Antitumor Immunotoxin Activity is Enhanced by Escherichia coli csrB-Promoter Expression

Background Cancer treatment is often hindered by tumor complexity, treatment resistance, and off-target toxicity. Bacterial oncotherapy offers a promising alternative, using tumor-targeting bacteria to deliver cytotoxins that selectively induce cancer-cell apoptosis while sparing healthy tissue. However, this approach is limited by its reliance on exogenous inducers to activate anticancer agents. To address this, we engineered a bacterial platform using the tumor-specific csrB promoter, activated by the acetate-rich tumor microenvironment, to selectively induce immunotoxins without external cues. Methods Acetate concentrations within CT26 tumors were quantified to assess tumor-specific metabolic enrichment. Transcriptomic profiling of tumor-colonizing Escherichia coli was performed by RNA sequencing to identify promoters responsive to the tumor microenvironment, identifying csrB . This promoter region was cloned into an expression vector to drive the production of a recombinant immunotoxin, TGFα–PE38, comprising TGFα, Pseudomonas exotoxin A (PE38), and a secretion tag. Promoter activity was characterized by qPCR and a β-galactosidase reporter assay under varying acetate levels. Cytotoxicity was evaluated in vitro using supernatants from the engineered bacteria. CT26-bearing BALB/c mice were intravenously administered with the engineered bacteria, and tumor growth and survival were monitored. To explore mechanisms underlying survival gain, tumor cytokine dynamics and apoptosis-related gene expression were analyzed. Results csrB , encoding a small regulatory RNA, was highly upregulated in a tumor-specific manner. Genes under csrB control participate in acetate metabolism, which is enriched in tumors. qPCR of in vitro cultures showed that csrB expression depended on acetate levels, with the csrB promoter driving acetate-responsive β-galactosidase expression. The csrB promoter regulated expression of the TGFα–PE38 recombinant immunotoxin. Under csrB control, this immunotoxin was strongly expressed in the presence of acetate, persisting in the tumors of CT26-bearing mice treated with E. coli expressing this immunotoxin. Application of the bacterial-culture supernatant reduced tumor-cell viability, markedly suppressing tumor growth and extending survival. This demonstrates that the csrB promoter is ideal for use in auto-inducing therapeutic platforms in bacterial oncotherapy. Conclusions These findings highlight the ability of tumor-colonizing bacteria to sense tumor conditions and alter antitumor protein expression, thus potentially improving outcomes in oncotherapy.