{"paper_id":"48a6e977-1799-4ffa-b29e-72c8c42f5f55","body_text":"aspA promoter as a tumor microenvironment-responsive autoswitch for anti- cancer agent expression in Escherichia coli | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article aspA promoter as a tumor microenvironment-responsive autoswitch for anti- cancer agent expression in Escherichia coli Dogeun Lee, Seyeon Hong, Jihyeon Kim, Miryoung Song, Daejin Lim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9515764/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract 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 ( E. 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 gene involved in fumarate respiration including frdA , dctA , dcuB , and aspA in the DcuSR regulon. aspA was most upregulated (65-fold) in tumors, suggesting that its promoter exhibits the strongest activity under TME. We used the aspA promoter (P aspA ) as a tumor-specific switch to control of TP expression, targeting secreted functional TP and induced cytotoxicity in vitro. TP was specifically expressed by P aspA in tumors, reducing tumor size by 2.8-fold and extending survival by 35 days in vivo. The results suggest that the P aspA as a tumor-specific expression system. E. coli Fumarate respiration aspA promoter Tumor-specific expression Tumor-microenvironment Nutrient heterogeneity Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Recent advances in cancer therapy have led to the development of targeted therapies and immunotherapies that aim to minimize the off-target side effects associated with conventional approaches such as surgery, chemotherapy, and radiotherapy. Although these modalities provide improved selectivity, they often exhibit limited efficacy in solid tumors due to poor tissue penetration. A critical factor in this limitation is the presence of metabolic barriers within the tumor microenvironment (TME), which restricts therapeutic access and fosters an immunosuppressive milieu that hinders anti-cancer immune responses. While these features are associated with reduced therapeutic efficacy in solid tumor, they may also create a selective niche for bacterial colonization. This unique property has made bacterial cancer therapy and attractive therapeutic strategy [ 1 , 2 ]. Historically, the concept of using bacteria for cancer treatment dates back to the late nineteenth century, when William B. Coley first used bacterial preparations in patients with malignant disease [ 3 ]. With advances in genetic manipulation, facultative anaerobes, such as Escherichia coli , have emerged as versatile vehicles for tumor-targeted delivery of diverse payloads, including small molecules, immunotoxins, immunomodulators, prodrug-converting enzymes, siRNAs, and nanobodies, to the TME [ 4 – 7 ]. Although bacteria have been widely explored as carriers for tumor-targeted delivery of therapeutic payloads, ensuring that these agents are expressed exclusively within tumor tissues without harming normal tissues remains a major challenge. Constitutive expression of anticancer agents by bacteria may cause off-target toxicity in healthy tissues, highlighting the need for precise control over therapeutic gene expression. Inducible systems may help to overcome this issue. However, such systems rely on the repeated administration of external inducers, which can complicate treatment regimens. This limitation prompted us to investigate whether metabolic features that distinguish the TME from normal tissues could be harnessed as endogenous signals for tumor-specific gene expression. Such regulation is important because constitutive expression of anticancer agents by bacteria may damage healthy tissues [ 8 – 10 ]. We hypothesized that the metabolic features of the TME could be exploited as endogenous signals for tumor-specific regulation of anticancer agent expression. The TME exhibits a metabolic profile that is distinct from that of normal tissues [ 11 ]. One of the key differences is the increased rate of glucose uptake by tumor cells, even under normoxic conditions, which was first observed by Otto Warburg and now known as the Warburg effect [ 12 , 13 ]. As tumors grow, these heightened metabolic demands, coupled with abnormal vascular architecture and disrupted tissue structure, aggravate nutrient insufficiency within the solid tumor tissues [ 14 ]. Unlike normal blood vessels, tumor vasculature branches irregularly, resulting in poor nutrient delivery and considerable heterogeneity in nutrient distribution within the TME [ 15 ]. This abnormal vascular architecture, combined with the metabolic reprogramming that is inherent to tumor cells, contributes to the accumulation of oncometabolites, such as succinate and fumarate [ 13 , 16 – 22 ]. The TME exhibits characteristic metabolic alterations, including low pH, hypoxia, glucose depletion, and the accumulation of oncometabolites. These features contribute to an immunosuppressive niche that undermines the effectiveness of conventional anticancer therapies. However, they also generate distinctive environmental cues that can be sensed by bacterial regulatory systems [ 5 , 9 , 23 ]. We hypothesized that intrinsic bacterial sensing systems can detect and respond to tumor-associated environmental cues, such as nutrient heterogeneity. These systems have the potential to enable tumor-specific regulation of gene expression in response to TME. In E. coli , adaptation of nutrient-limited or anaerobic environments involves of activation of fumarate respiration, key metabolic pathway that allow the utilization of alternative carbon sources such as C 4 -dicarboxylate when glucose and oxygen are scarce [ 24 ]. This process requires the coordinated expression of genes involved in C 4 -dicarboxylate transport and fumarate reduction such as aspA , frdABCD , dcuA and dcuB , which are transcriptionally regulated by the DcuSR two-component system, which enables E. coli to sense and respond to environmental changes in carbon availability under anaerobic conditions [ 25 – 30 ]. Promoters controlled by such a regulatory network can serve as precise biological switches, activating gene expression only under specific environmental condition. In this study, we aimed to develop an expression system in E. coli that exploits native metabolic sensing mechanisms responsive to tumor-specific nutrient cues, enabling selective induction of therapeutic protein expression within the TME. Materials and methods Bacterial strains, plasmid construction, and culture conditions Table 1 lists the bacterial strains and plasmids used in this study. E. coli K-12 MG1655 was used as the wild-type strain and was cultured in Luria–Bertani broth (LB, Cat. No. MB-L4488; MB Cell). To monitor aspA promoter (P aspA ) activity, a P aspA ::gfpOVA plasmid (pA-gfpOVA) was constructed using restriction enzyme-based cloning, as follows: first, genomic DNA extracted from E. coli K-12 MG1655 was used to amplify the aspA promoter region, which included DcuR-dependent binding sites located at positions − 67 to − 87 and − 169 to − 153 relative to the transcription start site [ 31 ]. The amplified promoter was cloned into a reporter gene (gfpOVA) by replacing the iroB promoter sequence with the piroB::gfpOVA plasmid [ 32 ] using the specific primer set listed in Supplementary Table S1 . To confirm the expression of the anti-cancer agent, the araBAD promoter in the psp-TGF⍺-PE38 plasmid was replaced with P aspA by Gibson assembly using the primer set listed in Supplementary Table S1 , consequently generating the P aspA ::psp-TGF⍺-PE38 plasmid (pA-TP). The plasmids were confirmed by DNA sequencing (Macrogen). Next, each plasmid was transformed into E. coli via heat shock. Bacterial strains carrying the plasmid were cultured in LB medium at 37°C with shaking at 200 rpm. Ampicillin (100 µg/mL, Cat. No. A9518; Sigma-Aldrich) was then added to the solution. Bacterial cells were grown in M9 medium (Cat. No. MM003-01; WELGENE) supplemented with casamino acid hydrolysate (0.1% w/v, Cat. No. MB-C1656; MB cell) and L-tryptophan (0.005% w/v, Cat. No. MB-T4863; MB Cell) [ 33 ]. Incubation was performed in an anaerobic bag (Cat. No. KS-B2002; Kisanbio, ROK), and the M9 medium was supplemented with 0.4% glucose (Cat. No. 50-99-7; Duksan) or 20 mM fumarate (Cat. No. A10976.36; Thermo Fisher Scientific) [ 34 ]. Fluorescence intensity analysis Carbon source-dependent P aspA activity was assessed using green fluorescent protein expression within the bacterial cell culture medium, and fluorescence intensity was measured hourly using a spectrophotometer (VLBL00D0; Thermo Fisher Scientific). Measurements were conducted at excitation and emission wavelengths of 488 and 513 nm, respectively, to detect gfpOVA accumulation. The absorbance was measured at 600 nm to standardize the fluorescence intensity of each