| 1. Aim and MeaningGlioma is a kind of malignant tumor which holds great threat to human helth. Although current surgery, radiotherapy and chemotherapy have grately improved, there remains high mortality and disability rates in patient with glioma. It’s mainly because of the invasive growth of glioma cells.Conventional chemotherapy currently used in clinical practice often damages the vital organs in patients like heart, liver, lung, kidney while killing tumor cells in the patient’s body. These have greatly affected the quality of life in patients. There for, the urgence remains in searching for new therapeutic means that’s safe and effective at the same time.In the late 1950s, academics proposed the concept of tumor stem cells (TSC), which believes that the innermost reason for tumor’s invasive growth, tendancy to relapse and tolerance of high doses of chemotherapy lies in a group of tumor cells that manifest the features of stem cells. With self-renewal capacity and pluripotency, these cells may well be the root of tumor growth, metastasis and recurrence. In 1997, this kind of tumor cells was separated from patients with leukemia by Bonnet and Dick. Afterwards, stem cell-like tumor cells were identified from a variety of solid tumors such as melanoma. In 2004, Singh etc. identified such stem cell-like tumor cells in glioma, naming them as glioma stem cells (GSC). In summary, searching for the methodological approach of targeted killing of BTSC or GSC, thus eliminating brain tumors to prevent its recurrence is meaningful research which could provide evidence for clinical study.In the latest research in 2015, it showed that tumor stem cells would become the new focus of cancer treatment strategies. Among all the approaches targeting TSC, such as induced differentiation and microwave therapy of TSC or therapies targeting its signaling pathways or microenvironment, immune therapy targeting TSC has received much concern due to its safety and theretical long-term protection. However, the immunotherapy of cancer stem cells are mostly concentrated in the study of the skin, liver, gastrointestinal and lung cancer treatment areas so far. And immunotherapy regarding GSC is still to be improved.In general, the immunotherapy of tumors consists of the following three types, namely passive immunotherapy, adoptive immunotherapy and specific active immunotherapy. Passive immunotherapy is based on monoclonal antibody technology which entails mainly on the treatment by monoclonal antibodies and immune-oriented monoclonal antibody conjugates. Adoptive immunotherapy is to enhance the immune function in patients by injection of immune cells or immune molecules that have been processed in vitro. Specificactive immunotherapy can be subdivided into tumor cell vaccine, tumor antigen vaccine, subcellular vaccine (such as exosome vaccine) and dendritic cell vaccine.As is known, dendritic cells (DC) are the most potent professional antigen-presenting cells in the human body. Immature DCs are in charge of active uptake and processing of antigen, while mature DC can effectively present antigens to T cells by expressing high levels of majorhistocompatibility complex (MHC)-I,II antigen and costimulatory molecules as CD80 and CD86. All the above information suggests that should GSC be killed, the brain tumor development/relapse can get virtual elimination and suppression. And DC can assume this effect by the presentation of GSC antigen.Because of the heterogeneity in glioma tissue, it has always been difficult to separate and purify specific antigen of glioma cells or glioma stem cells. So to elicit potent CTL reaction, a series of tumor antigen epitopes are required. That’s what makes total tumor cell antigens so important. The classic ways to pulse dendritic cells withtotal tumor cell antigens includes tumor lysates, protein extraction and apoptosis tumor cells etc. They allhave advantages and disadvantages and in our study, it is our aim to make comparisons of these methods by the evaluation of DC maturation and function in tumor vaccines.2. MethodsThis study comprises of three parts:part one, obtain total tumor antigen; part two, induction and culture of mouse bone marrow-derived dendritic cells; part three, induction of T lymphocytes and cytotoxic T lymphocytes reaction.In part one, we cultivated GSC using the classic serum free method, assisted by flow cytometric selection. Then we made GL261 glioma cells into tumor lysates antigens, apoptosis tumor cell antigens and tumor cell total RNA extraction antigens, respectively.In part two, we isolated mouse mononuclear cells from their bone marrow and induced them to be dendritic cells by adding 40ng/ml GM-CSF and 40ng/mL IL-4 into the culture system. We then obtained immature DCs at day 5 and turned them into mature DC by pulsing them with different GC antigens described above. As to positive control group, we used LPS as the stimulating factor, while in the negative control group, no stimulatory factors of any kind was added. At different time points after stimulation (12h,18h,24h), proliferation status of DCs in each group was measured. Moreover, after 24h of sensitization process, flow cytometry was performed to detect the major surface markers on DC(CD80, CD86, MHC-Ⅱ). Meanwhile, the classic ELISA method was used to measure the amout of cytokines secretioned (TNF-α, IFN-γ, IL-6, IL-10). After an elevulation of all the above three aspects, an optimal sensitization method was selected for following research.In part three, we isolated mouse peripheral blood mononuclear cells (PBMC) from their peripheral blood and induced them to be lymphocytes by adding 40ng/ml IL-2 into the culture system. We then performed flow cytometry to select CD3+ T lymphocytes. Detect the ecpression of CD3 on isolated cells to determine the purity of T cells. We then performed an in vitro cytotoxic T lymphocytes reaction elicited by GSC/GC apoptosis antigen+DC vaccines to assess the killing rate of glioma cells.3. ResultsGSC could be obtained from mouse GL261 glioma cell line by the method of serum free cultivation assisted by flow cytometric sorting method.Tumor lysates antigens, apoptosis tumor cell antigens and tumor cell total RNA extraction antigens of GC could be obtained in a stabilized way.Bone marrow mesenchymal cells and peripheral blood cells could be obtained from homologous C57 mice. And DC and T lymphocytes could be induced from the above mentioned cells respectively by adding appropriate cytokines into the culture system. Immature DC with stable features could be obtained at day 5 and turned into mature DC by pulsing them with different GC antigens or LPS (positive control group). By flow cytometry and ELISA method, the major functional surface markers of DC were detected and the secretion of immune active cytokines was mesured. From the above results, we were able to determine the apoptosis tumor method as the optimal sensitization method.And by using the optimal DC vaccine, we performed an in vitro cytotoxic T lymphocytes reaction elicited by GSC/GC apoptosis antigen+DC to assess the killing rate of glioma cells, and found that the GSC/GC apoptosis antigen+DC vaccine is quite potent. When the killing target is GSC, GSC+DC vaccine showed a more powerful effect than GC+DC vaccine.4. ConclusionGSC could be obtained from mouse GL261 glioma cell line by the method of serum free cultivation assisted by flow cytometric sorting method.Tumor lysates antigens, apoptosis tumor cell antigens and tumor cell total RNA extraction antigens of GC could be obtained in a stabilized way.Bone marrow mesenchymal cells and peripheral blood cells could be obtained from homologous C57 mice to induce into DC and T lymphocytes. By pulsing DC with different GC antigens or LPS (positive control group) for 24h, the proliferation status, major functional surface markers of DC were detected and the secretion of immune active cytokines was measured afterwards to determine that the apoptosis tumor method was the optimal sensitization method among all three total tumor antigens aensitization mehods in this study, and that the GSC/GC apoptosis antigen+DC vaccine is quite potent in eliciting an in vitro cytotoxic T lymphocytes reaction against glioma cells. However, the difference of glioma cells killing rate is not significant between GSC apoptosis antigen+DC vaccine and GC apoptosis antigen+DC vaccine, when the killing target is GC, which is probably due to the assessing standards we used in this study. However, when the killing target is GSC, GSC+DC vaccine showed a more powerful effect than GC+DC vaccine. In conclusion, further studies involving the observation of in vivo antitumor effect, especially long-term effect and anti-relapse effect are still required. |
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