Anti-Prostate Cancer Immune Response Induced By Dendritic Cells Modified With PSMA And4-1BBL Gene

Background and objective:Prostate cancer is the most frequently diagnosed cancer in old men and also the second leading cause of male cancer death in the Western countries. In addition, the incidence and mortality of carcinoma of prostate are increasing in China. Although radical prostatectomy and radiation therapy remain the primary choice for localized stage of prostate cancer, there is no effective treatment for patients with recurrences or metastatic disease, and patient who develop into hormone-refractory prostate cancer (HRPC). Therefore, development of new treatment modalities is urgently needed, especially for advanced prostate cancer. The publicated reports demonstrated that systemic immunotherapy with dendritic cells (DCs)-based vaccines are capable of inducing potent anti-tumor responses, which make cancer gene therapy and immunotherapy approaches more promising.Potent anti-tumor response require an effective cytotoxic T lymphocytes (CTL) response against cancer through recognizing tumor associated antigen (TAA). Prostate specific membrane antigen (PSMA) is an over-expressed membrane-bound cell surface protein on prostate cancer cells, and is a well-defined TAA. The properties of PSMA propose it as an ideal target of a variety of therapeutic approaches in prostate cancer including the delivery of immunoconjugates, immunotherapy, and prodrugs. Up-regulation of receptor-ligand pairs delivering costimulatory molecule signaling for The induction of CTL during anti-tumor immune response acquires activation signals offered by interaction of a peptide-bound MHC complex on dendritic cells (DCs) with cognate TCR on T cells, the other co-stimulatory signals between them are also essential. Up-regulated these co-stimulatory signals could improve T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily such as4-1BB and4-1BBL have gained importance as co-stimulatory molecules delivering signals, which have profound effects on T cells, including activation of both CD4+and CD8+T cells, enhanced expansion, increased long-term survival, and anti-apoptosis of activation-induced CD8+T cells.So, in this study, recombinant adenovirus vectors encoding tPSMA (the extracellular domain of human PSMA) and m4-1BBL (mouse4-1BBL) were used to infected DCs to induce CTL activity against prostate cancer cells in vitro and vivo. The study attempted to explore a new type of DCs vaccine for prostate cancer immunotherapy.Methods:At first, recombinant adenovirus encoding tPSMA and m4-1BBL and control adenovirus encoding enhanced green fluorescent protein (eGFP) were constructed using the AdMaxTM Expression System which is a replication-deficient adenovirus type5(Ad5) deleted for the genes E1and E3. A full-length m4-1BBL gene was amplified by PCR from plasmid pcDNA3-m4-1BBL and cloned into shuttle vecter pDC316-VP3-IRES-sEndo-HIS. tPSMA cDNA was amplified by PCR from pCR3.1(?)-Uni containing full-length of PSMA and inserted into shuttle vecter pDC316-IRES-EGFP. Then the fragments tPSMA were digested from pDC316-tPSMA-IRES-EGFP through restriction enzyme and subrate cloned into pDC316-VP3-IRES-m4-1BBL resulting in pDC316-tPSMA-IRES-m4-1BBL. After DNA sequence was assayed correctly, the recombinant plasmid pDC316-tPSMA-IRES-m4-BBL was co-transfected into293cells with adenovirus backbone plasmid pBHGlox_E1,3Cre using LipofectamineTM2000. Then recombinant adenoviruses Ad-tPSMA-IRES-m4-1BBL were packaged, amplified and purified. GFP was observed by fluorescenct microscope, and tPSMA and4-1BBL expression were detected by RT-PCR and western blot.Then, DCs vaccine tranducted with Ad-tPSMA-IRES-m4-1BBL were generated. Bone-marrow cells harvested from femurs and tibias of6-8week old C57BL/6mice were cultured in RPMI1640medium containing rmGM-CSF and rmIL-4at37℃and5%CO2On day7, non-adherent cells were collected as immature DCs, or were activated with lipopolysaccharide (LPS) for24h to obtain mature DCs. DCs were identified by morphological features, and infected with Ad-eGFP at various multiplicity of infection (MOI) to get optimal MOI by flow cytometry (FCM). After that, the DCs were infected with Ad-tPSMA-IRES-m4-1BBL at optimal MOI, and then the expression of4-1BBL on DCs was detected by western blot, as well the anti-apoptosis capacity and phenotypes of DCs were analyzed by FCM. Mixed leukocyte reaction (MLR) was performed by DCs co-cultured with Nylon wool-purified naive T cells derived from the spleen of allogeneic BALB/c mice, and the proliferation of T cells was determined by CCK-8method.At last, the efficacy of anti-prostate cancer immune response induced by DCs vaccine was analyzed. A eukaryotic expression vector pcDNA3.1-tPSMA was constructed and sequenced. After that, prostate cancer cell RM-1were transfected with plasmid pcDNA3.1-tPSMA using lipofectamineTM2000, and then screened by G418to obtain stable expression of tPSMA in RM-1cells (RM-1-tPSMA). The expression of tPSMA in RM-1cells was detected by RT-PCR, western blot and immunofluorescence. After three types of DCs transduced with Ad-tPSMA-IRES-m4-1BBL, Ad-eGFP or none were co-cultured with T cells for