group. Western blot analysis Western blot analysis To examine the expression of TP by P aspA in vitro , MG1655 cells carrying pA-TP were cultured in M9 medium with restrictively supplemented carbon sources for 24 h. Whole bacterial cells, pellets, and supernatants were collected at the indicated time points. Bacterial supernatants were filtered through a 0.2-µM filter (Cat. No. FJ13ASCCA002DL01; GVS Filter), mixed with 5× SDS, and boiled at 100°C for 5 min. To confirm TP expression in vivo , MDG003 (1 × 10 8 CFU/mouse) was injected into mice grafted with CT26 tumors through the tail vein when the tumors reached 100–130 mm 3 . Tumors were excised at the indicated time points and homogenized in 1 mL of RIPA buffer (Cat. No. IBS-BR002 (Intron Biotechnology) containing 1× Protease & Phosphatase Inhibitor Cocktail and 1× \\(\\:\\:\\) EDTA (Cat. No. 78440; Thermo Fisher Scientific). The filtered supernatants were mixed with 5× SDS and boiled at 100°C for 10 min. Total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Cat. No. IPVH00010; Sigma-Aldrich). The membrane was blocked with 5% skim milk for 1 h and probed with rabbit anti- Pseudomonas exotoxin A antibody (Cat. No. P2318; Sigma-Aldrich) or mouse anti-ß-actin antibody (Cat. No. sc-47778; Santa Cruz Biotechnology) overnight at 4°C. After washing off the primary antibodies with TBS (140 mM NaCl, 10 mM Tris-HCl; pH 8.0) containing 0.1% Tween 20, the membranes were incubated at room temperature (20 ± 5°C) for 1 h with goat anti-rabbit IgG (Cat. No. 7074s; Cell Signaling Technology, Danvers) and horse anti-mouse IgG (Cat. No. 7076s; Cell Signaling Technology) conjugated to horseradish peroxidase. The bound proteins were visualized using an ECL kit (Cat. No. 32209; Thermo Fisher Scientific). Cell lines and culture CT26 murine colon carcinoma, MC38 murine colon carcinoma, 4T1 murine mammary carcinoma, LLC1 lung carcinoma, and SW620 human colon carcinoma cells were purchased from the American Type Culture Collection. The CT26, MC38, 4T1, and LLC1 cells were grown in high-glucose Dulbecco’s modified Eagle medium (Cat. No. AL007A; HIMEDIA) containing 10% fetal bovine serum (FBS, Cat. No. SH30919.03; cytiva HyClone™) and 1% penicillin-streptomycin (Cat. No. 30-002CI; Corning Inc), whereas the SW620 cells were grown in Roswell Park Memorial Institute 1640 (Cat. No. AL028A; RPMI, HIMEDIA) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Animal ethics and experiments All animal procedures were performed in accordance with the National Guidelines for the Care and Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (approval numbers KW-240326-3). Female BALB/c mice (5–6 weeks old; body weight, 18–20 g) were purchased from Samtako Bio Company. Mice were maintained under specific-pathogen-free conditions in a biosafety level 2 facility with a 12-h light/dark cycle, controlled temperature (22 ± 2°C), and free access to standard chow and water. After an acclimation period of at least 7 days, mice were randomly assigned to experimental groups. Mice carrying subcutaneous tumors were generated as follows: tumor cells cultured in vitro were harvested, suspended in 30 µL phosphate-buffered saline (PBS), and injected subcutaneously into the right thigh at the indicated dose (1 × 10 6 cells for CT26). Bacterial injections were administered when the tumors reached a volume of 100–150 mm 3 . To evaluate the antitumor effects of TP immunotoxins, mice grafted with CT26 cells were injected with wild-type E. coli or E. coli carrying pA-TP through the tail vein. The tumor volume was calculated using the formula: ( L \\(\\:\\times\\:\\:\\) H \\(\\:\\times\\:\\) W )/2, where L is the length, W is the width, and H is the height of the tumor in millimeters. For bacterial distribution in vivo , solid tumors and other organs were extracted from mice and homogenized in 1× \\(\\:\\:\\) PBS using a homogenizer (Ultra–Turrax T10; IKA). Mice with tumor volumes \\(\\:\\:\\ge\\:\\) 1,500 mm 3 were humanely euthanized using a CO 2 chamber, in accordance with the guidelines of the Animal Research Committee of Kangwon National University. The survival rates were estimated using the Kaplan–Meier curve. Assessment of cell viability To evaluate the cytotoxic effects of TP on cancer cells, cell viability was assessed using an MTT assay kit (Cat. No. M3353; Tokyo Chemical Industry) after exposure to bacterial culture supernatant. Briefly, the bacterial supernatant was prepared from MG1655 cells carrying pA-TP and incubated in M9 medium supplemented with the indicated carbon sources. Subsequently, the supernatant was concentrated using a centrifugal filter (Amicon Ultra 10 K, #UFC901008; Millipore), and the total fraction concentration was measured using the Bradford method. The concentrated supernatant (0.1 mg/mL) was then added to SW620, CT26, MC38, 4T1, and LLC1 cells cultured in 96-well plates for 48 h at 37°C in a humidified chamber with 5% CO 2 . To assess cancer cell viability in response to TP exposure, MTT reagent was added to each well, followed by a 2-h incubation at 37°C to allow for formazan formation. Dimethyl sulfoxide (100 µL) was added to the plate and then resuspended to determine the dissolution of the purple crystals. A spectrophotometer (VLBL00D0; Thermo Fisher Scientific) was used to measure the OD of each well at 570 nm. Assessment of glucose and fumarate metabolites in vivo To measure the relative nutrient ratios, liver and tumor tissues were collected from CT26-bearing mice when tumor volumes reached 100–150 mm³, before bacterial injection. Glucose [ 35 ] (Cat. No. ab169559; Abcam) and fumarate [ 36 ] (Cat. No. ab102516; Abcam) concentrations were quantified using colorimetric assay kits according to the manufacturer’s instructions. Freshly harvested tissues were lysed, and metabolite levels were determined from standard curves to obtain the number of nanomoles of each metabolite per gram of tissue. The relative glucose-to-fumarate ratio was calculated by dividing the glucose concentration by the fumarate concentration in each tissue, whereas the relative fumarate-to-glucose ratio was calculated by dividing the fumarate concentration by the glucose concentration. Bacterial RNA, cDNA library preparation, and quantitative polymerase chain reaction Total RNA was extracted from in vitro cultured E. coli and mouse organs infected with E. coli (liver, spleen, and tumor) using a Monarch® Total RNA Miniprep Kit (Cat. No. T2010S; New England Biolabs®). cDNA was synthesized from 1–5 µg of total RNA using reverse transcriptase with random hexamer primers (TOPscript™ cDNA Synthesis Kit, Cat. No. EZ005S; Enzynomics). mRNA levels were quantified using quantitative polymerase chain reaction (qPCR) with a Rotor-Gene Q real-time PCR system (R072010103; Qiagen) with TOPreal™ qPCR 2× PreMIX (Cat. No. RT500S; Enzynomics). The expression levels of dcuS , aspA , dcuB , frdA , and dctA were normalized to those of the 16S rRNA. The primers used in the experiments are listed in Supplementary Table S2. Each experiment was performed in triplicate. Statistical analysis Data were analyzed using GraphPad Prism v.10.0.3 (GraphPad Software). Differences between the mean values of the two groups were analyzed using an unpaired two-tailed Student’s \\(\\:t\\) -test. Two-way analysis of variance (ANOVA) was used for the time-course studies. Survival rates were determined using the log-rank (Mantel–Cox) test and are shown by Kaplan–Meier curves. Statistical significance was set at \\(\\:\\text{P}\\:<0.05\\) . Results Nutrient heterogeneity in the TME drives the tumor-specific upregulation of E. coli aspA expression during the adaptive response We investigated nutrient heterogeneity to determine whether the TME provided a suitable environment for E. coli sensing, focusing on glucose depletion and fumarate accumulation in normal (e.g., the liver) and tumor tissues in CT26 tumor-bearing mice. The relative glucose-to-fumarate ratio was > 3-fold higher in the liver than in the tumor, whereas the relative fumarate-to-glucose ratio was > 5-fold lower in the liver than in the tumor (Fig. 1 A and B). These differences prompted us to explore whether E. coli responds to external carbon sources through its biological systems (Fig. 1 C). The relative expression of dcuS was 10-fold higher in tumors than in the liver, prompting further investigation into the regulation of the DcuSR regulon within the TME (Fig. 1 D). The expression of aspA , a gene within the DcuSR regulon, was remarkably higher in tumor tissues than in normal liver tissues, with expression levels increasing approximately 65-fold. The upregulation was > 3-fold higher than that of any other gene within the DcuSR regulon in the TME (Fig. 1 E), indicating that aspA was the most highly upregulated gene within the DcuSR regulon in tumor tissues. These findings