[Preparation of renal cancer vaccine of IL-12-anchored exosomes and its antitumor effect in vitro].
OBJECTIVE
To prepare a vaccine of IL-12-anchored exosomes derived from renal cancer cells and to evaluate its antitumor effect in vitro.


METHODS
A mammalian co-expression plasmid of glycolipid-anchor-IL-12 (GPI-IL-12) was constructed by subcloning IL-12A chain gene (P35 subunit) and a fusion gene containing GPI-anchor signal sequence and IL-12B chain gene (P40 subunit) in pBudCE4.1. Confocal laser scanning microscopy and flow cytometry were used to analyze the expression of the fusion proteins. Transmission electron microscopy and Western blot were used to identify the morphology and characteristic molecules of exosomes separated by ultrafiltration and sucrose gradient centrifugation. The function of IL-12-anchored exosomes was determined by IFN-gamma release assay.


RESULTS
Mammalian co-expression plasmids were successfully constructed. Confocal laser scanning microscopy and flow cytometric analysis of the RC-2-GPI-IL-12 transfectants showed the expression of IL-12 on the cell surface. Exosomes were purified by ultrafiltration and sucrose gradient centrifugation, which were 30-80 nm in diameter, typically saucer-shaped, and expressing HSP70, ICAM-1, G250 and GPI-IL-12. (80.0 +/- 9.6) pg/ml of IL-12 was detected in 10 microg/ml exosomes and it significantly induced the release of IFN-gamma. Stimulation with EXO-IL-12 could efficiently induce antigen-specific cytotoxic T lymphocytes (CTL), resulting in more significant cytotoxic effects in vitro.


CONCLUSIONS
A vaccine of exosomes-GPI-IL-12 can be obtained from the culture supernatant of renal cancer cells modified to express anchored IL-12. This vaccine expressing IL-12 and tumor associated antigen G250 may become a new strategy for the treatment of renal cancer.
Zhang
Yao
Y
Department of Urology, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Wu
Xiao-hou
XH
Chen
Gang
G
Luo
Chun-li
CL
Zhang
Jia-mo
JM
D000951:Antigens, Neoplasm / Q000378:metabolism; D019496:Cancer Vaccines / Q000276:immunology; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000378:metabolism; D045744:Cell Line, Tumor; D003602:Cytotoxicity, Immunologic; D055354:Exosomes / Q000235:genetics / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism*; D018664:Interleukin-12 / Q000235:genetics / Q000378:metabolism*; D007680:Kidney Neoplasms / Q000378:metabolism / Q000473:pathology*; D010957:Plasmids; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology*; D014162:Transfection
D000951:Antigens, Neoplasm; D019496:Cancer Vaccines; D017261:Glycosylphosphatidylinositols; D018840:HSP70 Heat-Shock Proteins; D018799:Intercellular Adhesion Molecule-1; D018664:Interleukin-12; D007371:Interferon-gamma; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases