Gemcitabine Enhances The Anti-pancreatic Cancer Effect Of Dendritic Cell-based Immunotherapy:an In Vitro Study

Backgrounds and aims:Pancreatic cancer is considered to have one of the worst prognoses among all the malignancies, with an overall5years survival rate of less than5%o As one of the most complex cancers to treat, pancreatic cancer is relatively resistant to chemotherapy and radiotherapy. So far, gemcitabine (GEM) monotherapy has been the only accepted treatment worldwide for advanced pancreatic cancer. Even so, it leads to only a5-week improvement in median survival duration in patients with advanced pancreatic cancer. Gemcitabine (GEM) is an S-phase-specific, fluorine-substituted pyrimidine analog, which is phosphorylated by deoxycytidine kinase to the active diphosphate and triphosphate metabolites. These metabolites inhibit ribonucleotide reductase and DNA synthesis.The efficacy and tolerability of GEM either alone or in combination with other treatments for advanced unresectable pancreatic cancer have been established, and GEM has become the standard treatment for unresectable pancreatic cancerImmunotherapy based on dendritic cell (DC) vaccine has become a new hotspot since Dr. Steinman attempted to treat his own pancreatic cancer and prolonged his life for four and a-half years. As the principal antigen-presenting cells (APCs) in the immune system, DCs can process and present cancer antigens on their surfaces to T cells, and thus result in antigen specific antitumor effect of cytotoxic T lymphocytes (CTLs). The efficacy and safety of DC vaccine on pancreatic cancer has been demonstrated in several clinical trials. And the dendritic cell vaccine Sipuleucel-T for advanced prostate cancer has been approved in clinical practice by the US Food and Drug Administration. All of above show that DC based immunotherapy will be a promising method for treatment of pancreatic cancer.In recent years, several studies have confirmed that combination therapy with DC based cancer vaccine and GEM exerts a better antitumor effect on pancreatic cancer, and GEM can enhance the efficacy of DC vaccine. However, the precise mechanism still cannot be explicitly explained. In the present study, we aimed to explore the potential mechanism that GEM enhanced immunotherapy based on dendritic cell.Methods:1. Preparation of DCs and T lymphocytes:Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by Ficoll-Hypaque density gradient centrifugation. After cultured in RPMI1640medium containing10%FCS for4h, DCs were generated from the adherent fraction of PBMCs as previously described elsewhere. The adherent cells were cultured for5days in RPMI1640medium containing10%FCS,20ng/mL human GM-CSF, and lOng/mL human IL-4. Culture medium and cytokines were refreshed every other day. On day5, the cells were incubated with lysate of pancreatic carcinoma cell lines at a final concentration of lOO?g of protein/ml for3h. Subsequently, DCs were activated with BCG for48h. Autologous CD3+T lymphocytes were magnetically isolated from the nonadherent fraction of PBMCs using CD3+MicroBeads. The isolated T lymphocytes were cultured in RPMI1640medium containing10%FCS, lOng/mL human IL2, and the medium was replaced every other day.2. CD11c, CD86, CD80, and HLA-DR were detected to analyze maturation of DCs by flow cytometric analysis. Cell counting kit-8assays were performed to determine the T lymph cell proliferation. CytoTox96Nonradioactive Cytotoxicity assay was used to determine T cell-mediated tumor cell lysis.3. After treated with different concentrations of GEM, the viability of SW1990cells was determined by CCK-8assays, and apoptosis and Fas expression were detected by flow cytometric analysis. Antagonistic an agonistic anti-Fas mAbs were used to assess the role of Fas-mediated pathway on the combination of GEM with DC vaccine based immunotherapy in pancreatic cancer.4. After DCs were incubated with GEM-treated medium, maturation of DCs was analyzed by flow cytometric analysis. T lymph cell proliferation were determined by cell counting kit-8assays, and T cell-mediated tumor cell lysis was detected by CytoTox96Nonradioactive Cytotoxicity assay.5. Western blotting was performed to detect the expression of Hsp70in SW1990cells, and Elisa was conducted to assess the level of Hsp70in the GEM-treated SW1990medium.Results:1. In the present study, BCG at multiplicity of infection (MOI) of1induced higher level of CD86, CD80, and HLADR on DCs compared to vehicle control. BCG-stimulated DCs promoted the proliferation of autologous T lymphocytes significantly, and the ability of proliferation increased with the increasing ratios of DC:T cells in a time-dependent manner. The T lymphocytes were collected after cocultured with BCG-stimulated DCs at the DC:T cell ratio of1:10for4days. The results showed that BCG-stimulated DCs induced more efficient CTLs anti-pancreatic cancer SW1990response compared to control at effector/target (E/T) ratios of100:1,50:1, and20:1. When a cholangiocarcinoma cell line QBC939was used as target, the ability of cytotoxicity decreased significantly.2. After treated with GEM for48h, the inhibition rate for SW1990cells increased with the increase of GEM concentrations (Fig.4A). In addition, GEM also induced tumor cell apoptosis in a concentration-dependent manner. The expression of Fas increased significantly after treated with GEM. When treated with0.025?M GEM for48h, the SW1990cells were harvested and cocultured with CTLs to detect the specific lysis. The results showed that GEM enhanced the tumor lysis at the E/T ratios of100:1,50:1, and20:1but10:1. When0.025?M GEM-treated SW1990cells were preincubated with antagonistic anti-Fas antibody ZB4prior to coincubation with CTLs, ZB4reduced the lysis of SW1990treated with0.025?M GEM at the E/T ratios of100:1,50:1, and20:1.3. Compared to control, G2.5medium induced more maturation of DCs. The proliferation rate of T Lymphocytes co-cultured with DCs pulsed with G2.5medium was increasing rapidly with the decreasing ratio of T cells:DCs. DCs pulsed with G2.5medium induced more efficient CTLs response compared to control at the E/T ratios of80:1,40:1,20:1.Conclusion:1. BCG could stimulate the maturation of DCs, and the mature DCs induced effective CTLs antitumor response.2. GEM sensitizes pancreatic cancer cells to the CTLs antitumor response, and the sensitization is associated with up-regulation of Fas on pancreatic cancer cells.3. Gemcitabine-treated pancreatic cancer cell medium induces the specific CTL antitumor activity by stimulating the maturation of dendritic cells. Further research showed stimulation of DCs maturation may be related to the elevated level of Hsp70induced by GEM.