indicate that the aspA promoter (P aspA ) may serve as a metabolically responsive auto-inducible switch for anticancer agent tumor-specific expression. Carbon source-dependent aspA promoter activation We assessed whether the P aspA is activated based on the availability of external carbon sources. First, wild-type E. coli was cultured in an anaerobic bag in M9 medium supplemented with the indicated carbon source for 5 h. Following incubation, we measured the relative expression of aspA . The expression of aspA was upregulated by > 3-fold in the presence of fumarate than in the presence of glucose (Fig. 2 A). To confirm the functionality of P aspA as a tumor-specific switch for anticancer agent delivery, we analyzed the P aspA region to evaluate promoter activity [ 31 , 34 , 37 ]. The promoter was primarily regulated by phosphorylated DcuR, which bound to two regions located at − 83 to − 67 and − 169 to − 153 upstream of the aspA transcriptional start site (Fig. 2 B). A gene reporter system was constructed using the unstable GFP variant gfpOVA, which was cloned downstream of P aspA in pBR322 to generate pA-gfpOVA. E. coli transformed with this plasmid was used to monitor P aspA activity. The fluorescence intensity was determined during in vitro growth in the aforementioned medium. The fluorescence intensity was > 4-fold higher in the fumarate medium than in the glucose medium (Fig. 2 C and Supplementary Fig. S1 ). Cytotoxic activity of psp-TGFα-PE38 expressed via the aspA promoter Next, we tested whether P aspA could drive the expression of anticancer agents. We selected a recombinant immunotoxin composed of transforming growth factor alpha (TGFα) and modified Pseudomonas exotoxin A (PE38) as a model therapeutic payload, with the psp signal peptide fused in-frame to the N-terminal of TGFα-PE38 to facilitate secretion [ 38 – 40 ]. Therefore, we cloned psp-TGFα-PE38 (TP) under the control of P aspA in pBR322 to generate pA-TP (Fig. 3 A). Subsequently, we observed whether TP was successfully controlled by P aspA under specific nutrient conditions similar to those in TME. TP was efficiently overexpressed with fumarate supplementation compared with glucose supplementation, supporting the functionality of P aspA as a switch for the tumor-specific expression of TP (Fig. 3 B). To evaluate the selective cytotoxicity of TP expressed by P aspA , MTT assay was performed using the supernatant from E. coli cultures carrying pA-TP supplemented with glucose or fumarate. The assay was applied to CT26 and MC38 murine colon cancer cell lines, LLC1 murine lung cancer, and 4T1 murine breast cancer cells, all of which exhibit high levels of epidermal growth factor receptor (EGFR) expression [ 41 , 42 ], compared with that in SW620 human colon cancer cells, which express low levels of EGFR [ 43 ]. The cytotoxic effect increased in proportion to the fumarate supplementation of the supernatant when 0.1 mg of the supernatant fraction was administered to tumor cells for 48 h, except for SW620 cells, which express low levels of EGFR (Fig. 3 C). The maximum cytotoxicity of TP was observed in LLC1, a tumor cell line that overexpresses EGFR, showing approximately a 90% increase in cytotoxicity relative to the mock group (Fig. 3 D). These results indicate that P aspA enables the selective expression of the anticancer agent TP under TME-like conditions while preserving its cytotoxic effect against EGFR-overexpressing tumor cells. Tumor-specific TP expression via P aspA suppressed tumor growth. The role of P aspA as a metabolically responsive promoter that enables tumor-specific expression of the immunotoxin TP was initially validated in vitro . Subsequently, we evaluated its ability to drive selective TP expression in the TME and minimize off-target expression in normal tissues in vivo . To this end, CT26 tumor-bearing mice were intravenously administered E. coli carrying pA-TP. TP expression was confirmed in the excised tumor tissue over the indicated days post-injection (dpi), with no significant expression detected in the liver, as shown using western blotting (Fig. 4 A). The results revealed that TP is selectively expressed within the TME under the control of P aspA . Furthermore, tumor volumes were used to evaluate the anti-tumor efficacy of this system. Mice treated with E. coli carrying pA-TP exhibited a 2.8-fold reduction in tumor growth compared with the mock treatment and a 2.1-fold reduction compared with wild-type E. coli (Fig. 4 B, with images of all tumors presented in Supplementary Fig. S2). Consistent with the tumor suppression results, survival rate analysis indicated that treatment with E. coli carrying pA-TP prolonged the lifespan of tumor-bearing mice by up to 35 days (Fig. 4 C). Although E. coli carrying pA-TP showed precise efficacy in delivering anticancer agents, the associated side effects of the bacteria should be carefully monitored. The body weight loss in the treated mice was < 6%, with a noticeable recovery after 4 dpi (Supplementary Fig. S3A). The bacterial loads in normal tissues were over 100,000-fold lower than those in tumor tissues after 3 days (Supplementary Fig. S3B). Correspondingly, serum IL-6 levels recovered significantly after 3 dpi compared with those observed after 1 dpi, indicating a reduction in systemic inflammatory responses (Supplementary Fig. S3C). Discussion Effective cancer therapy is limited by poor tumor selectivity and systemic toxicity. Bacterial cancer therapy has become a modern strategy that harnesses engineered microbes to colonize the TME and deliver therapeutic payload locally [ 5 – 7 ]. However, studies specifically exploiting the metabolic features of the TME to achieve tumor-selective expression of anticancer agents are limited [ 6 , 9 , 10 ]. In this study, we successfully developed and validated a TME-responsive gene expression system in E. coli by leveraging the distinctive metabolic characteristics of the TME as intrinsic cues for therapeutic gene expression. We began by systematically characterizing the nutrient heterogeneity within the TME of CT26 tumor-bearing mice. Consistent with previous reports [ 11 – 14 , 21 , 22 ], our metabolic analysis unequivocally showed significant glucose depletion and fumarate accumulation in tumor tissues compared with that in normal liver tissues (Fig. 1 A and B). This distinct metabolic signature provided the foundational rationale for exploiting TME-specific cues. Based on these findings, we investigated the DcuSR regulon as a promising metabolic-responsive system (Fig. 1 C) [ 25 – 28 ]. Crucially, the aspartase-encoding aspA gene exhibited the most pronounced tumor-specific upregulation (65-fold, Fig. 1 D), positioning its promoter, P aspA , as an ideal candidate for a tumor-specific, auto-switch. Our in vitro experiments confirmed robust activation of P aspA under tumor-like metabolic conditions and minimal activity under normal metabolic conditions (Fig. 2 ). To translate this into a therapeutic application, we engineered pA-TP, encoding the recombinant immunotoxin TP under P aspA control (Fig. 3 A). TP comprises two functional domains designed for selective cytotoxicity: a TGFα moiety that binds the epidermal growth factor receptor (EGFR), which is frequently overexpressed in malignant tumors, and a truncated Pseudomonas exotoxin A (PE38) domain, which inhibits protein synthesis upon internalization, leading to cell death. Through this design, TP combines receptor-ligand targeting specificity with the potent cytotoxicity of bacterial toxins. Despite the therapeutic promise of immunotoxins, their systemic administration often results in off-target toxicity and limited efficacy, highlighting the need for localized delivery strategies. In this context, the tumor-responsive P aspA system offers a strategy to localize TP expression within the tumor microenvironments. In vitro assays showed that TP expression was precisely induced under tumor-like conditions, with minimal expression in normal metabolic environments (Fig. 3 B). Furthermore, the secreted TP retained its EGFR-TGFα-mediated selective cytotoxicity against EGFR-overexpressing cancer cells (Figs. 3 C and D), confirming its functional integrity. The therapeutic relevance of the P aspA system was further validated in vivo . Intravenous administration of E. coli carrying pA-TP into CT26 tumor-bearing mice resulted in selective TP expression within tumors (Fig. 4 A). This selective expression led to remarkable tumor regression and a substantial prolongation of survival in treated mice (Fig. 4 B and C). These compelling in vivo results confirm that the P aspA system effectively leverages TME-specific metabolic conditions to enable the selective expression of therapeutic proteins. Our approach enhanced on-target accuracy while significantly reducing the risk of off-target