خاموش کردن محور PD-L1/PD-1 به منظور افزایش عملکرد واکسن بر پایه سلول دندرتیک در مدل تجربی سرطان

مجریان: فرهاد جدیدی نیارق

خلاصه روش اجرا: 1. Generation of nanoparticles: Chitosan lactate nanoparticles will be produced through ionic gelation method. We were used this nanocarrier in our previous study [29]. 2. Characterization of physicochemical features of nanoparticles including: a. Size and zeta potential by zeta sizer. b. Conjugation of chitosan with lactate by FTIR test. c. Assessment of siRNA loading by gel electrophoresis. d. Cellular uptake of nanoparticle s by confocal microscopy. 3. Treatment of 4T1 and B16 cells with siRNA loaded nanoparticles and investigation of: a. Antiproliferative effects of nanoparticles by BrdU incorporation assay. b. Apoptotic effects of siRNA nanoparticles by MTT assay. c. Antiangiogenic and anti-metastatic effects by investigation of expression of VEGF, CD31, MMP2, and MMP9. d. Investigation of cytokines IL-6 and IL-10 in supernatant of cells. 4. Generation of DCs based on our previous report protocol [29]. Briefly, DCs will be generated from precursor cells isolated from bone marrow of mice and treated with IL-4 and GM-CSF for five days. Cells were then pulsed with tumor lysate and incubated with LPS for final maturation. a. The phenotype and cytokine production will be performed for generated DCs. 5. Tumor inoculation; female, 6-8 weeks Balb/C mice will be S.C. inoculated with 700.000 4T1 cells in the right flank. 6. Mice will be divided into following groups: Group 1: PD-L1 suppressed DC+NP loaded with PD-L1 and PD-1 siRNA Group 2: DC+NP loaded with PD-L1 and PD-1 siRNA Group 3: PD-L1 suppressed DC +NP loaded with PD-L1 Group 4: PD-L1 suppressed DC +NP loaded with PD-1 siRNA Group 5: PD-L1 suppressed DC Group 6: DC Group 7: NP loaded with PD-L1 and PD-1 siRNA Group 8: NP loaded with irrelevant siRNA Group 9: Control N=11 mice per group Mice will be treated with optimized amounts of NPs (intravenously) at days 6, 9, 12, and 15 after tumor inoculation. DC vaccine will be injected beside the tumor at day 7. Mice (4 mice per group) will be sacrificed at day 18 after tumor inoculation and will be analyzed for immunologic assays. Frequency of Treg, Th17, and MDSCs in the tumor, spleen and lymph nodes will be investigated by flow cytometry. Function of anti-tumor lymphocytes will be assayed through CFSE, cytokine production, and granzyme B secretion, ex vivo. Function of Treg and MDSC will be analyzed by CFSE assay. Expression of tumorogenic genes in the tumor samples will be analyzed by real-time PCR. Seven mice per group will be analyzed for survival time.