IMMU-16. Development of high-purity bacterial extracellular vesicles for suppressive myeloid cell reprogramming in glioblastoma immunotherapy

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Abstract The tumor microenvironment of glioblastoma is dominated by immunosuppressive microglia/macrophages that dampen anti-tumor immunity, comprising a major barrier to effective immunotherapy. Developing strategies to reprogram these macrophages is essential to overcome treatment resistance. Bacterial extracellular vesicles (bEVs) are secreted nano-sized (~25–100 nm) particles that modulate myeloid cell activity in a species-dependent manner. We developed a scalable strategy to purify bEVs from the human commensal Escherichia coli Nissle strain consisting of: tangential-flow filtration, Benzonase depletion of free nucleic acids, and size-exclusion chromatography, yielding ~3.5 mg of bEV-associated protein per liter of culture. E. coli Nissle bEVs activated NF-κB and IRF pathways in THP-1 dual-reporter monocyte-derived cells in a dose-dependent manner, with an ED50 approximately seven-fold lower than that of purified LPS or heat-killed bacteria, indicating a distinctly higher potency. We further depleted free LPS that may co-isolate with bEVs, and noted a ~3000-fold reduction in LPS content, with less than 10% protein loss. LPS-depleted bEV bioactivity surpassed LPS amount-matched crude bEVs, supporting that bEV, not residual copurifying LPS, drove the observed in vitro bioactivity. bEVs activated peripheral-blood mononuclear cell-derived CD11b⁺CD68⁺ macrophage-like cells at concentrations as low as 1 ng/mL, while lymphocyte populations experienced minimal effects, in line with bEVs acting primarily on myeloid cells. We next treated M-CSF, IFNγ, and IL-4 polarized human monocyte-derived macrophages with bEVs. In IL-4 polarized (M2-like) macrophages, bEVs induced a more pronounced M1-like reprogramming compared to IFN-γ treatment, increasing CD38, CD80, and CD86 while decreasing CD47 and CD206. This reprogramming persisted even at sub-ED50 concentrations, as low as 0.6 ng/mL. Collectively, this workflow consistently yielded bioactive bEVs that robustly reprogram macrophages. Future work will test the ability of bEVs to reprogram GBM-associated microglia/macrophages, with the ultimate goal of combination with immunotherapies to overcome treatment resistance in the immunosuppressive GBM microenvironment.

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