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

Objective: Exosomes are small membrane vesicles that are secreted by various cells. Exosomes secretion was described in various cells where multivesicular bodies (MVBs) fused with plasma membrane in an exocytic manner. This exocytic pathway was shown to occur in a wide variety of cell types such as DCs, B lymphocytes, cytotoxic T lymphocytes and tumor cells. Tumor-derived exosomes have ubiquitous and cell-specific proteins such as antigen-presenting molecules, signal transduction proteins and tumor antigens. Exosomes play an important role in immune responses, immunomodulation and immunotherapy of tumors. It is a kind of non-cellular novel tumor vaccine. HCC is one of the common malignant cancer, and insensitive to chemotherapy and radiotherapy, only 15% may obtain a radical cure by surgery.The rate of recurrence and metastasis is high.In our study,we separated and identified exosomes derived from a mouse hepatoma cell line (H22), and detected its protein composition.We investigated the induction of antitumor response and its immune mechanism by H22 cell-derived exosomes.These were experimental works for further exploring immunity arosing by exosomes as vaccine to treat established tumors and their metastases and providing theoretic bases about exosomes as a kind of tumor vaccines.Methods:1.Separation and identification of H22 cell-derived exosomes and initial investigation of its protein composition: Exosomes were purified by serial ultracentrifugation and sucrose density ultracentrifugation,then observed and identified by electron microscope.Exosome underwent the peptide mass fingerprint and Western blot analyses.2.Immune response induced by H22 cell-derived exosomes as tumor vaccines: Female mice were vaccinated with exosomes or PBS(control group) three times 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 was determined by MTT . Infiltration of lymphocytes in tumor tissues was analysied by immunohistochemical staining of CD4+,CD8+ .Results:1. Separation and identification of H22 cell-derived exosomes and initial investigation of its protein composition: H22 cell-derived exosomes were 20~ 90nm menbrane vesicles.They were round or ellipse in shape and aggregated together.They showed low electron density components in the center. Exosomes expressed HSP70,ICAM-1,EF-G2,DLC-A,C-myc protein and Vav-2 protein. Both exosomes and H22 cell lysates had HSP70,ICAM-1 and C-myc protein.However, the contents of HSP70 and ICAM-1 in exosomes were more than that in H22 cell lysates by Western blot analyses .2. Immune response induced by H22 cell-derived exosomes as tumor vaccines:2.1 Four weeks after the injection with exosomes or PBS, the mice were challenged subcutaneously with H22 cell.The mice in control group were all died by the end of the 22nd day.On the contrary, at 80th days, the survival rate of immuned mice was 33% , and 22% of immuned mice remained H22 tumor- free.The tumor size was smaller,and the survival time was prolonged.2.2 Lymphocyte proliferation induced by exosomes was more than by PBS. Moreover, lymphocytes from exosomes immuned mice proliferated more and faster.2.3 Specific cytotoxic activity(%) of exosome 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). Specific cytotoxic activity (%) was 63.83±4.25 in exosome group.2.4 Obvious infiltrations of CD4+,CD8+T cells were observed in the tumor sites of exosome group.Conclusion:1. Serial ultracentrifugation and sucrose density ultracentrifugation can be used to purify H22 cell-derived exosomes. H22 cell-derived exosomes were 20~90nm menbrane vesicles. They were round or ellipse in shape with low electron density components in the center and aggregated together. It provides a morphological base about exosomes as a kind of tumor vaccines.2. H22 cell -derived exosomes expressed a discrete set of proteins involved in antigen presentation(HSP70,ICAM-1), migration (DLC-A), adhesion(ICAM-1), cytoskeleton(EF-G2)and tumour antigens(C-myc,Vav-2). The exosome proteins have immunogenicity. Moreover, the content of HSP70 and ICAM-1 in exosomes was more than that in H22 cell lysates. It indicated the enrichment of proteins on exosomes might facilitate the uptake of the exosomes by APCs and thereby induce an elevated immune response.3. Vaccination of H22 cell-derived exosomes was effective in the induction of protective immunity. The tumor size was smaller, and the survival time was prolonged.4. Exosomes could induce lymphocyte proliferation. Vaccination of H22 cell-derived exosomes can generate strong specific cytotoxic spleen lymphocyte response that kill the target H22 cells. Exosomes could activate CD4+ and CD8+T cells both in tumor sites. Exosomes might induce antitumor immunity through generating T cell responses, and further exploitation of it may lead to novel therapeutic intervention.