toxicity, thereby improving the overall efficacy of bacteria-mediated anti-cancer therapy. Despite these promising outcomes, complete tumor elimination was not achieved, likely because of the inherent challenge of stable plasmid maintenance in bacteria in vivo (Supplementary Fig. S4). This limitation, which can lead to a loss of therapeutic gene expression over time, represents a common hurdle in bacteria-mediated therapies. Future strategies to overcome this critical issue include employing essential gene complementation systems, such as asd and glmS , which can significantly promote stable plasmid inheritance and thus enhance the long-term therapeutic potential of the P aspA system. With the ongoing advancements in synthetic biology and genetic engineering, we anticipate that the P aspA system will evolve into a versatile and highly reliable platform. Its auto-inducible, tumor-specific nature makes it particularly promising for integration with other therapeutic strategies, potentially paving the way for more effective and precisely targeted cancer treatments. Conclusion We developed a tumor-responsive gene expression system based on the aspA promoter that enables tumor-selective expression of anticancer agents by exploiting metabolic features of the TME. Our findings demonstrate that TME-associated nutrient heterogeneity, particularly glucose depletion and fumarate accumulation, can be harnessed as intrinsic regulatory cues for controlling therapeutic gene expression in bacteria (Fig. 5). This strategy addresses a critical limitation in bacterial cancer therapy by enabling spatially restricted expression of therapeutic payloads, thereby improving targeting precision and reducing the risk of off-target toxicity. Importantly, the P aspA – based system represents a modular and adaptable platform that can be extended to a wide range of therapeutic protein beyond immunotoxins. Overall, our study provides a new framework for leveraging endogenous tumor-associated signals to achieve precise and context-dependent control of therapeutic gene expression in bacterial cancer therapy. Abbreviations ANOVA, analysis of variance; dpi, days post-injection; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; qPCR, quantitative polymerase chain reaction; TGFα, transforming growth factor alpha; TME, tumor microenvironment. Declarations Ethics approval and consent to participate All animal procedures were approved by the Institutional Animal Care and Use Committee of Kangwon National University (approval numbers KW-240326-3). Consent for publication: Not applicable Availability of data and materials The raw data supporting the conclusions of this article will be made available by the authors upon reasonable request. Competing interests The authors declare no competing interests. Funding This work was supported by the National Research Foundation (NRF) of Korea grants funded, Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry by the Korean government and the Regional Innovation System and Education program through the Gangwon RISE Center, [grant number NRF-RS-2023-00210053, RS-2025-02263715 and 2025-RISE-10-002 to Daejin Lim]; the NRF of Korea [grant numbers RS-2026-25487152 to Miryoung Song]; and the Hankuk University of Foreign Studies Research Fund of 2026 [to Miryoung Song]. Authors' contributions Conceptualization, D.L.; Data curation, D.L., M.S. and D.L.; Formal analysis, D.L.; Investigation, D.L, J.K., and S.H.; Methodology, D.L.; Project administration, D.L., and D.L.; Validation, D.L., J.K., and S.H.; Visualization, D.L.; Writing – original draft preparation, D.L.; Supervision, M.S., and D.L.; Writing – review & editing, M.S., and D.L.; Funding acquisition, D.L.; Project administration, D.L; Resources, D.L. 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EcoSal Plus. 2016;7. https://doi.org/10.1128/ecosalplus.ESP-0021-2015 Janausch IG, Garcia-Moreno I, Unden G. Function of DcuS from Escherichia coli as a fumarate-stimulated histidine protein kinase in vitro. J Biol Chem. 2002;277:39809–14. https://doi.org/10.1074/jbc.M204482200 Golby P, Davies S, Kelly DJ, Guest JR, Andrews SC. Identification and characterization of a two-component sensor-kinase and response-regulator system (DcuS-DcuR) controlling gene expression in response to C4-dicarboxylates in Escherichia coli. J Bacteriol. 1999;181:1238–48. https://doi.org/10.1128/JB.181.4.1238-1248.1999 Zientz E, Bongaerts J, Unden G. Fumarate regulation of gene expression in Escherichia coli by the DcuSR (dcuSR genes) two-component regulatory system. J Bacteriol. 1998;180:5421–5. https://doi.org/10.1128/JB.180.20.5421-5425.1998 Schubert C, Winter M, Ebert-Jung A, Kierszniowska S, Nagel-Wolfrum K, Schramm T, et al. C4-dicarboxylates and l-aspartate utilization by Escherichia coli K-12 in the mouse intestine: l-aspartate as a major substrate for fumarate respiration and as a nitrogen source. Environ Microbiol. 2021;23:2564–77. https://doi.org/10.1111/1462-2920.15478 Jones SA, Gibson T, Maltby RC, Chowdhury FZ, Stewart V, Cohen PS, et al. Anaerobic respiration of Escherichia coli in the mouse intestine. Infect Immun. 2011;79:4218–26. https://doi.org/10.1128/IAI.05395-11 Golby P, Kelly DJ, Guest JR, Andrews SC. Transcriptional regulation and organization of the dcuA and dcuB genes, encoding homologous anaerobic C4-dicarboxylate transporters in Escherichia coli. J Bacteriol. 1998;180:6586–96. https://doi.org/10.1128/JB.180.24.6586-6596.1998 Lim D, Kim KS, Jeong J-H, Marques O, Kim H-J, Song M, et al. The hepcidin-ferroportin axis controls the iron content of Salmonella-containing vacuoles in macrophages. Nat Commun. 2018;9:2091. https://doi.org/10.1038/s41467-018-04446-8 Kim OB, Lux S, Unden G. 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Proc Natl Acad Sci U S A. 2021;118:e2106947118. https://doi.org/10.1073/pnas.2106947118 Gencheva S, Dersch S, Surmann K, Wernet M, Antelo L, Hammer E, et al. Tight Complex Formation of the Fumarate Sensing DcuS-DcuR Two-Component System at the Membrane and Target Promoter Search by Free DcuR Diffusion. mSphere. 2022;7:e0023522. https://doi.org/10.1128/msphere.00235-22 Lim D, Kim KS, Kim H, Ko K-C, Song JJ, Choi JH, et al. Anti-tumor activity of an immunotoxin (TGFα-PE38) delivered by attenuated Salmonella typhimurium. Oncotarget. 2017;8:37550–60. https://doi.org/10.18632/oncotarget.17197 Grandis JR, Tweardy DJ. Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer. Cancer Res. 1993;53:3579–84. Weldon JE, Pastan I. A guide to taming a toxin--recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J. 2011;278:4683–700. https://doi.org/10.1111/j.1742-4658.2011.08182.x Turker NS, Heidari P, Kucherlapati R, Kucherlapati M, Mahmood U. An EGFR targeted PET imaging probe for the detection of colonic adenocarcinomas in the setting of colitis. Theranostics. 2014;4:893–903. https://doi.org/10.7150/thno.9425 Dykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, et al. miR-200 enhances mouse breast cancer cell colonization to form distant metastases. PLoS One. 2009;4:e7181. https://doi.org/10.1371/journal.pone.0007181 Balin-Gauthier D, Delord J-P, Rochaix P, Mallard V, Thomas F, Hennebelle I, et al. In vivo and in vitro antitumor activity of oxaliplatin in combination with cetuximab in human colorectal tumor cell lines expressing different level of EGFR. Cancer Chemother Pharmacol. 2006;57:709–18. https://doi.org/10.1007/s00280-005-0123-3 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-9515764\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":636081385,\"identity\":\"f993e22a-d7f4-4b84-83df-89ca543f66e7\",\"order_by\":0,\"name\":\"Dogeun Lee\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Kangwon National University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Dogeun\",\"middleName\":\"\",\"lastName\":\"Lee\",\"suffix\":\"\"},{\"id\":636081386,\"identity\":\"c8e3ad3c-ed02-4c02-931d-5c4fc5f95148\",\"order_by\":1,\"name\":\"Seyeon Hong\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Kangwon National University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Seyeon\",\"middleName\":\"\",\"lastName\":\"Hong\",\"suffix\":\"\"},{\"id\":636081387,\"identity\":\"e7e9a1b3-3b59-4909-92cc-882bfd3afb48\",\"order_by\":2,\"name\":\"Jihyeon Kim\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Kangwon National University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jihyeon\",\"middleName\":\"\",\"lastName\":\"Kim\",\"suffix\":\"\"},{\"id\":636081388,\"identity\":\"75ea3a14-9036-4c55-b711-d93bf343ab39\",\"order_by\":3,\"name\":\"Miryoung Song\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Hankuk University of Foreign Studies\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Miryoung\",\"middleName\":\"\",\"lastName\":\"Song\",\"suffix\":\"\"},{\"id\":636081389,\"identity\":\"161f76d7-4ac1-4472-814f-7fdb193e6e5e\",\"order_by\":4,\"name\":\"Daejin