| Background:Lung cancer is one of the most dangerous cancers in the world because of its high incidence rate（2.206 million cases,11.4%）and high mortality rate（1.796million cases,18%）[1].At present,the treatment methods of lung cancer are mainly as below,surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy.Immunotherapy includes active immunotherapy and passive immunotherapy.Currently,immunocheckpoint inhibitors（ICIs）,which are commonly used in clinical,belong to active immunotherapy which target human immune system.Data show that approximately 30%of patients benefit from ICIs after clinical application[2].Monoclonal antibody and adoptive cell therapy are categerized as passive immunotherapy which target on tumors.In recent years,more and more targeted therapies have been approved for lung adenocarcinoma[3-6].However,there is no genotype-matched targeted therapy for lung squamous cell carcinoma[7].In addition,because of the targeted specificity of adoptive cell therapy,it has not achieved the idealized clinical efficacy and has been carried out slowly in clinical practice,so it is one of the urgent problems to improve the sensitivity of adoptive immunotherapy to the treatment of lung squamous cell carcinoma by improving the modification of targeted specificity.The anti-tumor immune response takes cellular immunity as the core[8].Cellular immunity is closely to“Hot Tumor”which infiltrated by lymphocytes,however,even in“Hot Tumor”,merely 10%of tumor infiltrating lymphocytes（TILs）were able to identify tumor cells,while the rest of TILs which named bystander cells are failed.TILs can not only recognize tumor cells but also can kill tumor cells.Furthermore,TILs can also mediate tumor immune microenvironment,which weakens the anti-tumor immunogenicity and leads to the escape of tumor cells.In the process of anti-tumor cellular immunity,only by antigen-presenting cell presented tumor-associated antigens to CD8+T cells through major histocompatibility complex-I,and meanwhile provide co-stimulation and cytokine signals to activate the specific immune response can thus achieve the killing of tumor cells.Therefore,improving the immunogenicity of tumor is one of the key problems to enhance the killing power of tumor.Dendritic cell is the most important APC in vivo for antigen acquisition,processing and presentation.DC has the strongest ability of antigen presentation.In recent years,it has been found that modified DC can enhance immunogenicity,furthermore,cytotoxic T lymphocyte can be identified and specifically targeted to kill tumor cells.Although researchers have made great progress in understanding the role and function of DC,it remains to be further studied that specific antigen-loaded DC enhances the targeting of anti-tumor immunity.Glypican-3 is a member of sulfated heparan proteoglycan anchored on the cell membrane.GPC3 was found overexpressed in tumor tissues,including lung cancer,and almost not expressed in normal tissues[9,10].The expression rates of GPC3 in lung squamous cell carcinoma were about 45%～58%[9,11]and about 3%～10%[9,11]in lung adenocarcinoma.Many interested tumor antigens of lung cancer include melanoma-associated antigen-A3（MAGE-A3）,melanoma-associated antigen-A4（MAGE-A4）,and New-York esophal squamous cell carcinoma antigen-1（NY-ESO-1）,expressed more frequently in squamous tumors than in non-squamous[12-15].Furthermore,CD8+T lymphocyte infiltrates lung squamous cell carcinoma more extensively than non-squamous cell tumors[16].Genomic analysis of lung squamous cell carcinoma revealed that many samples showed HLA-I gene inactivation,which may lead to loss of immune function and reduce expression of tumor antigens thus cause immune escape[17].Because of the low expression of GPC3 in lung adenocarcinoma,the low expression of GPC3 in normal tissues and the high expression in lung squamous cell carcinoma,we hypothesis that GPC3 may play an important role in immunotherapy as a tumor-associated antigen of LUSC so as to be a reasonable immunotherapy target for LUSC.Objective:To analyze the differential expression of GPC3 in LUSC and the relationship between GPC3 and the clinical pathological features of LUSC.To analyze the effect and mechanism of GPC3 on the malignant biological behavior of LUSC at cytological level.To analyze the cytotoxicity of specific CTL induced by dendritic cells pulsed with epitope peptides of HLA-A2 restricted GPC3 antigen in vitro.In order to prove that GPC3 interact with signaling pathway by regulating cell cycle related proteins,and GPC3 epitope peptides sensitized dendritic cells induce CTL specifically activing anti-tumor immune response.The aim of this study was to explore a new target of immunotherapy for LUSC in order to overcome the MHC mediated immune escape,thus to improve the killing effect of immune cells on LUSC.Methods:1.The differentially expressed gene GPC3 in LUSC was analyzed by bioinformatics method.2.The expression of GPC3 in human LUSC tissues was confirmed by immunohistochemical staining and the prognosis was analyzed.3.Construction of lentiviral vector mediated GPC3 silenced and overexpressed transfected human LUSC cell lines,which