Glioma is the most common tumor in neurosurgery with poor outcome. Immunotherapy is an effective biological treatment for malignant glioma, of which dendritic cell (DC)-mediated immunotherapy is now commonly used to prolong the survival of glioma patients. Our previous study found that glioma stem cell-like antigen (whole tumor antigen extracted from stem cell-like cells) has stronger immunogenicity than traditional antigen, and the vaccine prepared by loading autologous DC with this antigen has been proved to be effective in both in vitro and animal tests. Materials and methods 1. Patient selection criteria: patients with recurrent glioma, clinical diagnosis of recurrent malignant glioma (WHO pathology grade III-IV); complete tumor resection or resection degree > 80% (confirmed by postoperative enhanced MRI); expected survival > 3 months; long-term quality of life KPS (Karnofsky Performance Scale) score ≥ 60 for glioma patients. Patients’ hematological indexes met the following conditions: white blood cells > 3×109/L, hemoglobin > 90 g/L; normal liver and kidney function (portal amino transferase and glutamate alanine aminotransferase ≤ 2 times the upper limit of standard normal values, creatinine ≤ 150 μmol/L, urea nitrogen ≤ 9 mmol/L); agreed to join the study and signed the informed consent form. The study was approved by the ethics committee of Huashan Hospital, and the relevant patients and their families were informed in detail and signed the informed consent form. 2. Primary culture of glioma cells: Fresh tumor specimens were taken at the time of surgery and primary culture was performed in vitro. The steps were briefly described as follows: fresh glioma tissue blocks were washed and cut up, added 5-10 times the volume of 0,2% collagenase IV (Sigma) for 10 min in 37°C incubator, centrifuged and lysed with ammonium chloride erythrocyte lysis solution, filtered through a steel mesh (pore size 30 μm) to make a single cell suspension The cells were inoculated with DMEM/F12 culture medium (with 20 ng/μl bFGF, 20 ng/μl EGF, 20 ng/μl B27) at a density of 3×105 cells/cm2 and incubated in a 5% CO2 incubator at 37℃. 2-3 d fluid changes were performed once a week and the morphology was observed under a phase contrast microscope. 3, CD133-positive stem cell-like cell sorting: Some primary cultured cells (5×107~1×108 cells) were taken to make tumor monocytes and labeled with CD133/1 (Miltenyi Biotec) flow and magnetic bead antibodies respectively according to the kit instructions, and the proportion of CD133+ cells was detected by flow cytometry, and CD133/1 magnetic bead antibodies ( Miltenyi Biotec) labeling and then sorted. The magnetic bead sorting method was as follows: CD133/1 magnetic bead antibody was co-incubated with cells for 10 min at 4°C, washed twice with PBS; 500 μl PBS was used to resuspend the cells, and the cell suspension was slowly passed through the column; 1 ml PBS was washed twice to collect CD133- cells; the magnetic field was removed, and CD133+ cells were collected by PBS washing. CD133+ and CD133- cells were labeled with CD133+ and CD133- cells were analyzed by CD133/2 flow antibody labeling for CD133+ cell ratio. 4, Stem cell-like antigen and DC vaccine preparation: CD133+ cells were collected after the above sorting, irradiated by X-ray (6 Gray, Varian 600C linear gas pedal, Shanghai Gamma Hospital), collected cells were repeatedly freeze-thawed in liquid nitrogen and 37°C water bath three times, repeatedly blown with a pipette, and the supernatant obtained after high-speed centrifugation was the glioma stem cell-like antigen solution. The concentration was determined by standard Bradford protein assay, adjusted to 1 μg/μl by PBS, and stored at -20 ℃ for backup. Patient’s autologous blood was collected in 15-20 ml, and mononuclear cells were separated by Ficoll lymphocyte isolation solution (Cedarlane) and inoculated in RPMI1640 culture solution (Gibco) containing GM-CSF and IL-4 (R&D Systems). 