Lim\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACPmYgwVORgCTEQ0ALG1jLGZK0gAjeNpK0sHMnMLydlyZvzr/G8HEBg508A8/ZBwQcxruBce62HMOdM94YG89gSDZs4G03IKiFmXdbBeOGG2e3SfMwMCcw8LMRcBhYy5wKe6iWemK1NOQkbjjfC9JyOAEYGIS1HJxzLC15ww3+z8Y8BscN23iO4dfCz39244M3Ncm2G84fS3zMU1Etz8+Thl8LCBwAkxIJQMIAGlPEAf4DxKsdBaNgFIyCkQUAtKM51sikVacAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Kangwon National University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Daejin\",\"middleName\":\"\",\"lastName\":\"Lim\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-04-24 10:10:22\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-9515764/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-9515764/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":109067865,\"identity\":\"bcf7bc76-b736-4099-8f43-eecd4983b72e\",\"added_by\":\"auto\",\"created_at\":\"2026-05-12 10:02:04\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":81106,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eNutrient heterogeneity in the CT26 tumor-microenvironment (TME) and the adaptive response of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eEscherichia coli\\u003c/strong\\u003e\\u003c/em\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A–E) \\u003c/strong\\u003eBALB/c mice were subcutaneously implanted with 1 × 10\\u003csup\\u003e6\\u003c/sup\\u003e CT26 colon carcinoma cells in the right thigh. \\u003cstrong\\u003e(A and B)\\u003c/strong\\u003e The relative nutrient ratio was measured in the indicated tissue (the white and blue bars represent the liver and tumor, respectively) collected when the tumor size reached 100–150 mm\\u003csup\\u003e3\\u003c/sup\\u003e before injecting the bacteria into the mice. \\u003cstrong\\u003e(A) \\u003c/strong\\u003eThe relative glucose-to-fumarate ratio, as determined by dividing the glucose concentration (pmol/g) by the fumarate concentration (pmol/g) in the indicated tissue. Data represent mean ± SD of n = 5 biological replicates. \\u003cstrong\\u003e(B)\\u003c/strong\\u003e The relative fumarate-to-glucose ratio, as determined by dividing the fumarate concentration (pmol/g) by the glucose concentration (pmol/g) in the indicated tissue. Data represent mean ± SD of n = 5 biological replicates. \\u003cstrong\\u003e(C) \\u003c/strong\\u003eRepresentation of the \\u003cem\\u003eE. coli \\u003c/em\\u003eDcuSR two-component signal transduction system that responds to the presence of external fumarate and induces the expression of genes for its utilization. \\u003cstrong\\u003e(D and E) \\u003c/strong\\u003eCT26 tumor-bearing BALB/c mice were intravenously injected with wild-type MG1655 when the tumor size reached 100 mm\\u003csup\\u003e3\\u003c/sup\\u003e. \\u003cstrong\\u003e(D) \\u003c/strong\\u003eRelative gene expression levels of \\u003cem\\u003edcuS\\u003c/em\\u003e, as measured at 3 days post-bacterial injection (dpi) in the indicated tissue (white and blue bars represent \\u003cem\\u003eE.coli \\u003c/em\\u003egene expression in the liver and tumor, respectively). Data represent mean ± SD of n = 4 biological replicates. \\u003cstrong\\u003e(E) \\u003c/strong\\u003eRelative expression levels of DcuSR regulon genes measured at 3 dpi in the indicated tissues. Data represent mean ± SD of n = 4 biological replicates. ***P \\u0026lt; 0.001, ****P \\u0026lt; 0.0001; Unpaired two-tailed Student’s t-test for (A), (B), and (D); two-way ANOVA for (E) with Dunnett’s comparisons test (\\u003cem\\u003easpA\\u003c/em\\u003e vs other genes within tumor).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9515764/v1/b573640dc66c0085a776a35f.png\"},{\"id\":109003486,\"identity\":\"fe174ac0-2023-4e56-b08f-08f238515705\",\"added_by\":\"auto\",\"created_at\":\"2026-05-11 15:21:23\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":61337,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAssessment of \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003easpA\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e promoter (P\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003easpA\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e) activity under TME-like nutrient availability\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A and C) \\u003c/strong\\u003eBacteria were cultured in M9 medium supplemented with the indicated carbon source for 5 h in an anaerobic bag. \\u003cstrong\\u003e(A) \\u003c/strong\\u003eRelative gene expression levels of \\u003cem\\u003easpA\\u003c/em\\u003e, as measured after 5 h of incubation at the indicated culture conditions\\u003cem\\u003e \\u003c/em\\u003e[black, glucose alone; gray, glucose + fumarate; blue, fumarate alone]. Data represent mean ± SD of n = 4 biological replicates \\u003cstrong\\u003e(B) \\u003c/strong\\u003eRepresentation of the promoter region of \\u003cem\\u003easpA \\u003c/em\\u003e(P\\u003csub\\u003easpA\\u003c/sub\\u003e); dashed boxes indicate the C\\u003csub\\u003e4\\u003c/sub\\u003e-dicarboxylate-inducible DcuR-dependent activation site whereas solid boxes represent the ribosome-binding site. Solid underlines indicate the −35 to −10 RNA polymerase-binding region. \\u003cstrong\\u003e(C) \\u003c/strong\\u003eP\\u003csub\\u003easpA\\u003c/sub\\u003e activity was measured using an unstable variant of green fluorescent protein (gfpOVA), which was expressed by \\u003cem\\u003eE. coli \\u003c/em\\u003ecarrying pA-gfpOVA (MDG001) [black, glucose alone; gray, glucose + fumarate; blue, fumarate alone]. Fluorescence was detected after 5 h of incubation under the indicated culture conditions, using excitation and emission wavelengths of 488 and 513 nm, respectively. The measured fluorescence was normalized to the absorbance at 600 nm. Data represent mean ± SD of n = 4 biological replicates. ****P \\u0026lt; 0.0001; Unpaired two-tailed Student’s t-test for (A) and (C).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9515764/v1/3aaf57c8e423bdb72a669604.png\"},{\"id\":109068105,\"identity\":\"bb23ccbf-1b05-4f89-bde9-59fe81f32963\",\"added_by\":\"auto\",\"created_at\":\"2026-05-12 10:03:38\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":130536,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eSelective cytotoxicity of the immunotoxin\\u003c/strong\\u003e \\u003cstrong\\u003epsp-TGFα-PE38 (TP),\\u003c/strong\\u003e \\u003cstrong\\u003econtrolled by the \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003easpA \\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003epromoter (P\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003easpA\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e), against EGFR-overexpressing cancer cells\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003e A schematic of the plasmid (pA-TP) carrying psp-TGFα-PE38 (TP) controlled by P\\u003csub\\u003easpA\\u003c/sub\\u003e. \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e carrying pA-TP (MDG003) was cultured in M9 medium supplemented with the indicated carbon source for 24 h in an anaerobic bag. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eExpression and secretion of TP from MDG003 were detected via western blot analysis.\\u003cstrong\\u003e \\u003c/strong\\u003eMDG003 was cultured anaerobically in M9 medium supplemented with the indicated carbon sources (+Glu/-Fum; +Glu/+Fum; -Glu/+Fum) for 24 h. After incubation, cultures were fractionated into whole-cell lysate, pellet, and supernatant. Supernatants were clarified by centrifugation and filtration. \\u003cstrong\\u003e(C and D) \\u003c/strong\\u003eCytotoxic activity of TP within the supernatant from MDG003 was measured using the MTT assay in the indicated cell lines. MDG003 was cultured anaerobically in M9 medium supplemented with glucose and/or fumarate (+Glu/-Fum; +Glu/+Fum; -Glu/+Fum) as indicated, and clarified culture supernatants were applied to cells. Cell viability was calculated using the phosphate-buffered saline (PBS)-treated control (100%). \\u003cstrong\\u003e(C)\\u003c/strong\\u003e SW620 cells were used as an EGFR-negative control cell line. \\u003cstrong\\u003e(D)\\u003c/strong\\u003e CT26, MC38, LLC1, and 4T1 cells were used as EGFR-positive cell lines. Data represent mean ± SD of n = 3 biological replicates. ***P \\u0026lt; 0.001, ****P \\u0026lt; 0.0001; one-way ANOVA for (C) and (D) with Dunnett’s comparisons test (-Glu/+Fum vs other incubated conditions).