was validated by q RT-PCR,Western Blotting and immunofluorescence assay.4.CCK-8 assay and clonogenic assay were used to detect the effects of GPC3 on the proliferation of LUSC.5.Annexin-V/PI double staining method was used to detect the effects of GPC3 on LUSC apoptosis by flow cytometry.6.The effects of GPC3 on the cell cycle distribution of LUSC were detected by flow cytometry.7.The effects of GPC3 on the migration of LUSC were examined by wound healing test.8.Transwell assay was used to examine the effects of GPC3 on the migration and invasion of LUSC.9.Western blotting method was used to detect the changes in protein level of GPC3 on PI3K/Akt signaling pathway.10.Prediction of HLA-A2 restricted GPC3 epitope peptides was accomplished from the bioinformatics database,which synthesized by Sangon Biotech?.11.Isolation,induction and identification of mature DC in vitro.12.The secretion level of DC cytokines was detected by ELISA assay.13.Induction of CTL in vitro and CD8+、CD28+CTL sorting by flow cytometry.14.Determination of the optimal killing ratio of CTL cells to target cells and analysis of cytotoxicity to human LUSC cells.15.The experiment was repeated three times in each group.T test,Chi-square test or rank sum test were performed by SPSS 23.0 statistical software.The results were expressed by mean±standard deviation（`x±s）,P<0.05was considered to be statistically significant.Results:1.Bioinformatics analysis was performed to screen out GPC3 as differentially expressed gene in LUSC tissues.Enrichment analysis of differentially expressed genes indicated that GPC3 was associated with cell proliferation of LUSC.STRING database was used to construct PPI network,and GPC3 was found to have a direct interaction with IHH and WNT2.The expression of GPC3 in TCGA database was not correlated with the clinical pathological features of LUSC.KM-plotter method was carried out for survival analysis,which showed that GPC3,IHH and WNT2 were significantly correlated with the prognosis of LUSC（P<0.05）.2.Immunohistochemical staining of GPC3 was carried out in LUSC and paracancerous normal lung tissues.The positive staining rate of GPC3 in normal tissues was 5.8%（20/342）,and in LUSC tissues was 50.6%（173/342）.The positive staining rate of GPC3 in metastatic lymph nodes was 12.7%（20/157）,and negative in normal lymph nodes（0/342）.The positive expression of GPC3 was negatively correlated with survival time in LUSC patients,and also has interaction with smoking status,clinical stage and the expression of GPC3 in metastistic lymph nodes.3.The expression of GPC3 in human LUSC cell lines were detected by Western Blotting and q RT-PCR.The expression location of GPC3 was confirmed by immunofluorescene assay.The silencing of GPC3 gene can reduce the proliferation of LUSC cells and promote cell apoptosis（P<0.05）,the overexpression of GPC3 gene can promote cell proliferation and inhibit cell apoptosis（P<0.05）.GPC3 silencing could induce G1 phase arrest and S phase decrease in human LUSC cells（P<0.05）,while overexpression of GPC3 could induce S phase increase（P<0.05）.GPC3 silencing decreased migration and invasion of human LUSC cells（P<0.05）,and overexpression of GPC3 increased migration and invasion（P<0.05）.In vivo experiments in BALB/c mice with subcutaneous tumor-bearing models confirmed that GPC3 could promote the development of LUSC.GPC3 may be involved in the malignant biological behavior of LUSC by regulating the distribution of cell cycle through the PI3K/Akt signaling pathway.4.We predicted and synthesized three immunogenic epitopes of GPC3,which were naturally processed by HLA-A2 restriction,and their sequences were 169-177ELFDSLFPV,522-530 FLAELAYDL and 102-110 FLIIQNAAV.Peripheral blood mononuclear cells were isolated in vitro.Tested by Flow cytometry,the mature DC can highly express HLA-DR,CD11C,CD83 and CD80/86 costimulatory molecules.Furthermore,peptide loading did not affect the induction of mature DC by TNF-α.The secretion of IL-12 and IL-2 were increased by mature antigen-loaded DC,but decrease of IL-10 and IL-6（P<0.05）.ELISA assay was used to detect the increased secretion of IFN-γfrom CTL after activation,and the secretion level of IFN-γincreased with the elevated of efficiency target ratio.The cytotoxicity of CTL to the target cells was detected by flow cytometry assay and clonogenic assay.The results showed that the mature DC loaded with antigen peptide could activate CTL to kill LUSC cells,among them,HLA-A2-restricted GPC3 immunogenic peptide 522-530 FLAELAYDL,102-110FLIIQNAAV were the most lethal sequences with the best efficency target ratio of 80:1.Conclusions:The positive GPC3 expression was negatively correlated with survival time,and also has interaction with smoking status,clinical stage and survival time in LUSC patients.GPC3 may be involved in the regulation of Cyclin A,E2F1 and c-Myc in PI3K/Akt signaling pathway to affect the distribution of cell cycle so as to promote the occurrence and development of LUSC.Specific CTL induced by DC modified by HLA-A2 restricted GPC3 immunogenic peptide enhances targeted killing of LUSC. |
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