Utilizing engineered endosymbionts to modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment
Modulating gene expression in macrophages can be used to improve tissue regeneration and redirect tumor microenvironments (TME) toward positive therapeutic outcomes. We have developed Bacillus subtilis as an engineered endosymbiont (EES) that resides in the host cell cytoplasm and controls the fates of macrophages. Secretion of mammalian transcription factors (TFs) from B. subtilis that expresses listeriolysin O (LLO; allowing the EES to escape destruction by the macrophage) modulated expression of surface markers, cytokines and chemokines indicating functional changes in a macrophage/monocyte cell line. The engineered B. subtilis LLO TF strains were evaluated in murine bone marrow-derived macrophages (BMDMs) by flow cytometry, chemokine/cytokine profiling, metabolic assays and RNA-Seq. Secretion of TFs by the EES shifted BMDM gene expression, production of cytokine and chemokines and metabolic patterns indicating that the TF strains could guide primary macrophage function. Therefore, the ability of the TF strains to alter the TME was characterized in vivo, in a murine 4T1 orthotopic breast cancer model to assess therapeutic effects. The TF strains altered the TME by shifting immune cell composition and attenuating tumor growth. Additionally, multiple doses of the TF strains were well tolerated by the mice. The use of B. subtilis LLO TF strains as EES showed promise as a unique therapeutic approach for cancer immunotherapy. The uses of EES could be expanded to modulate other mammalian cells over a range of biomedical applications. Overall design: To investigate the ability of the EES to influence primary macrophage function, we delivered two strains of B. subtilis LLO secreting pairs of transcription factors to direct the macrophage function. The populations of the primary macrophages that were influenced by the transcription factor secreting strains were compared to the populations stimulated by just the B. subtilis LLO strain, typical pro- and anti-inflammatory control stimuli and controls for sugar induced transcription within the bacteria in biological triplicate at 12 and 24 hours. Bulk RNA-seq was then performed to characterize genome wide gene expression shifts after exposure to stimuli.