aspA promoter as a tumor microenvironment-responsive autoswitch for anticancer agent expression in Escherichia coli

Bacterial cancer therapy has emerged as a promising strategy for the local delivery of anticancer agents to solid tumors because bacteria preferentially colonize the tumor microenvironment (TME). However, strategies for exploiting metabolic features of the TME to achieve tumor-selective expression of anticancer agents remain limited. Here, we present a tumor-responsive gene expression system in Escherichia coli for tumor-selective expression of the recombinant immunotoxin psp-TGFa-PE38 (TP), exploiting its intrinsic ability to sense metabolic cues in the TME. To identify such TME-associated metabolic cues, we examined tumor metabolites in CT26 tumor-bearing mice and confirmed that tumors contained > 3-fold lower glucose and > 5-fold higher fumarate levels than those in the liver. In response to this nutrient heterogeneity, E. coli upregulated genes involved in fumarate respiration, including frdA, dctA, dcuB, and aspA, in the DcuSR regulon. aspA was most upregulated (65-fold) in tumors, which is indicative of the strongest activity of its promoter in the TME. We used the aspA promoter (P_aspA) as a tumor-specific switch to control TP expression, targeting secreted functional TP and inducing cytotoxicity in vitro. TP was specifically expressed by P_aspA in tumors, reducing the tumor size by 2.8-fold and extending survival by 35 days in vivo. Overall, these results suggest that the potential of P_aspA as a tumor-specific expression system.