Switching Macrophages to Cancer-Killing Machines using Recombinant Type-VI Secretion System in “Tumor-Seeking” Bacteria: A Novel Delivery System for Cancer Treatment

Abstract

Introduction: Cancer is a complex, heterogeneous disease that is challenging to treat. Immunotherapy, which involves activating or suppressing the immune system, is emerging as a option for many resistant cancers. However, one persistent obstacle is ensuring that the drug reaches the appropriate immune cells as to minimize off-target cellular cytotoxicity. Recent studies have demonstrated the efficacy of some anaerobe bacteria to specifically “seek” the hypoxic tumor microenvironment and are thus valuable vessels to carry anti-tumor drugs. CD206+ tumor associated macrophages (TAMs) are characteristically anti-inflammatory, innate immune cells that foster a supportive microenvironment to support tumorigenesis. The cGAS-STING pathway can be activated within macrophages, allowing a switch to a pro-inflammatory phenotype. We propose that tumor-seeking bacteria can be effectively manipulated to deliver cGAS to TAMs, resulting in decreased tumor load and minimum toxicity.

Methods: We propose to design a plasmid carrying a constitutively active cGAS, inserted into E.Coli. E.Coli will contain an injection system, modified with nanobodies specific to CD206 receptors on TAMs. To deliver this drug into the intracellular compartment of the macrophage, here we propose to incorporate a recombinant Type-VI injection system, expressed by these tumor-seeking bacteria, to deliver constitutively active cGAS-STING to the TAMs. In vitro cell culture experiments using mouse and human macrophages will be used to initially test the efficacy of binding and insertion of cGAS sting, followed by intra-tumor or systemic delivery in an in vivo melanoma mouse tumor model.

Results: We anticipate that the E.Coli would specifically target CD206+ macrophages both in vitro and within the tumor. The Type-VI injection system would effectively deliver the cGAS into the macrophages, resulting in the activation of the cGAS-STING pathway and the expression of pro-inflammatory cytokine genes, as measured in a cytokine assay. The frequencies and absolute numbers of pro-inflammatory macrophages will be increased over time. We expect that there will be no toxic, or excessive inflammatory effects on the mice administered with the recombinant tumor-seeking bacteria, and the tumor size will be reduced at endpoint.

Conclusion: This proposal explores a novel cancer immunotherapy product exploiting the potential of tumor-seeking recombinant bacteria as a drug carrier.