two times, the culture supernatants were harvested and analyzed for IL-4, IL-10and IFN-y by enzyme-linked immunosorbent assay (ELISA), and activated T cells were collected to be detected expression of P38and IκkB-α by western blot, as well the expression of anti-apoptosis protein Bcl-xL by FCM. In addition, the activated T cells harvested were used to culture with RM-1-tPSMA or RM-1cells to assay the cytotoxic activities of T cells using Cell Counting Kit-8, as well the apoptosis of T cells determined by FCM using Annexin-FITC/PI kit. In mice modality, RM-1-tPSMA cells suspension were inoculated into mice subcutaneously, and then tumor volume was calculated, as well the tumor tissues were evaluated by hematoxylin and eosin (H&E) and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) separately in cancer protective study and cancer therapeutic study.Results:A replication-deficient adenovirus vector carrying the tPSMA gene and mouse4-1BBL gene (Ad-tPSMA-IRES-m4-1BBL) or enhanced green fluorescent protein (Ad-eGFP) was constructed correctly, which was identified by PCR and DNA sequencing. Virus granules were appeared in293cells, and the titre of Ad-tPSMA-IRES-m4-1BBL and Ad-eGFP were6.3xlO9PFU/ml and7.5×109PFU/ml respectively. Western blot showed the tPSMA and4-1BBL could be expressed correctly in RM-1cells tranducted with Ad-tPSMA-IRES-m4-1BBL, not in RM-1cells.DCs derived from bone-marrow cells of C57BL/6mice were successfully induced outside exposed to GM-CSF and IL-4. The typical morphology of DCs with elongated dendritic processes was viewed by inverted microscope and electron microscope. To obtain optimal MOI, DCs were infected with Ad-eGFP at various values. The data suggested that the efficiency of infection increased in a MOI-dependent manner, the viability of DCs, however, in opposite. When MOI was300, the efficiency of infection and viability achieved balance, with about65.5%of the cells showed strong fluorescence and93%cells viability. Western blot demonstrated that DCs tranducted with Ad-tPSMA-IRES-m4-1BBL expressed higher level of4-1BBL than Ad-eGFP-tranducted DCs and none-tranducted DCs. The expression of costimulatory molecules (CD80(81.6±5.4)%and CD86(80.13±2.81)%) up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, higher than that in Ad-eGFP-tranducted DCs and none-tranducted DCs (P<0.05). The results of FCM showed that Ad-tPSMA-IRES-m4-1BBL-tranducted DCs possess enhanced anti-apoptosis capacity with apoptosis rate of16.4%, lower than that in Ad-eGFP-tranducted DCs and control DCs (P<0.05). Data of MLR revealed DCs transduced with Ad-tPSMA-IRES-m4-1BBL induced stronger allogeneic T-cell proliferative responses in vitro than untreated DCs and DCs tranduced with Ad-eGFP.RM-1-tPSMA, a cell of stable expression of tPSMA, was successfully generated, which was identified by RT-RCR, western blot and immunofluorescence. The results of ELISA revealed that Ad-eGFP-transduced DCs and none-transduced DCs induced a basal level of IFN-y in co-culture supernatants. However, a significant increase of IFN-y production (1330.16±251.02)pg/ml was induced by Ad-tPSMA-IRES-m4-1BBL-transduced DCs (P<0.05). The level of IL-4and IL-10among three groups did not show any obvious discrepancy. Western blot illustrated that Ad-tPSMA-IRES-m4-1BBL-tranducted DCs induced decreased IκB-α expression but enhanced phosphorylation of P38without influence of total expression of P38. FCM showed that the mean fluorescence intensities of anti-apoptosis protein Bcl-xL (93.2±10.36)%in T cells induced by Ad-tPSMA-IRES-m4-1BBL-trandutced DCs was higher than that in T cells induced by Ad-eGFP-transduced DCs (65.8±5.17)%and none-tranducted DCs (53.46±6.04)%(P<0.05). Data revealed that the cytotoxic activity was enhanced with increased ratio of effector-to-target cells. T cells pulsed with Ad-tPSMA-IRES-m4-1BBL-tranducted DCs displayed higher cytotoxic activity (54%specific killing; E:T ratio=50) against RM-1-tPSMA than that pulsed with Ad-eGFP transfected DCs or un-transfected DCs (P<0.05). However, none of these cells showed detectable cytotoxic activities against the RM-1cells. In addition, Ad-tPSMA-IRES-m4-1BBL-transduced DCs induced enhanced anti-apoptosis of prostate cancer cells-induced T cells with apoptosis rate of (10.51±1.02)%, compared to that of T cells induced by Ad-eGFP-transduced DCs (22.16±2.3)%and none-transduced DCs (26.85±3.04)%(P<0.05). Moreover, in tumor model, vaccination of Ad-tPSMA-IRES-m4-1BBL-transduced DCs showed a significant inhibition of tumor growth and apoptosis of tumor cells in mice compared to those vaccinated with Ad-eGFP-transduced DCs, none-transduced DCs, or PBS (P<0.05). In the therapeutic study, tumor growth was also inhibited and tumor cells was apoptotic heavily in mice vaccinated with Ad-tPSMA-IRES-m4-1BBL-transduced DCs when compared to those of Ad-eGFP-transduced DCs, or other control groups (P<0.05).Conclusions:The development of DCs engineered to express tPSMA and m4-1BBL by recombinant adenovirus-mediated gene transfer could enhance activation capacity of T cells, resulting specific CTL against tumor cells that express tPSMA in vitro and in vivo, may offer a potential immunotherapy strategy for prostate cancer.