3d Multicellular Coculture Platform Enables Rapid Engineering Of Tumor-Homing Bacteria

Engineering tumor-homing bacteria to locally deliver therapeutics is an emerging approach for next-generation living therapy for cancer. However, the lack of physiologically-relevant in vitro testing environments to rapidly and quantitatively characterize complex multicellular interactions of bacteria and cancer cells is a major bottleneck for clinical translation. We recently developed a 3D multicellular coculture platform that grow various bacteria within the necrotic core of multicellular spheroids, recapitulating selective bacterial growth in solid tumor in vivo. Tumor spheroids were generated from CT26 colorectal cell lines in a round-bottom low-adhesion 96 well plate. Bacteria were inoculated into the media containing tumor spheroids and gentamicin, a poorly diffusible antimicrobial agent, were added to confine bacterial growth to spheroid cores. Time-lapse microscopy was utilized to track growth dynamics and spatial profiles. To validate efficacy, mice with hind flank tumors were treated with engineered bacteria and measured for the therapeutic effect on tumor size. The multicellular coculture platform allowed for parallel and continuous culture of bacteria in tumor spheroids. Notably, bacteria stably grew within the spheroid necrotic cores, representing tumor colonization in animals and patients. Leveraging the high throughput nature of the platform, we screened a library of ten cytotoxic molecules delivered by clinically relevant tumor-homing S. typhimurium. We validated similarities in efficacies using a syngeneic mouse model and identified novel therapeutic candidates that reduced tumor growth significantly. Additionally, we utilized the tumor spheroid to construct bacterial biosensors. As tumor microenvironment such as oxygen, pH, and metabolite gradients are recapitulated in the 3D spheroid setting, we tested hypoxia, acid and lactate sensors and confirmed their activity in tumor environment. Constructing an AND-gate logic circuit for bacterial growth using synthetic biology tools, we rapidly tested the bacterial containment capability to the spheroid cores. This strain demonstrated a 100-fold reduction in off-target colonization compared to the wildtype strain in an animal model. Rapid and quantitative characterization of dynamical bacteria-cancer cell interactions can predict therapeutic performance more accurately. We hope this scalable approach serves to accelerate future clinical development of bacterial applications for cancer therapy. Citation Format: Tetsuhiro Harimoto, Zakary Singer, Oscar Velazquez, Joanna Zhang, Samuel Castro, Taylor Hinchliffe, William Mather, Tal Danino. 3D multicellular coculture platform enables rapid engineering of tumor-homing bacteria [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6317.