Lactococcal Extracellular Vesicles as In Situ Vaccine Activators in Combination with Doxorubicin for Cancer Therapy

Background/Objectives: In situ vaccines that directly release endogenous tumor antigens in situ to elicit anti-tumor immune responses without exogenous antigen preparation have emerged as a promising cancer immunotherapy strategy, due to their enhanced safety by local immunization that minimizes systematically adverse reactions. However, the anti-tumor efficacy of most in situ vaccines is affected by their limited access to tumors in distant sites and the toxicity of the adjuvants contained. Methods: To overcome these shortcomings, the present study explored the feasibility of utilizing extracellular vesicles from the probiotic bacteria Lactococcus lactis as both immune activators and drug carriers, which were formulated into nanoparticles to target distant tumors. Results: Using confocal microscopy and flow cytometry, we confirmed that the Lactococcus lactis-derived extracellular vesicles possess adjuvant activity that promoted the maturation of dendritic cells without affecting their viability or apoptosis rate. Moreover, the Lactococcus lactis-derived extracellular vesicles, both alone and when carrying the drug doxorubicin, could target and accumulate in solid tumor tissues via the enhanced permeability and retention effect. Interestingly, compared to healthy cells, the Lactococcus lactis-derived extracellular vesicles tended to be taken up more by tumor cells and readily released their encapsulated doxorubicin in the acidic tumor environment, which resulted in their enhanced reactive oxygen species production and immunogenic cell death. Ultimately, systemic administration of Lactococcus lactis-derived extracellular vesicle-encapsulated doxorubicin greatly increased the anti-tumor efficacy by boosting the number of infiltrating dendritic cells and CD8⁺ T cells in the tumor tissues and doxorubicin-mediated immunogenic cell death. Conclusions: Collectively, this study demonstrated that the probiotic Lactococcus lactis-derived extracellular vesicles are both safer adjuvants and effective drug carriers with immunostimulatory activity and tumor-targeting capability, shedding an interesting light on this vaccine design platform for future cancer immunotherapy.