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9515764/v1/3297285820155bcef4f76a85.png\"},{\"id\":109067792,\"identity\":\"25948456-456c-4b78-8fbd-24fe8118d136\",\"added_by\":\"auto\",\"created_at\":\"2026-05-12 10:01:04\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":266538,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnti-cancer effect of TP, driven by P\\u003c/strong\\u003e\\u003csub\\u003e\\u003cstrong\\u003easpA\\u003c/strong\\u003e\\u003c/sub\\u003e\\u003cstrong\\u003e-mediated tumor-specific regulation in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003eEscherichia coli\\u003c/strong\\u003e\\u003c/em\\u003e\\u003cstrong\\u003e, was evaluated using BALB/c mice grafted with CT26 colon carcinoma\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A–C) \\u003c/strong\\u003eBALB/c mice were subcutaneously implanted with 1 × 10\\u003csup\\u003e6 \\u003c/sup\\u003eCT26 colon carcinoma cells in the right thigh. \\u003cstrong\\u003e(A) \\u003c/strong\\u003eTo assess the selective expression of TP by P\\u003csub\\u003easpA\\u003c/sub\\u003e \\u003cem\\u003ein vivo\\u003c/em\\u003e, tumor-bearing mice were intravenously injected with \\u003cem\\u003eE. coli\\u003c/em\\u003e carrying pA-TP (MDG003) (n = 3 per group; 10\\u003csup\\u003e8\\u003c/sup\\u003e CFU/mouse) when tumor size approached approximately 100–150 mm\\u003csup\\u003e3\\u003c/sup\\u003e. TP was extracted by homogenizing the tissue in RIPA lysis buffer. The lysates were centrifuged and filtered. The \\u003cem\\u003ein vivo\\u003c/em\\u003e expression of TP was determined through western blot analysis at 1, 3, and 5 dpi. Supernatant collected from a previous \\u003cem\\u003ein vitro \\u003c/em\\u003eexperiment under fumarate-induction conditions was used as control. \\u003cstrong\\u003e(B–C) \\u003c/strong\\u003eCT26-bearing BALB/c mice were treated with PBS (Mock; black line circle), wild-type \\u003cem\\u003eE. coli \\u003c/em\\u003e(\\u003cem\\u003eE. coli\\u003c/em\\u003e only; green line triangle), or MDG003 (pA-TP; blue line square) by intravenous injection at a dose of 1 × 10\\u003csup\\u003e8\\u003c/sup\\u003e CFU/mouse when tumor size approached approximately 100–150 mm\\u003csup\\u003e3\\u003c/sup\\u003e. \\u003cstrong\\u003e(B) \\u003c/strong\\u003eTumor volumes (left) were measured every 2 days following bacterial injection until they reached \\u0026gt; 1500 mm\\u003csup\\u003e3\\u003c/sup\\u003e. Data represent mean ± SD of n = 5 biological replicates. Representative images (right) show CT26 carcinoma progression in each group. \\u003cstrong\\u003e(C) \\u003c/strong\\u003eSurvival of CT26-grafted mice was monitored until death when tumor volume exceeded 1500 mm\\u003csup\\u003e3\\u003c/sup\\u003e. Data represent mean ± SD of n = 5 biological replicates. The survival of CT26 tumor-bearing mice was determined using Kaplan–Meier curves. **P \\u0026lt; 0.01, ***P \\u0026lt; 0.001; Mixed-effects model (REML) for (B) with Geisser–Greenhouse correction and Holm–Šídák’s multiple comparisons test (\\u003cem\\u003eE. coli\\u003c/em\\u003e pA-TP vs other group at each time point); Log-rank (Mantel–Cox) test for (C) using Kaplan–Meier survival curves (\\u003cem\\u003eE. coli\\u003c/em\\u003e pA-TP vs \\u003cem\\u003eE. coli\\u003c/em\\u003e only).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9515764/v1/d09c878ee666af3918552cb2.png\"},{\"id\":109296073,\"identity\":\"687bf302-f1b2-4fb1-8f7b-b28abc51bdc7\",\"added_by\":\"auto\",\"created_at\":\"2026-05-15 08:45:15\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":743632,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9515764/v1/53efc48a-0808-44c8-a15c-e103d68b5c3c.pdf\"},{\"id\":109003485,\"identity\":\"ad895765-1ee2-4b50-a58c-e23c263ceea9\",\"added_by\":\"auto\",\"created_at\":\"2026-05-11 15:21:23\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":1918898,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementarydata.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-9515764/v1/7cc2cc16d792b35cf38dd3f2.docx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"aspA promoter as a tumor microenvironment-responsive autoswitch for anti- cancer agent expression in Escherichia coli\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eRecent advances in cancer therapy have led to the development of targeted therapies and immunotherapies that aim to minimize the off-target side effects associated with conventional approaches such as surgery, chemotherapy, and radiotherapy. Although these modalities provide improved selectivity, they often exhibit limited efficacy in solid tumors due to poor tissue penetration. A critical factor in this limitation is the presence of metabolic barriers within the tumor microenvironment (TME), which restricts therapeutic access and fosters an immunosuppressive milieu that hinders anti-cancer immune responses. While these features are associated with reduced therapeutic efficacy in solid tumor, they may also create a selective niche for bacterial colonization. This unique property has made bacterial cancer therapy and attractive therapeutic strategy [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. Historically, the concept of using bacteria for cancer treatment dates back to the late nineteenth century, when William B. Coley first used bacterial preparations in patients with malignant disease [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. With advances in genetic manipulation, facultative anaerobes, such as \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e, have emerged as versatile vehicles for tumor-targeted delivery of diverse payloads, including small molecules, immunotoxins, immunomodulators, prodrug-converting enzymes, siRNAs, and nanobodies, to the TME [\\u003cspan additionalcitationids=\\\"CR5 CR6\\\" citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. Although bacteria have been widely explored as carriers for tumor-targeted delivery of therapeutic payloads, ensuring that these agents are expressed exclusively within tumor tissues without harming normal tissues remains a major challenge. Constitutive expression of anticancer agents by bacteria may cause off-target toxicity in healthy tissues, highlighting the need for precise control over therapeutic gene expression. Inducible systems may help to overcome this issue. However, such systems rely on the repeated administration of external inducers, which can complicate treatment regimens. This limitation prompted us to investigate whether metabolic features that distinguish the TME from normal tissues could be harnessed as endogenous signals for tumor-specific gene expression. Such regulation is important because constitutive expression of anticancer agents by bacteria may damage healthy tissues [\\u003cspan additionalcitationids=\\\"CR9\\\" citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eWe hypothesized that the metabolic features of the TME could be exploited as endogenous signals for tumor-specific regulation of anticancer agent expression. The TME exhibits a metabolic profile that is distinct from that of normal tissues [\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e]. One of the key differences is the increased rate of glucose uptake by tumor cells, even under normoxic conditions, which was first observed by Otto Warburg and now known as the Warburg effect [\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e]. As tumors grow, these heightened metabolic demands, coupled with abnormal vascular architecture and disrupted tissue structure, aggravate nutrient insufficiency within the solid tumor tissues [\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e]. Unlike normal blood vessels, tumor vasculature branches irregularly, resulting in poor nutrient delivery and considerable heterogeneity in nutrient distribution within the TME [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. This abnormal vascular architecture, combined with the metabolic reprogramming that is inherent to tumor cells, contributes to the accumulation of oncometabolites, such as succinate and fumarate [\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e, \\u003cspan additionalcitationids=\\\"CR17 CR18 CR19 CR20 CR21\\\" citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. The TME exhibits characteristic metabolic alterations, including low pH, hypoxia, glucose depletion, and the accumulation of oncometabolites. These features contribute to an immunosuppressive niche that undermines the effectiveness of conventional anticancer therapies. However, they also generate distinctive environmental cues that can be sensed by bacterial regulatory systems [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eWe hypothesized that intrinsic bacterial sensing systems can detect and respond to tumor-associated environmental cues, such as nutrient heterogeneity. These systems have the potential to enable tumor-specific regulation of gene expression in response to TME. In \\u003cem\\u003eE. coli\\u003c/em\\u003e, adaptation of nutrient-limited or anaerobic environments involves of activation of fumarate respiration, key metabolic pathway that allow the utilization of alternative carbon sources such as C\\u003csub\\u003e4\\u003c/sub\\u003e-dicarboxylate when glucose and oxygen are scarce [\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e]. This process requires the coordinated expression of genes involved in C\\u003csub\\u003e4\\u003c/sub\\u003e-dicarboxylate transport and fumarate reduction such as \\u003cem\\u003easpA\\u003c/em\\u003e, \\u003cem\\u003efrdABCD\\u003c/em\\u003e, \\u003cem\\u003edcuA\\u003c/em\\u003e and \\u003cem\\u003edcuB\\u003c/em\\u003e, which are transcriptionally regulated by the DcuSR two-component system, which enables \\u003cem\\u003eE. coli\\u003c/em\\u003e to sense and respond to environmental changes in carbon availability under anaerobic conditions [\\u003cspan additionalcitationids=\\\"CR26 CR27 CR28 CR29\\\" citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003ePromoters controlled by such a regulatory network can serve as precise biological switches, activating gene expression only under specific environmental condition. In this study, we aimed to develop an expression system in \\u003cem\\u003eE. coli\\u003c/em\\u003e that exploits native metabolic sensing mechanisms responsive to tumor-specific nutrient cues, enabling selective induction of therapeutic protein expression within the TME.