The in vivo tumorigenicity of murine B16 melanoma cells engineered to secret TNF-a was observed. The retrovirus containing mouse TNF-a cDNA was generated by the virus-packing cell PA317 transfected with plasmid pXT-TNF. The B16 cell clone secreting the highest TNF-a level was obtained after G418 resistance selection, limiting dilution and the assay of TNF-a activity. After the mice were inoculated subcutaneously with the cell clone, we found the tumor growth was inhibited and the survival period of the mice extended when compared with the mice inoculated with the wild-type B16 cells . We also found that the tuinorigenicity of B16-TNF-a+ cell was associated with the cell number inoculated. At or above the 1.25? 104 cells, the percentage of the mice with detectable tumor correlated negatively with the cell number inoculated: however, at the 6.25 ? 103 cells, the percentage was higher than that at 2.5?10~(4) cells. These results encourage us to do further experiments on the following tumor cell-targeted TNF-a gene therapy.

Objective:To study the in vivo anti-tumor effect of exosomes (Exo) combined with bacillus Calmette-Gu?rin vaccine(BCG). Methods:Exo was isolated and purified from culture supernatant of E.G7-OVA tumor cells by density gradient centrifugation. Protein components of Exo were detected by Western blotting. Exo,BCG,Exo combined with BCG (Exo+BCG) or PBS were pre-injected into mice before injection of E.G7-OVA cells,and the anti-tumor effects were observed in each group. Mouse model bearing E.G7-OVA cells was established to examine the immuno-therapy effects of Exo with or without BCG. Cytotoxity of spleen CTL was measured by LDH in different groups. Results:Exo derived from E.G7-OVA cells contained HSP60,OVA,HSC70 and CD63 as detected by Western blotting. Tumor-free rate at 90 d was significantly higher in Exo+BCG vaccinated mice than those in Exo or BCG vaccinated mice as measured by immuno-protective assay (60% vs 20% or 0%,P

Objective To study the antitumor effects of exosomes derived from heat-shocked E.G7-OVA tumor cells in vivo. Methods Exosomes derived from E.G7-OVA tumor cells were isolated and purified by serial centrifugation and sucrose gradients ultracentrifugation. Exosomes from heat-shocked or non-heat-shocked E.G7-OVA tumor cells were named as Exo/HS and Exo correspondingly. Exosomes were viewed by electron microscopy. Protein components of exosomes were detected by Western blot. Exo, Exo/ HS or PBS were injected into mice before injection of E.G7-OVA tumor cells, and antitumor effects were ob-served in each group. Mouse model bearing E.G7-OVA tumor cells were established to examine immunother-apy effects of Exo or Exo/HS. Cytotoxity of spleen CTL were measured by LDH. Results Exosomes con-tained bi-layer membrane and their diameters are between 40 nm and 100 nm under electron microscopy. The Western blot results showed that HSC70, HSP70, HSP60, HSP90, MHC Ⅰ and OVA were present in both Exo and Exo/HS. However, Exo/HS contained more HSP70 and MHC Ⅰ than Exo. Protective antitu-mor immunity suggested that tumor-free survival (90 days) rate in Exo/HS vaccinated mice was significantly higher than those in Exo or PBS vaccinated mice (50%, 20%, 0%, P<0.01). Therapeutic antitumor effects showed that immunization by Exo/HS resulted in dramatically enhanced antitumor effects when com-pared to the Exo- or PBS-treated groups (P<0.01). CTL results showed that immunization with Exo/HS in-duced higher level of OVA-specific CTL responses as compared with those from Exo or PBS (P<0.01). Conclusion Exosomes derived heat-shocked E.G7-OVA tumor cells may be used as potent cancer vaccine.