In Vitro Study Of ERBB2 Gene-modified Lentiviral Vector Mediated Dendritic Cells In Lung Cancer Treatment

Objective:Constructed a recombinant lentiviral vector(LV)encoding ERBB-2 gene,to explored the effect of cytokine induce killer's(CIK)proliferation and anti-tumor immune responses by co-culture with DCs transduced with lentiviral vectors encoding ERBB2 gene and untreated DCs in Her-2 positive lung cancer.Methods:1.Construction of full-length Her-2 gene containing lenti-virus vector(LV-Her2):ERBB2 gene was directly amplified from pcDNA3.1-ERBB2-EGFP plasmid by PCR.The PCR products were visualized on ethidium bromide-stained agarose gel,and then inserted into pCDH-CMV-MCS-EF1-GFP-T2A-Puro vector,which encoded a GFP gene for monitoring transduction efficiency.Human embryonic kidney 293 T cells were triple transfected using the calcium phosphate method with the lentiviral expression vector and three packaging vectors: pMD 2.G,pMDL-G/P-RRE and pRSV-REV.The supernatants containing the viral particles were collected at 36 h post-transfection and filtered through a 0.45-um filtration column.Viral titers were determined by infecting 293 T cells with serial dilutions of the vector stock.2.Preparation of DCs and transduction with LV-Her2(LV-Her2-DC): Dendritic cells were isolated and cultured from peripheral blood.On day 5,immature DCs(imDCs)were transduced with LV-Her2 at 10 multiplicities of infections(MOI)for 72 h at 37? with 5 % CO2 incubator.The efficiency of transduction of DCs was analyzed for green fluorescent protein(GFP)-positive cells by fluorescence microscope.On day six,tumor necrosis factor alpha(TNF-?)was added to the culture medium for 48 h to induce DCs maturation.Cells were harvested on day 8 for subsequent experiments.Cells were subjected to flow cytometry(FCM)for the detection of CD1a/83/86 and HLA-DR expression.DCs transduced with empty vector(LV-DC)and untreated DCs were used as controls.3.Detection of CIK proliferation:CIK was isolated and cultured from peripheral blood according to the protocol.CIK cells were co-cultured with autologous DCs inthree parallel groups consisting of LV-Her2(LV-Her2-DC-CIK),empty vector(LV-DC-CIK),or untreated DCs(DC-CIK).CIK were subjected to flow cytometry for the detection of CD3/56 expression.CIK proliferation was measured by cell counting Kit8(CCK8)assay.Absorbance was measured at 450 nm using a Microplate Reader.4.Cytotoxicity assays: The activity of CIK against the target tumor cells was tested by CCK8 assay.One string of lung cancer cell line expressed Her-2 protein was incubated with CIK stimulated by DCs at effector/ target ratios of 10:1,20:1,40:1,respectively.After 48 h co-incubation,CCK8 reaction solution at ten percent of total volume was added to each well.The OD values were measured at 450 nm,by which to calculate the tumor killing activity of CIKs.Result:1.LV-Her2 construction: Green fluoresce was observed in 293 T cell after transfection,suggesting that the vector containing Her-2 sequence had been transferred into the target cells.The virus titer was 2x108TU/ml as tested by hole dilution method.2.DCs preparation and transduction with LV-Her2:We obtained dendritic-like morphology upon the incubation of GM-CSF and IL-4.LV-Her2 and empty vector were transfected into DCs successfully and the transfection rate were 41.8% and40.2% at MOI of 10,suggested that the fraction of GFP cells did not differentiate significantly(p=0.293),and the Her-2 gene had no difference effort on lentiviral transduction efficiency in DC.After adding TNF-? for 48 h to the culture medium,the results showed that Her-2 could be effectively expressed in LV-Her2-DC.Surface markers on DCs transduced with LV-Her2,empty vector and untreated were tested using flow cytometry.There were no significant differences in CD1a/83/86 and HLA-DR on DCs expression after transduced with LV-Her2,empty vector or untreated,suggested that LV-Her2 transduction did not alter the ability of DC maturation.3.Peripheral blood was isolated and cultured to product CIK,with a form of cluster growth,and was easy to stir into single cells.There were no significant differences in the surface marker CD3/56 of CIKs co-cultured with LV-Her2-DC,empty vector or untreated DCs.The activation and proliferation capacity of CIK co-culture with LV-Her2-DC is about twice than the empty vector DCs and untreated DCs(p=0.00),the proliferation of CIK co-culture with empty vector DCs and untreated DCs had no significant difference(p=0.06),the proliferation of CIK co-culture with LV-Her2-DC was significantly higher than untreated DC(p=0.007).4.A549 lung cancer cell showed weak and incomplete membrane staining for Her-2 immunohistochemistry test.When effector/ target ratios were10:1,20:1,40:1,respectively,the percentages of specific lysis in LV/Her2-DC-CIK against A549 lung cancer cells were: 26.9%,43.8%,52%,respectively.LV-Her2-DC-CIK was markedly enhanced than DC-CIK(p<0.05),but there was no significant difference between LV-DC-CIK and DC-CIK(p>0.05).These data suggested that lytic activity of antigen-specific CIK induced by LV-Her2-DCs had more effort than other methods,and was specific to Her2-positive cells.Conclusion1.We successfully constructed a recombinant lentiviral vector encoding Her-2gene,which could effectively induce Her-2 expression in transfected cells.2.The activation and proliferation capacity of CIK transduced with LV-Her2-DCs was markedly enhanced.3.LV-Her2-DC-CIK had stronger anti-tumor effect than LV-DC-CIK and DC-CIK.4.LV-Her2-DC vaccine could be a promising target in the treatment of Her-2positive lung cancer.This study made a certain experimental and theoretical basis to developan innovating individualized treatment for patients with Her-2 positive lung cancer.