Rapid screening of engineered microbial therapies in a 3D multicellular model

Abstract

Synthetic biology is transforming therapeutic paradigms by engineering living cells and microbes to intelligently sense and respond to diseases including inflammation, infections, metabolic disorders, and cancer. However, the ability to rapidly engineer new therapies far outpaces the throughput of animal-based testing regimes, creating a major bottleneck for clinical translation. In vitro approaches to address this challenge have been limited in scalability and broad applicability. Here, we present a bacteria-in-spheroid coculture (BSCC) platform that simultaneously tests host species, therapeutic payloads, and synthetic gene circuits of engineered bacteria within multicellular spheroids over a timescale of weeks. Long-term monitoring of bacterial dynamics and disease progression enables quantitative comparison of critical therapeutic parameters such as efficacy and biocontainment. Specifically, we screen Salmonella typhimurium strains expressing and delivering a library of antitumor therapeutic molecules via several synthetic gene circuits. We identify candidates exhibiting significant tumor reduction and demonstrate high similarity in their efficacies, using a syngeneic mouse model. Last, we show that our platform can be expanded to dynamically profile diverse microbial species including Listeria monocytogenes, Proteus mirabilis, and Escherichia coli in various host cell types. This high-throughput framework may serve to accelerate synthetic biology for clinical applications and for understanding the host–microbe interactions in disease sites.

Magnetospirillum magneticum as a Living Iron Chelator Induces TfR1 Upregulation and Decreases Cell Viability in Cancer Cells

“…Additionally, safe administration of Salmonella typhimurium (VPN20009) has been shown for animal models and patients with metastatic melanoma [ 2 , 3 ]. Bacteria can act therapeutically by secreting innate or engineered toxins in situ (e.g., hemolysin E), transporting attached nanodrug formulations, or stimulating an immune response [ 4 , 5 , 6 , 7 ]. Colonizing bacteria can also engage in nutrient competition within the tumor microenvironment [ 8 , 9 , 10 ].…”

Multiplexed biosensors for precision bacteria tropism in vivo

“…We next tested whether multiplexed containment circuits can enhance specificity for tumor environments. We employed a recently designed three-dimensional bacteria spheroid co-culture system (BSCC) that recapitulates properties of the tumor microenvironment including oxygen and nutrient gradients, mammalian cell metabolism, and local 3D growth of the bacterial population in tumors (51) (Fig. 3A).…”

A programmable probiotic encapsulation system enhances therapeutic delivery in vivo

“…5f). To engineer bacteria to deliver antitumor payloads, we cloned a gene encoding pore-forming toxin, theta toxin (TT), previously shown to be effective as a bacterial cancer therapy 42 , in a high copy number plasmid (ColE1) with a stabilization mechanism for in vivo applications (Axe/Txe system 43 ). In a syngeneic CT26 colorectal cancer model, we intravenously administered engineered EcN at the corresponding MTD of each strain (EcN, EcN ΔkfiC, and EcN iCAP at 5x10 6 , 1x10 7 , and 5x10 7 CFU, respectively), along with a low dose of EcN iCAP at 5x10 6 CFU to match the MTD of EcN.…”