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eBacterial strains, plasmid construction, and culture conditions\\u003c/h2\\u003e \\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003eTable\\u0026nbsp;1 lists the bacterial strains and plasmids used in this study. \\u003cem\\u003eE. coli\\u003c/em\\u003e K-12 MG1655 was used as the wild-type strain and was cultured in Luria\\u0026ndash;Bertani broth (LB, Cat. No. MB-L4488; MB Cell). To monitor \\u003cem\\u003easpA\\u003c/em\\u003e promoter (P\\u003csub\\u003easpA\\u003c/sub\\u003e) activity, a P\\u003csub\\u003easpA\\u003c/sub\\u003e::gfpOVA plasmid (pA-gfpOVA) was constructed using restriction enzyme-based cloning, as follows: first, genomic DNA extracted from \\u003cem\\u003eE. coli\\u003c/em\\u003e K-12 MG1655 was used to amplify the \\u003cem\\u003easpA\\u003c/em\\u003e promoter region, which included DcuR-dependent binding sites located at positions\\u0026thinsp;\\u0026minus;\\u0026thinsp;67 to \\u0026minus;\\u0026thinsp;87 and \\u0026minus;\\u0026thinsp;169 to \\u0026minus;\\u0026thinsp;153 relative to the transcription start site [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. The amplified promoter was cloned into a reporter gene (gfpOVA) by replacing the \\u003cem\\u003eiroB\\u003c/em\\u003e promoter sequence with the piroB::gfpOVA plasmid [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e] using the specific primer set listed in Supplementary Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e. To confirm the expression of the anti-cancer agent, the araBAD promoter in the psp-TGF⍺-PE38 plasmid was replaced with P\\u003csub\\u003easpA\\u003c/sub\\u003e by Gibson assembly using the primer set listed in Supplementary Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e, consequently generating the P\\u003csub\\u003easpA\\u003c/sub\\u003e::psp-TGF⍺-PE38 plasmid (pA-TP). The plasmids were confirmed by DNA sequencing (Macrogen). Next, each plasmid was transformed into \\u003cem\\u003eE. coli\\u003c/em\\u003e via heat shock. Bacterial strains carrying the plasmid were cultured in LB medium at 37\\u0026deg;C with shaking at 200 rpm. Ampicillin (100 \\u0026micro;g/mL, Cat. No. A9518; Sigma-Aldrich) was then added to the solution.\\u003c/p\\u003e \\u003cp\\u003eBacterial cells were grown in M9 medium (Cat. No. MM003-01; WELGENE) supplemented with casamino acid hydrolysate (0.1% w/v, Cat. No. MB-C1656; MB cell) and L-tryptophan (0.005% w/v, Cat. No. MB-T4863; MB Cell) [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. Incubation was performed in an anaerobic bag (Cat. No. KS-B2002; Kisanbio, ROK), and the M9 medium was supplemented with 0.4% glucose (Cat. No. 50-99-7; Duksan) or 20 mM fumarate (Cat. No. A10976.36; Thermo Fisher Scientific) [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e].\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eFluorescence intensity analysis\\u003c/h3\\u003e\\n\\u003cp\\u003eCarbon source-dependent P\\u003csub\\u003easpA\\u003c/sub\\u003e activity was assessed using green fluorescent protein expression within the bacterial cell culture medium, and fluorescence intensity was measured hourly using a spectrophotometer (VLBL00D0; Thermo Fisher Scientific). Measurements were conducted at excitation and emission wavelengths of 488 and 513 nm, respectively, to detect gfpOVA accumulation. The absorbance was measured at 600 nm to standardize the fluorescence intensity of each group.\\u003c/p\\u003e\\n\\u003ch3\\u003eWestern blot analysis\\u003c/h3\\u003e\\n\\u003cdiv class=\\\"Heading\\\"\\u003eWestern blot analysis\\u003c/div\\u003e \\u003cp\\u003eTo examine the expression of TP by P\\u003csub\\u003easpA\\u003c/sub\\u003e \\u003cem\\u003ein vitro\\u003c/em\\u003e, MG1655 cells carrying pA-TP were cultured in M9 medium with restrictively supplemented carbon sources for 24 h. Whole bacterial cells, pellets, and supernatants were collected at the indicated time points. Bacterial supernatants were filtered through a 0.2-\\u0026micro;M filter (Cat. No. FJ13ASCCA002DL01; GVS Filter), mixed with 5\\u0026times; SDS, and boiled at 100\\u0026deg;C for 5 min. To confirm TP expression in \\u003cem\\u003evivo\\u003c/em\\u003e, MDG003 (1 \\u0026times; 10\\u003csup\\u003e8\\u003c/sup\\u003e CFU/mouse) was injected into mice grafted with CT26 tumors through the tail vein when the tumors reached 100\\u0026ndash;130 mm\\u003csup\\u003e3\\u003c/sup\\u003e. Tumors were excised at the indicated time points and homogenized in 1 mL of RIPA buffer (Cat. No. IBS-BR002 (Intron Biotechnology) containing 1\\u0026times; Protease \\u0026amp; Phosphatase Inhibitor Cocktail and 1\\u0026times;\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003eEDTA (Cat. No. 78440; Thermo Fisher Scientific). The filtered supernatants were mixed with 5\\u0026times; SDS and boiled at 100\\u0026deg;C for 10 min. Total proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Cat. No. IPVH00010; Sigma-Aldrich). The membrane was blocked with 5% skim milk for 1 h and probed with rabbit anti-\\u003cem\\u003ePseudomonas\\u003c/em\\u003e exotoxin A antibody (Cat. No. P2318; Sigma-Aldrich) or mouse anti-\\u0026szlig;-actin antibody (Cat. No. sc-47778; Santa Cruz Biotechnology) overnight at 4\\u0026deg;C. After washing off the primary antibodies with TBS (140 mM NaCl, 10 mM Tris-HCl; pH 8.0) containing 0.1% Tween 20, the membranes were incubated at room temperature (20\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5\\u0026deg;C) for 1 h with goat anti-rabbit IgG (Cat. No. 7074s; Cell Signaling Technology, Danvers) and horse anti-mouse IgG (Cat. No. 7076s; Cell Signaling Technology) conjugated to horseradish peroxidase. The bound proteins were visualized using an ECL kit (Cat. No. 32209; Thermo Fisher Scientific).\\u003c/p\\u003e\\n\\u003ch3\\u003eCell lines and culture\\u003c/h3\\u003e\\n\\u003cp\\u003eCT26 murine colon carcinoma, MC38 murine colon carcinoma, 4T1 murine mammary carcinoma, LLC1 lung carcinoma, and SW620 human colon carcinoma cells were purchased from the American Type Culture Collection. The CT26, MC38, 4T1, and LLC1 cells were grown in high-glucose Dulbecco\\u0026rsquo;s modified Eagle medium (Cat. No. AL007A; HIMEDIA) containing 10% fetal bovine serum (FBS, Cat. No. SH30919.03; cytiva HyClone\\u0026trade;) and 1% penicillin-streptomycin (Cat. No. 30-002CI; Corning Inc), whereas the SW620 cells were grown in Roswell Park Memorial Institute 1640 (Cat. No. AL028A; RPMI, HIMEDIA) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.