A Bioluminescent Transposon Reporter-Trap Identifies Tumor-Specific Microenvironment-Induced Promoters in Salmonella for Conditional Bacterial-Based Tumor Therapy

Abstract

Salmonella specifically localize to malignant tumors in vivo, a trait potentially exploitable as a delivery system for cancer therapeutics. To characterize mechanisms and genetic responses of Salmonella during interaction with living neoplastic cells, we custom designed a promoterless transposon reporter containing bacterial luciferase. Analysis of a library containing 7,400 independent Salmonella transposon insertion mutants in co-culture with melanoma or colon carcinoma cells identified five bacterial genes specifically activated by cancer cells: adiY, yohJ, STM1787, STM1791, and STM1793. Experiments linked acidic pH, a common characteristic of the tumor microenvironment, to a strong, specific and reversible stimulus for activation of these Salmonella genes in vitro and in vivo. Indeed, a Salmonella reporter strain encoding a luciferase transgene regulated by the STM1787 promoter, which contains a tusp motif, showed tumor-induced bioluminescence in vivo. Furthermore, Salmonella expressing Shiga toxin from the STM1787 promoter provided potent and selective anti-tumor activity in vitro and in vivo, demonstrating the potential for a conditional bacterial-based tumor-specific therapeutic.

Synthetic biology expands chemical control of microorganisms

“…Although therapies using S. typhimurium have shown limited efficacy [56-58], an on-going phase I clinical trial using intratumoral injections of attenuated Clostridium novyi has shown promising anti-tumor activity [59**]. In addition, the natural anti-tumor activity of Salmonella typhimurium has been expanded by giving it the ability to invade cancer cells or to secrete toxins when in the vicinity of tumors [8,9,18,43]. These strategies take advantage of promoters that induce gene expression under conditions indicative of the tumor microenvironment.…”

“…These strategies take advantage of promoters that induce gene expression under conditions indicative of the tumor microenvironment. For example, Flentie et al 2012 [43] discovered a Salmonella typhimurium promoter responsive to low pH and placed Shiga Toxin 2 under its control in S. typhimurium . When injected into mouse xenograph tumors, this strain decreased tumor cell viability within 5 days of injection [43].…”

“…Sensors with different specificities can be mined from the literature, discovered through screens for small molecule responsive promoters [16,43], or computationally designed [36,44,45**]. Once a suitable sensor is defined, its function can be tested by placing the expression of a reporter gene under the sensor’s control (Figure 1A).…”

Bugging Tumors

“…In the current issue of Cancer Discovery, Flentie et al (4) designed a promoterless transposon luciferase reporter to analyze a library containing 7,400 independent Salmonella transposon insertion mutants and identified five Salmonella promoter specifically activated by cancer cells, as well as by acidic pH often found in tumors.…”

“…Flentie et al (4) utilized the most pH-sensitive promoter ( STM1787 ) they identified, to demonstrate the utility of tumor-specific Salmonella promoters to drive the Shiga toxin-2 (Stx-2) transgene in a wild-type strain of S. typhimurium . Salmonella expressing Stx-2 from the STM1787 promoter had anti-tumor activity in vivo as well as in vitro.…”