Simple SummaryIn situ vaccination (ISV) envisages the intratumoral injection of immunostimulatory molecules, which inflame the tumor and induce anti-tumor immune responses. Since bacterial outer membrane vesicles (OMVs) are naturally decorated with components which stimulate innate immunity, we tested whether OMVs can be used in ISV. Using three different tumor mouse models, we demonstrated the effectiveness of OMVs in inhibiting tumor development and in curing a large fraction of treated mice. We also show that if combined with tumor-specific neoantigens, the anti-tumor activity of OMVs is further enhanced. These latter results are particularly relevant since they support the use of a general strategy to optimize any in situ vaccination protocol. Considering their potency and the ease with which are produced, OMV-based ISV has the potential to become a standard of care for most solid tumors, particularly as a neoadjuvant therapy to be performed before surgery.AbstractIn situ vaccination (ISV) is a promising cancer immunotherapy strategy that consists of the intratumoral administration of immunostimulatory molecules (adjuvants). The rationale is that tumor antigens are abundant at the tumor site, and therefore, to elicit an effective anti-tumor immune response, all that is needed is an adjuvant, which can turn the immunosuppressive environment into an immunologically active one. Bacterial outer membrane vesicles (OMVs) are potent adjuvants since they contain several microbe-associated molecular patterns (MAMPs) naturally present in the outer membrane and in the periplasmic space of Gram-negative bacteria. Therefore, they appear particularly indicted for ISV. In this work, we first show that the OMVs fromE. coliBL21(DE3)Δ60 strain promote a strong anti-tumor activity when intratumorally injected into the tumors of three different mouse models. Tumor inhibition correlates with a rapid infiltration of DCs and NK cells. We also show that the addition of neo-epitopes to OMVs synergizes with the vesicle adjuvanticity, as judged by a two-tumor mouse model. Overall, our data support the use of the OMVs in ISV and indicate that ISV efficacy can benefit from the addition of properly selected tumor-specific neo-antigens.