Background Checkpoint inhibitors work only in cancers that host inflammatory cells, and ‘cold’ tumors normally do not respond. Therefore, making ‘cold’ tumors ‘hot’ is required to increase the response rate to immunooncology therapies in general. Bacteria and bacterial products have been utilized for cancer immunotherapy for more than 100 years, but currently no such treatment is available because of the severe side effects that are observed. In this study, we produced artificial outer membrane vesicles (aOMVs) from Escherichia coli outer membrane, and injected them together with cancer tissue-derived exosomes to booster an immune response to the malignancy.
Methods Outer membranes were obtained from E. coli by chemical means, followed by ionic stress and applied mild energy to generate aOMVs. The yield and purity of aOMVs were analyzed by nanoparticle tracking analysis and transmission electron microscopy. The protein and RNA contents were examined by label-free quantitative mass spectrometry and bioanalyzer. Inflammation was evaluated in macrophage cell line (RAW 264.7) and mice in vivo, and bone marrow-derived dendritic cells were used to assess the immunomodulatory functions of the aOMVs. For the study of antitumor activity, mice were subcutaneously inoculated with B16F10 cells and then subcutaneously immunized with aOMVs and melanoma exosomes five times at 3-day intervals. Also, anti-mouse PD-1 antibody was intraperitoneally injected into mice 1 day prior to immunization to investigate the effects of combination therapy. To elucidate the immunogenic mechanism, blood and spleen were obtained for antibody titer and splenocyte function study.
Results Bacterial aOMVs presented nanosized spherical shape with closed membranes and exhibited high yield and purity with very few cytosolic components. These aOMVs do not cause pro-inflammatory cytokine responses in RAW 264.7 cells and mice in vivo, despite high exposure levels. The aOMVs could be taken up by dendritic cells to stimulate cytokine and maturation marker expression. Co-immunization with aOMVs and melanoma tissue-derived exosomes elicited tumor regression in melanoma-bearing mice through Th-1 type T cell immunity and anti-tumor exosome IgG antibody production. Also, the immunotherapeutic effect of aOMVs was synergistically enhanced by anti-PD-1 inhibitor.
Conclusions Bacterial aOMVs can be produced in a large quantities with high purity, but are ‘detoxified’ compared to naturally released OMVs. The non-toxic aOMVs are powerful adjuvants for eliciting specific anti-tumor response, suggesting that aOMVs may be novel bacterial vesicle-mimetics clinically applicable as cancer treatment.
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