Dual-targeted bacterial outer membrane vesicles enhance glioblastoma immunotherapy by regulating tumor microenvironment and inducing IFN-γ-mediated ferroptosis.

Glioblastoma (GBM) immunotherapy is limited by the blood-brain barrier (BBB) and the tumor immune resistance. Here, we develop an engineered bacterial outer membrane vesicle (OMV) through safe and straightforward genetic modification of Escherichia coli, termed OMV-C-C, enabling co-expression of cell-penetrating peptides (CPP) and chlorotoxin (CLT) on its surface. The OMV-C-C efficiently crosses BBB and specifically targets tumor cells both in vitro and in vivo. A single intravenous administration of OMV-C-C significantly inhibit GBM growth by enhancing the infiltration of CD8+ T cells in orthotopic brain tumor models. Mechanistically, interferon-gamma (IFN-γ) released by CD8+ T cells induces ferroptosis-specific lipid peroxidation in tumor cells through inhibiting cystine-glutamate exchanger (system Xc-) and downregulating glutathione peroxidase 4 (GPX4), a pathway demonstrated to be extremely activated in human GBM samples. Furthermore, IFN-γ facilitates erastin- and RSL-3-induced ferroptosis of tumor cells, particularly in temozolomide (TMZ)-resistant cells. Additionally, OMV-C-C@RSL-3 synergistically suppresses GBM growth in vivo. Thus, biosynthetically engineered OMV-C-C integrates intrinsic immunomodulatory activity with ferroptosis enhancement to strengthen glioblastoma immunotherapies, offering a versatile platform to overcome limitations in brain tumor immunotherapy.