Reprogramming the tumor immune microenvironment using engineered dual-drug loaded Salmonella

In the present study, we asked whether engineering of attenuated Salmonella with dual payloads of ClyA and FlaB (Salmonella-based Armed Microbe, SAM) might potentiate antitumor activity in primary and metastatic tumor models by inducing ICD and improving macrophage-activating capacity. ClyA is a potentially immunogenic pore-forming protein that can trigger ICD, which is characterized by release of damage-associated molecular patterns (DAMPs) and neoantigens. FlaB is a promising adjuvant that activates tumor-associated macrophages (TAMs)* that may have the potential to inhibit metastasis. Our Major Findings • Bacteria expressing ClyA and FlaB produced strong anti-tumor effects in diverse tumor-bearing mouse models. • Localized secretion of ClyA from bacteria promoted release of TSAs/TAAs and DAMPs by inducing ICD of cancer cells, resulting in the establishment of long-term anti-tumor memory. • Bacteria expressing FlaB promoted M2-to-M1 polarization in the tumor microenvironment via TLR4 and TLR5 signaling pathways, which markedly inhibited tumor metastasis. • Intratumoral injection of engineered SAM exerted a tumor-suppressive effect in both SAM-treated and untreated tumors, which was similar to that elicited by intravenous injection of bacteria. Novelty, Advantages, and Translational Potential • To the best of our knowledge, this is the first report to describe the rational design of ClyA- and FlaB-secreting bacteria as immunoadjuvants (by increasing DAMPs/TSA levels and TAM activation) for the boosting of antitumor T cell and TAM responses, leading to the prevention of tumor recurrence and distant metastasis. We verified the role of SAM-FC in HMGB1 release and cross-presentation of TSAs in various models in vitro and in vivo. • Taking advantages of bacteria’s inherent immunostimulatory nature, secretion of ClyA and FlaB complementarily elicited a very effective anticancer immune response. • SAM-FC exhibited high translational potential with excellent and long-lasting antitumor immunity and low systemic toxicity. Overall, our results indicate that the engineered SAM can activate both the innate and adaptive branches of immunity and could be used in a rational design for the targeted delivery of multiple immunotherapeutic payloads for the induction of potent and long-lasting antitumor immunity.