gemcitabine(GEM),against pancreatic cancer cells.Methods The recombinant oncolytic virus OvFlu-GM-CSF was successfully rescued using RG technology in our previous study.The virus was then comprehensively characterized through chicken red blood cell hemagglutination assay,transmission electron microscopy,and viral replication assay.CCK-8 assay was utilized to determine the impact of OvFlu-GM-CSF viruses[multiplicities of infection(MOI):0,0.1,1.0,3.0]on the survival rate of pancreatic cancer cell lines(Panc02,PANC-1,SW1990,BxPC-3)and normal pancreatic ductal epithelial cells(HPDE6-C7)after treatment for 24,48 or 72 h.Using a subcutaneous tumor-bearing mouse model of pancreatic cancer,36 female C57BL/6 mice(6 weeks old)were randomly divided into PBS group,recombinant oncolytic virus group,GEM group,and the combined treatment group,with 9 mice in each group.PBS(100 μL/animal)or OvFlu-GM-CSF virus(1× 107PFU/100 μL)was given to the mice of the corresponding groups through intratumoral injection,while GEM(100 mg/kg)was injected intraperitoneally,once per 3 days,for totally 9 times.Changes in tumor volume and survival rate were monitored.Multi-immunofluorescence staining was employed to analyze T cell infiltration and proliferation in the tumor tissues.HE staining was performed to observe the pathological changes in major organs(heart,liver,lungs,kidneys and brain),and the serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were measured to evaluate the safety of the recombinant oncolytic virus.Results The recombinant oncolytic influenza virus OvFlu-GM-CSF has a hemagglutination titer of 28,typical morphological features of influenza virus,and can selectively replicate within pancreatic cancer cells.At the cellular level,the viruses demonstrated a significant selective cytotoxic effect on Panc02,PANC-1,SW1990,and BxPC-3 cells under the conditions of 48 h post-infection and MOI=3.0,when compared to 48 h post-infection and MOI=0(P＜0.01).The cell survival rate was gradually decreased with the increase in MOI value and the extension of infection time(P＜0.01),but the viruses showed no significant effect on normal pancreatic ductal epithelial cells(HPDE6-C7).In the pancreatic cancer tumor-bearing mouse model,the combined treatment of the viruses+GEM significantly reduced the tumor volume than simple virus treatment and simple GEM treatment(P＜0.01),and enhanced the infiltration of T cells in the tumor tissues.No obvious pathological changes were observed in the above-mentioned major organs.Additionally,there were no significant differences in the serum levels of ALT and AST in the OvFlu-GM-CSF group,GEM group,and OvFlu-GM-CSF+GEM group compared to the PBS group.Conclusion RS technology-constructed recombinant oncolytic influenza virus OvFlu-GM-CSF,when combined with the chemotherapeutic agent GEM,enhances the cytotoxic efficacy against pancreatic cancer cells and effectively activates the host's anti-tumor immune response.

Objective To investigate the correlation between the expression level of glypican-3(GPC3)and the immune therapy response in clinical liver cancer patients.Methods Clinical data of 232 liver cancer immunotherapy response/tolerance group patients from January 2019 to May 2023 at the General Hospital of the People's Liberation Army were collected,and the correlation between GPC3 expression levels and the efficacy of immunotherapy in liver cancer patients was statistically analyzed;TCGA database validation of immune cell infiltration in GPC3 high and low expression groups of liver cancer patients;Further,real-time fluorescence quantitative PCR(qPCR)and immunohistochemical staining methods were used to detect the expression of GPC3 and immune cell infiltration in paired tissues of liver cancer immunotherapy response/tolerance group patients,and multi-color immunofluorescence was applied to detect the expression of GPC3 and related molecules.Results Clinical evidence shows that the GPC3 positive group of liver cancer patients has a low response rate to immunotherapy.Univariate/multivariate analysis results indicate that GPC3 is an independent risk factor for tumor recurrence after liver cancer immunotherapy;The analysis of the TCGA database revealed that high expression of GPC3 in liver cancer tissue leads to increased infiltration of regulatory T cells(Tregs);Paired tissue testing of liver cancer patients and adjacent tissues revealed that the immunotherapy effect was worse in the GPC3 high expression group,and Tregs cell infiltration increased,consistent with the results of database analysis.The TCGA database analysis results showed that GPC3 was positively correlated with CCL20 and its ligand CCR6,and the multi-color immunohistochemistry results were consistent with the database analysis results.Conclusion GPC3 is highly expressed in tumor tissues of liver cancer patients and is positively correlated with immune therapy tolerance in liver cancer.GPC3 regulates Tregs cell infiltration through the CCL20-CCR6 signaling axis and is expected to serve as a biomarker for predicting the efficacy of immune therapy in clinical liver cancer patients.

Objective To investigate the correlation between the expression level of glypican-3(GPC3)and the immune therapy response in clinical liver cancer patients.Methods Clinical data of 232 liver cancer immunotherapy response/tolerance group patients from January 2019 to May 2023 at the General Hospital of the People's Liberation Army were collected,and the correlation between GPC3 expression levels and the efficacy of immunotherapy in liver cancer patients was statistically analyzed;TCGA database validation of immune cell infiltration in GPC3 high and low expression groups of liver cancer patients;Further,real-time fluorescence quantitative PCR(qPCR)and immunohistochemical staining methods were used to detect the expression of GPC3 and immune cell infiltration in paired tissues of liver cancer immunotherapy response/tolerance group patients,and multi-color immunofluorescence was applied to detect the expression of GPC3 and related molecules.Results Clinical evidence shows that the GPC3 positive group of liver cancer patients has a low response rate to immunotherapy.Univariate/multivariate analysis results indicate that GPC3 is an independent risk factor for tumor recurrence after liver cancer immunotherapy;The analysis of the TCGA database revealed that high expression of GPC3 in liver cancer tissue leads to increased infiltration of regulatory T cells(Tregs);Paired tissue testing of liver cancer patients and adjacent tissues revealed that the immunotherapy effect was worse in the GPC3 high expression group,and Tregs cell infiltration increased,consistent with the results of database analysis.The TCGA database analysis results showed that GPC3 was positively correlated with CCL20 and its ligand CCR6,and the multi-color immunohistochemistry results were consistent with the database analysis results.Conclusion GPC3 is highly expressed in tumor tissues of liver cancer patients and is positively correlated with immune therapy tolerance in liver cancer.GPC3 regulates Tregs cell infiltration through the CCL20-CCR6 signaling axis and is expected to serve as a biomarker for predicting the efficacy of immune therapy in clinical liver cancer patients.