Antitumor Immune Responses Induced By Gene Transfer Of B7-H3 Into Squamous Cell Carcinoma Tca8113 In Vitro

ObjectiveTumors may present antigens to T-cells, however they lack costimulatory signals which is necessary to initialize an effective immunologic response. The present study was purposed to develop a tumor cell-based cancer vaccine by genetically modifying oral squamous cell cancer (OSCC) cell line Tca8113 with human B7-H3 immunoglobulin, and detect gene expression in transfected cells, evaluate its efficacy on enhancing the tumor specific immune response.MethodsThe plasmid pEGFP-C1-B7-H3 was transfected into Tca8113 with Lipofectamine2000.Then the expression of GFP was observed under fluorescent microscope after 24h,14d and 30d. The expression stability and efficiency of the target molecule was identified by RT-PCR,Western Blot. Selected by G418 after transfection,a stable cell line B7-H3/Tca8113 expressing human B7-H3 was established.Tumor cell vaccine TCV-hB7-H3 and mock control were prepared by treating B7-H3/Tca8113 cells with MMC. After being stimulated with TCV-hB7-H3 or mock control, the proliferation, IFN-γexpression and cytotoxicity of the T cells were assessed.Results The total RNA was isolated by using Trizol from the Tca8113 cell transfected pEGFP-C1-B7-H3, and detected the expression of B7-H3.PCR products analysis have showed that the amplified product was about 215 bp,consisted with the previous prediction.however, we failed to find PCR product of 215bp in Tca8113 transfected with mock vector pEGFP-C1. Western blot have showed that a differential belt was consisted of about 84～90KD protein molecules, but the lack of differential belt belt in the mock/Tca811 cell.After 24 hours,14days and 30days of transferring of pEGFP-C1-B7-H3 on Tca8113 cell lines by lipofectamine 2000, observing cell transfection efficacy under fluorescent microscope at 480nm or 513nm after incubation, the expression of B7-H3 was detected by RT-PCR and Western blot respectively. Then the transfected cells were selected in medium containing G418 (600μg/ ml) and termed as Tca8113-hB7-H3. Through monoclone-picking method, we have constructed the cell line of Tca8113/hB7-H3. B7-H3 gene highly expressed was detected in the Tca8113 cell which has been passed several times. Tca8113 cells transfected with human B7-H3 significantly enhanced the proliferation, IFN-γexpression, and cytotoxicity of the T cells.ConclusionsThe recombined expression vector, pEGFP-C1-B7-H3, was transfected into Tca8113 by Lipofectamine 2000 and the Tca8113 cells were identifying the expression of B7-H3.with RT-PCR and Western blot. The cell line of Tca8113/hB7-H3 has been constructed through monoclone-picking method. B7-H3 gene had an important significance and wide application in the future.In our study, The cell line of Tca8113/hB7-H3 has been constructed and cancer vaccines have been made too, Tca8113 cells transfected with human B7-H3 significantly enhanced the proliferation, IFN-γexpression, and cytotoxicity of the T cells, the genetically modified OSCC cells encoding B7-H3 enhance the induction of tumor specific immune response.This will give us a good level to research cancer in vivo and lay a base of making further study its antitumor function.