Cytosolic bacterial pathogens activate TLR pathways in tumors that synergistically enhance STING agonist cancer therapies

Intracellular bacterial pathogens are distinctive tools for fighting cancer, as they can proliferate in tumors and deliver therapeutic payloads to the eukaryotic cytosol. Cytosol-dwelling bacteria have undergone extensive preclinical and clinical testing, yet the mechanisms of activating innate immunity in tumors are unclear. We report that phylogenetically distinct cytosolic pathogens, including <i>Listeria</i>, <i>Rickettsia</i>, and <i>Burkholderia</i> species, elicited anti-tumor responses in poorly immunogenic melanoma and lymphoma in mice. Although the bacteria required cytosolic access, anti-tumor responses were largely independent of the cytosolic sensors cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING), but instead required Toll-like receptor (TLR) signaling. Combining pathogens with STING agonists elicited profound, synergistic anti-tumor effects with complete responses in >80% of mice. Small molecule TLR agonists also synergistically enhanced STING agonists. The responses required RAG2 but not interferons, and cured mice developed immunity to cancer rechallenge requiring CD8⁺ T cells. These studies provide a framework for enhancing microbial and small molecule innate agonists for cancer, via co-activating STING and TLRs.