Controlled Live Dendritic Cell Vaccine Induce Antitumor Immunity Against Ovarian Cancer

Live cancer vaccine can elicit stronger tumor-specific cytotoxic of T-lymphocyte (CTL) responses in vitro, and induce more efficient immunopreventive and immunotherapeutic immunity against tumor challenge in vivo than irradiated vaccine. However, using live vaccine to treat cancer has been controversial, mainly due to they have the potential to induce cancer in some vaccinated individuals. Our researches suggested that live vaccine modified with suicide gene might be effective and controllable in the therapy of ovarian cancer. The studies were proceded in 3 steps.Part I: Preparation of a controllable live vaccine based on ovarian cancer/ dendritic fusion cellsObjective: To establish the methods of preparation of a controllable live vaccine based on ovarian cancer / dendritic fusion cells, and study the morphological structure and bio-immunological characteristics of the live vaccine.Methods: 1. Dendritic cell (DC) sprecursors were isolated from Fischer344 rat bone marrow and cultured with granulocyte-macrophage colony-stimulating factor and interleukin-4. 2. The rat ovarian tumor cell line NuTu-19 was genetically modified by retroviral-mediated suicide gene (HSV₁- tk) , and the positive clones were selected using G418. 3. Controllable live vaccine was then fused with DC and NuTu-19/TK cell by polyethylene glycol. 4. The characteristics of live vaccine were assayed with light microscopy, electron microscopy, Confocal laser scanning microscopy and flow cytometry (FCM). The specific expression of HSV₁-tk gene in live vaccine was evaluated by RT-PCR and Western Blot. Mixed lymphocyte reaction and specific CTL were set up by coculturing cells of T cell with live vaccine or irradiated vaccine.Results: 1. About 5×10⁷ DC with typical morphology were harvested from bone marrow per rat. After 10 days culture, DC expressed high level of surface molecules and strongly stimulated T cell proliferation; 2. HSV₁-tk gene transfection of NuTu-19/TK was confirmed by RT-PCR and Western blot; 3. The fusion efficiencywas approximately (23.43±13.80)%. Live vaccine displayed an up-regulated expression of major histocompatibility complex (MHC)-II (OX6), costimulatory molecule (B7-2), integrin (OX62), and adhesion molecules (ICAM-1), while the irradiated vaccine low expressed all these surface molecules (P<0.05). The tumor cell apoptosis of irradiated vaccine was much higher than that of live vaccine(P<0.01). The capacity of stimulating T lymphocyte proliferation of live vaccine was increased dramatically compared with the irradiated vaccine groups(P<0.01). The specific CTL activity induced by live vaccines against NuTu-19 cells was significantly higher than irradiated vaccine groups too(P<0.01).Conclusions: The results demonstrate that live vaccine modified by HSV₁-tk gene can induce antitumor immunity in vitro.Part II. The antitumor immunopreventic and immunotherapeutic effect induced by the controllable live vaccineObjective: To study the antitumor immunopreventic and immunotherapeutic effect on ovarian cancer induced by the controllable live vaccine.Methods: 1. CTL activity was then examined from the spleen T lymphocyte cells of live vaccine-treated rats in LDH cytotoxicity assays. 2. In immunopreventic studies in vivo, rats vaccinated twice with live vaccine were compared to which vaccinated with irradiated vaccine, or PBS. Seven days following the final immunization, the rats were challenged with NuTu-19, and then seven days after challenge, the rats of live vaccine plus prodrug ganciclovir (GCV) group and GCV group were intraperitoneally treated with GCV for continuing 7 days. The tumor incidence and volume were measured in 90 days after challenge, and the vaccine injection site, tumor tissue, lungs, and lymph nodes were examined histologically. The tumor-inhibitory rate was calculated and FCM analysis was performed to access cell apoptosis. 