7 d later, suspended DC cells were collected, labeled with CD11c flow antibody, analyzed by flow cytometry, and additionally incubated with the above antigen Co-cultured at 37°C for 18 h (final concentration of antigen was 150 μg/μl), the expression of surface molecules such as HLA-Ⅰ, HLA-Ⅱ, CD80 and CD86 on DCs was detected after antigen stimulation [2] to understand the maturation of DCs. Counted after physiological saline washing and adjusted the volume to 0,5 ml.5. DC vaccine treatment combined with chemotherapy and related indexes detection: head-enhanced MRI was performed within 3 d after surgery to observe the surgical resection. The tumor DCs vaccine prepared by the above method was injected intramuscularly into the triangle within 4-8 weeks after surgery. One injection was given every 1 week for a total of 3 injections. Vital signs and neurological symptoms were observed during immunotherapy. Before immunotherapy (1st blood draw) and after treatment (3rd vaccination), 5 ml of blood was collected and tested at the Department of Laboratory Medicine of Huashan Hospital (accredited by College of American Pathologists) for blood count (including white blood cell count, lymphocyte ratio, etc.), liver and kidney function, blood sodium, potassium, calcium, etc. After immunotherapy, temozolomide (TMZ) chemotherapy was continued with a regimen of TMZ 200 mg?m-2?d orally for 5 d, 28 d for 1 cycle. Immunotherapy was followed by head-enhanced MRI examination and survival follow-up at intervals of about 3 months. RESULTS 1. Summary of patient data: All five patients met the inclusion criteria, and the pathology was malignant glioma after the second surgery. Postoperative enhanced MRI confirmed total resection in 4 cases and partial resection in 1 case. Three patients received radiotherapy and chemotherapy after the first surgery, one patient underwent local gamma knife treatment, and one patient did not receive radiotherapy or chemotherapy. After the 2nd operation, all 5 patients received immunotherapy with 3 vaccinations and temozolomide chemotherapy. Detailed information is shown in Table 1. 2. CD133+ cells in glioma cells: tumor cells grew in suspension sphere-like in serum-free culture medium (Figure 1a), and tumor spheres were seen rolling when the culture bottle was shaken. Some cells were taken on day 3 of primary culture to make single cell suspension, and the proportion of CD133+ cells was detected by flow cytometry after CD133-1/PE antibody labeling, and the results showed that all the recurrent gliomas in the above patients contained CD133+ cells, but the proportions were different. Magnetic bead sorting was able to increase the proportion of CD133+ cells in each glioma cell line to varying degrees (Table 2).3. Criteria for DC vaccine preparation: Stem cell-like antigen stimulation of DCs resulted in increased expression of molecules such as HLA and CD86 on their surface, suggesting mature DCs [2], and most cells were seen to be aggregated, suspended and with finger-like protrusions under the microscope, with characteristics of mature DCs. The flow detection results showed that the purity of CD11c positive cells reached (80±6,34)% after induction (Figure 1b-e), and the number of DCs cells could reach 3×106 to 5×106 (Table 2). 4. Relevant indexes before and after immunotherapy: there was no significant difference in liver and kidney function and electrolytes before and after immunotherapy, and they were all within the normal range. The percentage of lymphocytes in blood routine increased from (38±6,72)% before immunotherapy to (44,6±8,61)%, but the difference was not statistically significant (n=5, P=0,0592). 1 case complained of dizziness after DC vaccination in 5 patients, which resolved on its own within 24 h. All patients had no headache and no fever. All patients had no headache, no fever, no diarrhea, no rash, no change in neurological signs, and no redness or swelling at the injection site. One patient with partially resected tumor had a grade IV glioblastoma, and the tumor recurrence was confirmed by MRI and MRS at 15 weeks after surgery and 7 weeks after immunotherapy. MRS confirmed residual tumor growth to the contralateral side, and significant tumor shrinkage was seen at 30 weeks postoperatively and 22 weeks after immunotherapy, but the residual tumor progressed again at 53 weeks postoperatively (Figure 2), and he died of tumor progression at 57 weeks postoperatively. Discussion With the rapid development of immunology, the understanding of central nervous immunity has changed from “immune-privilege” to “immune-special” [4]. Immunotherapy of glioma is gaining attention. Highly malignant glioma itself has a strong immune escape ability, and surgical resection of the tumor helps to