Exosomes Derived From Il-12-anchored Renal Cancer Cells Increase Induction Of Specific Antitumor Response In Virto

PART ONE Separation and identification of exosomes derived from renal cancer cells and primary study of protein compositionObjective:To separate and purify exosomes derived from renal cancer cells and detect their immune associated protein in order to investigate their potential clinical value.Methods:Exosomes derived from RC-2 cells culture supematants were separated and purified by ultrafiltration and sucrose gradient centrifugation, which were identified by transmission electron microscope. The expressions of HSP70, ICAM-1 and G250 were detected by western blot.Results:Exosomes were successfully purified by ultrafiltration and sucrose gradient centrifugation, which were 30~80 nm in diameter with typical saucer-shape morphology, and expressed HSP70,ICAM-1 and G250 antigen. Conclusion:Ultrafiltration and sucrose gradient ultracentrifugation can be used to purify renal cancer cells-secreted exosomes. Exosomes, expressing HSP70, ICAM-1 and G250, might be the novel vaccine for renal cell cancer.PART TWO Construction of mammalian co-expression plasmid of chemric IL-12-GPI cDNAObjective:To construct a mammalian co-expression plasmid of pBudCE4.1/IL-12A/IL-12B-GPI.Methods:A mammalian co-expression plasmid of GPI-anchor- IL-12 (human) were constructed by subcloning IL-12A (P35) gene and a fusion gene containing GPI-anchor signal sequence of hPLAP-1 and IL-12B (P40) in pBudCE4.1. .Results:Plasmid pBudCE4.1/IL-12A was digested with KpnI and XhoI, and resulted in band of IL-12A-chain(762 bp). The correct sequence was identified by DNA sequencing. The recombinant plasmids pBudCE4.1/IL-12A/IL-12B-GPI were digested with KpnI, XhoI, BamHI andXbaI, and resulted in two bands, 1104 and 762 bp, named IL-12A-chain and IL-12B-GPI, respectively. The correct sequence was identified by DNA sequencing, showing that co-expression plasmid of pBud-CE4.1/IL-12A/IL-12B-GPI was successfully constructed.Conclusion The recombinant plasmid may provide foundation for further preparation of tumor vaccine.PART THREE The preparation and identification of renal-cancer vaccine of exosomesObjective:To prepare IL-12-anchored exosomes(EXO/IL-12) derived from renal cancer cells and identify their structure and function.Methods:RC-2 transfectants expressing GPI-IL-12 (RC-2-GPI-IL-12) were established by transfecting pBudCE4.1/IL-12A /IL-12B-GPI plasmid. Confocal laser scanning microscopy and flow cytometry analyzed the expression of fusion protein. TEM and Western blot identified the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of anchored IL-12 exosomes was determined by IFN-γrelease assay and CFSE-based cell proliferation.Results:Confocal laser scanning microscopy and flow cytometry analysis of the RC-2-GPI-IL-12 transfectants showed expression of IL-12 on the cell surface via a GPI-moiety. EXO/IL-12 were purified by ultrafiltration and sucrose gradient centrifugation, which were 30~80 nm in diameter with typical saucer-shape morphology, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. 80±9.6pg/ml of IL-12 was detected in 10μg EXO/IL-12 and it significantly induced cell proliferation and the release of IFN-γ.Conclusion:The vaccine of EXO/IL-12 can be obtained from the culture supernatant of IL-12-anchored RC-2 cells, exprssing IL-12 and tumor associated antigen G250, could efficiently increase induciont of cell proliferation and the release of IFN-γof T lymphocytes in vitro.PART FOUR EXO/IL-12 increases induction of specific antitumor response in vitro:Objective:To evaluate antitumor effect of EXO/IL-12 in vitro.Methods:Lymphocytes and dendritic cells were isolated from human peripheral blood mononuclear for cytotoxicity assay. The morphology of DC was identified by SEM. Exosome-loaded DCs promoted activation of autologous T cells used as effector cells. Autologous DCs and T cells were co-inoculated with EXO/IL-12 (10μg) for 48h, PBS, IL-12, EXO was used as control. Target cells were RC-2, T-24 (bladder cancer cell line) or SW480 (colon cancer cell line). Cytotoxicity assays based on PI costaining of CFSE-labeled target cells were performed. All cells were harvested and resuspended in PBS and analyzed by flow cytometry. Killing rate was assessed by the percentage of PI- costaining cells out of gated CFSE-labeled cells.Results:The results showed that initiation by EXO and EXO/IL-12 efficiently killed tumor cells as compared with PBS and IL-12 group (P<0.01); however, initiation by EXO/IL-12 exhibited the highest killing rate (P<0.01). To further explore whether EXO/IL-12 could induce antigen-specific cytotoxic effect, we used T-24, SW480 and RC-2 as target cells and evaluated the difference in killing rate. These results suggested that exosomes derived from renal cancer cell could activate T cells in antigen-specific manner through DCs, resulting in more efficient killing of renal cancer cells as compared with T-24 and sw480 cells (P<0.01).Conclusion:The vaccine of EXO/IL-12 could more effectively increase induction of antigen-specific antitumor immune response in vitro.