Anti-tumor Effects Of Exosomes Derived From Tumor Cells Modifieded By Interleukin-2 Gene And Their Mechanisms

Exosomes are small membrane vesicles secreted by many types of cells. Dendritic cell (DC)-derived exosomes (DEXs) and tumor-derived exosomes (TEXs) are two kinds of exosomes used in tumor therapy. Clinical trial using DEXs for treatment of cancer patients have been undertaken already. But their anti-tumor activities were not satisfactory. To further improve their efficiency, we transfected the E.G7-OVA tumor cells which express OVA as model antigen by adenovirus vector carrying interleukin 2 (IL-2) gene and then isolated their secreted exosomes (named as Exo/IL-2). IL-2 was found to be within those exosomes detected by both ELISA and Western blot. Vaccination with these exosomes induced tumor antigen-specific Thl-polarized immune response and cytotoxic T lymphocyte (CTL) that resulted in significant inhibition of tumor growth and protected the mice from the wild type tumor challenge. 70% of mice immunized with Exo/IL-2 were tumor free and survived beyond 90 days and their tumor growth was significantly delayed than those of control groups. In addition, Exo/IL-2 significantly inhibited the tumor growth in the established E.G7-OVA tumor model. We also found that the T cells from Exo/IL-2 immunized mice proliferated significantly upon stimulation of E.G7-OVA cells (MLTR), secreted higher levels of IL-2 and IFN-γ. In addition, NK cells and CTL activities were also augmented significantly. IFN-y ELISPOT assay for the splenocytes stimulated with OVA, OVA _257-264 or VSV324-332 demonstrated thatExo/IL-2 immunization markedly increased the antigen-specific immune response. The immune subsets depletion assay demonstrated that CD8⁺T cells, CD4⁺ T cells and NK cells were all involved in the induction of anti-tumor activity of EXO/IL-2, however, CD8⁺T cells played critical role in the anti-tumor activity by immunization with Exo/IL-2. Taken together, these results demonstrated that Exo/IL-2 vaccine may represent a promising way of exosome-based tumor vaccine.Main methodsPreparation of Exo/IL-2. Exo/IL-2 were purified from the culture supernatants of E.G7-OVA tumor cells transfected by recombinant adenovirus encoding IL-2 (AdIL-2). Briefly, E.G7-OVA cells were infected with AdIL-2 at a multiplicity of infection (MOI) of 100:1. 2 h later the cells were washed three times with PBS and then supplemented with complete medium. After 48 h culture, the culture supernatants were collected and the exosomes were isolated by sequential centrifugation (4°C) at 800g for 10 min, l,200g for 30 min, 10,000g for 30 min. After concentrated by ultrafiltration through a 500-kDa MWCO hollow fiber membrane (Millipore, Bedford, MA, USA), the post-ultrafiltered supernatant was pelleted at 100,000 g for 1 h. To further purify exosomes, the pellet was resuspended in 0.25M sucrose and were floated into a discontinuous density cushion containing of 20mM Tris/30%sucrose/deuterium oxide pH7.2 (density 1.210g/ml) at 100,000g for 2h in a SW-32 swinging bucket rotor (Beckman, Optima L-80XP). We also prepared the exosomes from E.G7-OVA cells transfected with AdLacZ and E.G7-OVA without any transfection respectively, those exosomes were named as Exo/LacZ and Exo correspondingly. The protein concentrations of exosomes were measured by Bradford assay (Pierce Biotechnology, Rockford, USA). 200μg of exosomes was usually obtained from 1×10⁸ E.G7-OVA tumor cells cultured for 48 h.Electron Microscopy (EM). For EM observation, exosome pellets were fixed in 4 % paraformaldehyde. The pellets were then loaded onto eletro-microscopy grids coated with formvar carbon, followed by contrasted and embedded in a mixture of uranyl acetate and methylcellulose. Sections were observed by a Philips Tecnai-10 transmission electron microscope operating at 80 kV (Phillips Electronic Instruments, Mahway, NJ). Exosome size was measured by the scale bar.Western blot Analysis of Exosomes. Western blot was preformed routinely. Briefly, exosomal or celluar lysate proteins were first separated by 10% SDS-PAGE gel, and transferred to polyvinylidene difluoride membrane. The m...