reduce tumor-induced local and systemic immunosuppression [5]; the blood-brain barrier around glioma often has increased permeability due to upregulation of vascular endothelial growth factor, increased expression of aqueous phase channel protein 4, and broken tight junctions, which facilitate the entry of systemic immune cells and other cells into the brain [4], and these immune properties have triggered interest in immunotherapy of gliomas. DCs are the most potent specialized antigen-presenting cells known to take up, process and deliver antigens, have a powerful ability to activate T cells, participate in the immune response in vivo, and are central to the tumor immune response. In order to enhance the immune response of the body, tumor antigens are often introduced into DCs in vitro to stimulate the immune response against tumor cells, i.e. tumor DC vaccine. The advantages are: (1) specific recognition of tumor cells, strong targeting, and low adverse effects; (2) combined with radiotherapy and chemotherapy, it can produce better effects than single treatment; (3) memory cells generated during immunotherapy can help prevent recurrence. Sensitized DC cells, patients had significantly longer survival and CD8+ T-cell infiltration and memory cells in tumor specimens for reoperation. In China, Xue Delin et al [7] inoculated glioma patients with DC vaccine prepared from tumor tissue homogenate, which also prolonged patient survival. Nevertheless, the lack of glioma-specific antigens has limited the further development of glioma immunotherapy. Although gliomas express a variety of relevant antigens such as variant EGFR, IL-13R, and tenascin [4], the above antigen expression is not a commonly expressed glioma antigen. It is now recognized that tumor mixed antigen-sensitized DC is one of the safer and more effective methods for the treatment of glioma [5], but mixed antigens themselves have the disadvantage of weak antigenicity. Our previous study found that tumor mixed antigens derived from glioma stem cell-like cells, i.e., stem cell-like antigens, are more immunogenic than conventional antigens [2]. Stem cell-like antigen-sensitized DC vaccines are currently available only in in vitro experiments [2] and mouse animal experiments [3] (whose stem cell-like antigens are obtained from unsorted cells), and no clinical studies are currently available. Based on this, this study applied glioma stem cell-like antigen-sensitized DC vaccine to patients with malignant glioma to observe its feasibility, safety, and efficacy. All patients enrolled in this study were recurrent and postoperative pathologically confirmed malignant gliomas. Surgery is preferred for the treatment of malignant gliomas, and immunotherapy as postoperative adjuvant therapy is suitable for microscopic lesions or scattered tumor cells. Temozolomide has also been reported to prolong the survival of glioma patients [8], and vaccine can enhance the efficacy of chemotherapy for glioma [9], so we combined the two. However, since chemotherapy significantly reduces leukocytes, immunotherapy is recommended to precede chemotherapy. In this study, patients with relapsed glioma treated with DC vaccine had no significant adverse reactions, no autoimmunity, etc. The survival of two grade III patients was 72 and 75 weeks, and the survival of three grade IV patients was 57, 77, and 80 weeks, with a mean survival time of 72, 2 weeks, and follow-up work and this study is still in progress. yU et al [10] studied the mean survival of DC vaccine group was about 133 weeks; the control group was recurrent glioma and the average survival was about 33 weeks after surgery and radiotherapy. Zhang X. S. [5] synthesized the literature and found that the current survival period after surgery for recurrent glioma is about 25, 7 weeks. This shows that the modified DC vaccine in this study is safe and effective, and that DC vaccine combined with temozolomide chemotherapy prolongs patient survival. Although immunotherapy for glioma is effective, there are some drawbacks of the existing DC vaccines and it is still difficult to improve their efficacy. In addition, efforts are needed to simplify the process and industrialize them. In addition, due to the limited number of cases in this paper, the safety and efficacy of DC vaccines for glioma need to be more carefully and thoroughly observed.