3. In immunotherapeutic studies in vivo, Fischer344 rats were injected subcutaneously with NuTu-19 cells, followed by treatment of live vaccine on days 7 and 14, with the controls of irradiated vaccine, or PBS, respectively. Seven days following the final treatment, the rats of live vaccine plus GCV group and GCV group were intraperitoneally treated with GCV for continuing 7 days. All rats were sacrificed 90 days after the NuTu-19 challenge and the tumors' volume were measured and tumor-inhibitory rate was calculated after that. The necropsy and histological examination were performed, and FCM was analyzed to access cell apoptosis.Results: 1. The spleen CTL activity against NuTu-19 cells in rats vaccined with live vaccine was significantly higher than control groups (P<0.01). 2. The results showed that rats vaccined with live vaccine significantly prolonged tumor latent period and reduced tumor development, compared with rats vaccined with irradiated vaccine (P <0.05). The tumor-inhibitory rate of the live vaccine group was 83.02%, while the irradiated vaccine group was 25.47% (P<0.01). There is extensive necrosis and lymphocytes aggregated in the tumor tissue of the live vaccine group. The tumor cell apoptosis of the live vaccine group was much higher than that of irradiated vaccine group (P <0.05). 3. The data indicated that live vaccine could significantly inhibit the tumor growth, compared with irradiated vaccine, and PBS (P<0.05). The tumor-inhibitory rate of the live vaccine group was 86.24%, while that of the irradiated vaccine group was 34.39% (P<0.01). There is extensive necrosis and lymphocytes aggregated in the tumor tissue of the live vaccine group. The tumor cell apoptosis of the live vaccine group was much higher than that of irradiated vaccine group (P <0.05).Conclusion: Our researches suggest that live vaccine based on tumor/DC fusion cells is an effective therapy for ovarian cancer.Part III: Study on the safety of the controllable live vaccineObjective: To detect the cell-death of live vaccine induced by GCV in vitro and in vivo.Methods: 1.The cytotoxic efficacy of GCV on live vaccine was evaluated by MTT assay. Apoptosis in live vaccine was analyzed by FCM and Hoechst 33258 staining after GCV treatment. 2. To determine the GCV induced death of live vaccine in vivo, rats were vaccinated with live vaccine, and were intraperitoneally treated with GCV 7 days after vaccination. The necropsy and histological examination were performed 90d later. 3. To determine the killing effect of live vaccine in vivo, live vaccine pre-stained with DiI were injected subcutaneously into hind foot pads of rats, and then the GCV intraperitoneally injection to the rats for next 7 days. Cryostat sections of popliteal and inguinal lymph nodes, and spleens were analyzed for TUNEL-positive cells using an in situ cell death detection kit.Results: 1. In vitro experiment showed a dose-dependent and time-dependent cell death of live vaccine by GCV treatment, and 88.42% of the live vaccine was killed by GCV at a concentration of 100μg/ml. In vitro, GCV treatment induced apoptosis wasobserved in up to 80% of live vaccine cells who manifested the typical features of apoptosis, including cell shrinkage, chromatin condensation. 2. In the rat models, the tumor was developed in the injection site in 3 of 5 the rats vaccinated with live vaccine and metastasized to lung and lymph node in one of those rats, but no tumorigensis was observed in the rats vaccinated with live vaccine followed by GCV administration. 3. Data under fluorescence microscope showed that fluorescence-stained live vaccine migrated to spleen also displayed TUNEL-positive, which suggested the live vaccine were killed by GCV in vivo.Conclusion: HSV₁-tk/GCV is likely to kill the live vaccine by triggering apoptosis of cancer vaccine both in vitro and in vivo.In summary, our data showed that immunization of rats with live vaccine modified with suicide gene solicited stronger ovarian tumor-specific CTL responses in vitro and induced more potent immunopreventive and immunotherapeutic effect against parental tumor cells challenge in vivo. Live vaccine could be dead after GCV treatment in vitro and in vivo, which could ensure the safety of the live vaccine.