Antitumor Immunity Of Exosomes Derived From Heat Shocked Mouse Hepatoma Cell Line (H₂₂)

Objective: Exosomes are compounds which contain at least ten necessary proteins.It had been a new subcellular tumor vaccine because it stimulates CD₈⁺ T cell-dependent antitumor immunity.In order to increase its production and make better use of it in tumor immunotherapy ,we separated and identified H₂₂ cell-derived Exosomes and heat shocked Exosomes (HS-Exo), then detected their protein compositon.We investigated the effect of heat shock to the induction of antitumor response and the immune mechanism of HS-Exo.These works may provide a new route to develop neotype and high-performance tumor vaccine.Methods:1.Heat shock mouse hepatoma cell line(H₂₂): Mouse hepatoma cell line(H₂₂) was carried out for 2 hours at 43℃in water bath and recovery in 37℃5% CO2 incubator for 4 hours,then collect the culture supernatants.2.Seperation and identification of H₂₂ cell-derived Exosomes and HS-Exo and initial investigation of their protein composition: Exosomes and HS-Exo were purified by serial ultracentrifugation and sucrose density ultracentrifugation,then observed and identified by electron microscope. Quantitate the protein content by Bradford's method and their immunological factors were detected by Western blot.3.Immune response induced by H₂₂ cell-derived Exosomes and HS-Exo as tumor vaccine: Female BALB/c mice were vaccinated with Exosomes (control group) or HS-Exo three times at the root of the left hind leg and were rechallenged subcutaneously with parental H₂₂ cell 4 weeks later. Anti-tumor effect was evaluated by tumor size. Lymphocyte proliferation and specific cytotoxic activity of mice splenic cells were determined by MTT . Infiltration of lymphocytes in tumor tissues was analysied by immunohistochemical staining of CD₄⁺,CD₈⁺ . Seven days after the injection with H₂₂ cell in the right costal part, the BALB/c mice were challenged subcutaneously with Exosomes or HS-Exo .Then observed tumor size and survival time of mice.Results:1.Separation and identification of H₂₂ cell-derived Exosomes and HS-Exo and initial investigation of its protein composition: H₂₂ cell-derived Exosomes and HS-Exo were 20⁹0 nm membrane vesicles.They were round or ellipse in shape and aggregated together. They showed low electron density components in the center. The production was increased in HS-Exo by SDS-PAGE analyses:the average concentration of Exosomes is ( 1.13±0.13 ) mg/ml while the average concentration of HS-Exo is (1.23±0.06)mg/ml.And the expressions of immunological factors such as HSP70,HSC70,HSP60,ICAM-1 in HS-Exo were significantly up-regulated than those in Exosomes by Western blot analyses .2.Immune response induced by H₂₂ cell-derived Exosomes and HS-Exo as tumor vaccines:2.1 Four weeks after the injection with Exosomes or HS-Exo, the mice were challenged subcutaneously with H₂₂ cells.And HS-Exo could exert better effect to restrain tumor growth(P<0.05). At the 22th day,the average tumor size was 4.3346cm3 in Exosomes group while the average tumor size was 2.5122 cm3 in HS-Exo group.2.2 Lymphocyte proliferation induced by HS-Exo was more than that by Exosomes(P <0.05). Moreover, lymphocytes from HS-Exo immuned mice proliferated more and faster(P <0.05).2.3 Specific cytotoxic activity(%) of HS-Exo group was higher than that of control group respectively at E/T=12.5∶1, 25∶1,50∶1,100∶1, there is a significant difference within these groups(P <0.05).2.4 Obvious infiltrations of CD₄⁺,CD₈⁺ T cells were more observed in the tumor sites of HS-Exo group(P <0.05).2.5 The tumor size was smaller in HS-Exo group(P <0.05).And the survival time was prolonged(P <0.05).At the 100th day,the survival rate was 30%.Conclusion: 1. Serial ultracentrifugation and sucrose density ultracentrifugation can be used to purify H₂₂ cell-derived Exosomes and HS-Exo. It provides a morphological basis about Exosomes as a kind of tumor vaccines.2. The production was increased in H₂₂ cell-derived HS-Exo.It provides a new route to develop neotype and high-performance tumor vaccine.3. HSP70,HSC70,HSP60,ICAM-1 were more enriched in HS-Exo than Exosomes.These data may suggest that HS-Exo would have more effective immunostimulatory functions.4. Vaccination of H₂₂ cell-derived HS-Exo was more effective in the induction of protective immunity. The tumor size was smaller.5. HS-Exo could induce more lymphocyte proliferation. Vaccination of H₂₂ cell-derived HS-Exo can generate stronger specific cytotoxic spleen lymphocyte response that kill the target H₂₂ cells. HS-Exo could activate more CD₄⁺ and CD₈⁺ T cells both in tumor sites. HS-Exo might induce better antitumor immunity through generating T cell responses, and further exploitation of it may lead to novel therapeutic intervention.6. H₂₂ cell- derived HS-Exo could exert better effect to treat tumor. So it could be a new route to develop neotype and high-performance tumor vaccine.