\\u003c/p\\u003e\\n\\u003ch3\\u003eAnimal ethics and experiments\\u003c/h3\\u003e\\n\\u003cp\\u003e All animal procedures were performed in accordance with the National Guidelines for the Care and Use of Laboratory Animals and the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines were approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (approval numbers KW-240326-3). Female BALB/c mice (5\\u0026ndash;6 weeks old; body weight, 18\\u0026ndash;20 g) were purchased from Samtako Bio Company. Mice were maintained under specific-pathogen-free conditions in a biosafety level 2 facility with a 12-h light/dark cycle, controlled temperature (22\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;2\\u0026deg;C), and free access to standard chow and water. After an acclimation period of at least 7 days, mice were randomly assigned to experimental groups. Mice carrying subcutaneous tumors were generated as follows: tumor cells cultured \\u003cem\\u003ein vitro\\u003c/em\\u003e were harvested, suspended in 30 \\u0026micro;L phosphate-buffered saline (PBS), and injected subcutaneously into the right thigh at the indicated dose (1 \\u0026times; 10\\u003csup\\u003e6\\u003c/sup\\u003e cells for CT26). Bacterial injections were administered when the tumors reached a volume of 100\\u0026ndash;150 mm\\u003csup\\u003e3\\u003c/sup\\u003e. To evaluate the antitumor effects of TP immunotoxins, mice grafted with CT26 cells were injected with wild-type \\u003cem\\u003eE. coli\\u003c/em\\u003e or \\u003cem\\u003eE. coli\\u003c/em\\u003e carrying pA-TP through the tail vein. The tumor volume was calculated using the formula: (\\u003cem\\u003eL\\u003c/em\\u003e\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\times\\\\:\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003e\\u003cem\\u003eH\\u003c/em\\u003e\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\times\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003e\\u003cem\\u003eW\\u003c/em\\u003e)/2, where L is the length, W is the width, and H is the height of the tumor in millimeters. For bacterial distribution \\u003cem\\u003ein vivo\\u003c/em\\u003e, solid tumors and other organs were extracted from mice and homogenized in 1\\u0026times;\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003ePBS using a homogenizer (Ultra\\u0026ndash;Turrax T10; IKA). Mice with tumor volumes\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\:\\\\ge\\\\:\\\\)\\u003c/span\\u003e\\u003c/span\\u003e 1,500 mm\\u003csup\\u003e3\\u003c/sup\\u003e were humanely euthanized using a CO\\u003csub\\u003e2\\u003c/sub\\u003e chamber, in accordance with the guidelines of the Animal Research Committee of Kangwon National University. The survival rates were estimated using the Kaplan\\u0026ndash;Meier curve.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eAssessment of cell viability\\u003c/h2\\u003e \\u003cp\\u003eTo evaluate the cytotoxic effects of TP on cancer cells, cell viability was assessed using an MTT assay kit (Cat. No. M3353; Tokyo Chemical Industry) after exposure to bacterial culture supernatant. Briefly, the bacterial supernatant was prepared from MG1655 cells carrying pA-TP and incubated in M9 medium supplemented with the indicated carbon sources. Subsequently, the supernatant was concentrated using a centrifugal filter (Amicon Ultra 10 K, #UFC901008; Millipore), and the total fraction concentration was measured using the Bradford method. The concentrated supernatant (0.1 mg/mL) was then added to SW620, CT26, MC38, 4T1, and LLC1 cells cultured in 96-well plates for 48 h at 37\\u0026deg;C in a humidified chamber with 5% CO\\u003csub\\u003e2\\u003c/sub\\u003e. To assess cancer cell viability in response to TP exposure, MTT reagent was added to each well, followed by a 2-h incubation at 37\\u0026deg;C to allow for formazan formation. Dimethyl sulfoxide (100 \\u0026micro;L) was added to the plate and then resuspended to determine the dissolution of the purple crystals. A spectrophotometer (VLBL00D0; Thermo Fisher Scientific) was used to measure the OD of each well at 570 nm.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eAssessment of glucose and fumarate metabolites in vivo\\u003c/h3\\u003e\\n\\u003cp\\u003eTo measure the relative nutrient ratios, liver and tumor tissues were collected from CT26-bearing mice when tumor volumes reached 100\\u0026ndash;150 mm\\u0026sup3;, before bacterial injection. Glucose [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e] (Cat. No. ab169559; Abcam) and fumarate [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e] (Cat. No. ab102516; Abcam) concentrations were quantified using colorimetric assay kits according to the manufacturer\\u0026rsquo;s instructions. Freshly harvested tissues were lysed, and metabolite levels were determined from standard curves to obtain the number of nanomoles of each metabolite per gram of tissue. The relative glucose-to-fumarate ratio was calculated by dividing the glucose concentration by the fumarate concentration in each tissue, whereas the relative fumarate-to-glucose ratio was calculated by dividing the fumarate concentration by the glucose concentration.\\u003c/p\\u003e\\n\\u003ch3\\u003eBacterial RNA, cDNA library preparation, and quantitative polymerase chain reaction\\u003c/h3\\u003e\\n\\u003cp\\u003eTotal RNA was extracted from \\u003cem\\u003ein vitro\\u003c/em\\u003e cultured \\u003cem\\u003eE. coli\\u003c/em\\u003e and mouse organs infected with \\u003cem\\u003eE. coli\\u003c/em\\u003e (liver, spleen, and tumor) using a Monarch\\u0026reg; Total RNA Miniprep Kit (Cat. No. T2010S; New England Biolabs\\u0026reg;). cDNA was synthesized from 1\\u0026ndash;5 \\u0026micro;g of total RNA using reverse transcriptase with random hexamer primers (TOPscript\\u0026trade; cDNA Synthesis Kit, Cat. No. EZ005S; Enzynomics).\\u003c/p\\u003e \\u003cp\\u003emRNA levels were quantified using quantitative polymerase chain reaction (qPCR) with a Rotor-Gene Q real-time PCR system (R072010103; Qiagen) with TOPreal\\u0026trade; qPCR 2\\u0026times; PreMIX (Cat. No. RT500S; Enzynomics). The expression levels of \\u003cem\\u003edcuS\\u003c/em\\u003e, \\u003cem\\u003easpA\\u003c/em\\u003e, \\u003cem\\u003edcuB\\u003c/em\\u003e, \\u003cem\\u003efrdA\\u003c/em\\u003e, and \\u003cem\\u003edctA\\u003c/em\\u003e were normalized to those of the 16S rRNA. The primers used in the experiments are listed in Supplementary Table S2. Each experiment was performed in triplicate.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eData were analyzed using GraphPad Prism v.10.0.3 (GraphPad Software). Differences between the mean values of the two groups were analyzed using an unpaired two-tailed Student\\u0026rsquo;s \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:t\\\\)\\u003c/span\\u003e\\u003c/span\\u003e-test. Two-way analysis of variance (ANOVA) was used for the time-course studies. Survival rates were determined using the log-rank (Mantel\\u0026ndash;Cox) test and are shown by Kaplan\\u0026ndash;Meier curves. Statistical significance was set at \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\text{P}\\\\:\\u0026lt;0.05\\\\)\\u003c/span\\u003e\\u003c/span\\u003e.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e \\u003cdiv class=\\\"BlockQuote\\\"\\u003e \\u003cp\\u003e \\u003cb\\u003eNutrient heterogeneity in the TME drives the tumor-specific upregulation of E. coli aspA expression during the adaptive response\\u003c/b\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eWe investigated nutrient heterogeneity to determine whether the TME provided a suitable environment for \\u003cem\\u003eE. coli\\u003c/em\\u003e sensing, focusing on glucose depletion and fumarate accumulation in normal (e.g., the liver) and tumor tissues in CT26 tumor-bearing mice. The relative glucose-to-fumarate ratio was \\u0026gt;\\u0026thinsp;3-fold higher in the liver than in the tumor, whereas the relative fumarate-to-glucose ratio was \\u0026gt;\\u0026thinsp;5-fold lower in the liver than in the tumor (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA and B). These differences prompted us to explore whether \\u003cem\\u003eE. coli\\u003c/em\\u003e responds to external carbon sources through its biological systems (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). The relative expression of \\u003cem\\u003edcuS\\u003c/em\\u003e was 10-fold higher in tumors than in the liver, prompting further investigation into the regulation of the DcuSR regulon within the TME (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD). The expression of \\u003cem\\u003easpA\\u003c/em\\u003e, a gene within the DcuSR regulon, was remarkably higher in tumor tissues than in normal liver tissues, with expression levels increasing approximately 65-fold. The upregulation was \\u0026gt;\\u0026thinsp;3-fold higher than that of any other gene within the DcuSR regulon in the TME (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eE), indicating that \\u003cem\\u003easpA\\u003c/em\\u003e was the most highly upregulated gene within the DcuSR regulon in tumor tissues. These findings indicate that the \\u003cem\\u003easpA\\u003c/em\\u003e promoter (P\\u003csub\\u003easpA\\u003c/sub\\u003e) may serve as a metabolically responsive auto-inducible switch for anticancer agent tumor-specific expression.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCarbon source-dependent aspA promoter activation\\u003c/h2\\u003e \\u003cp\\u003eWe assessed whether the P\\u003csub\\u003easpA\\u003c/sub\\u003e is activated based on the availability of external carbon sources. First, wild-type \\u003cem\\u003eE. coli\\u003c/em\\u003e was cultured in an anaerobic bag in M9 medium supplemented with the indicated carbon source for 5 h. Following incubation, we measured the relative expression of \\u003cem\\u003easpA\\u003c/em\\u003e. The expression of \\u003cem\\u003easpA\\u003c/em\\u003e was upregulated by \\u0026gt;\\u0026thinsp;3-fold in the presence of fumarate than in the presence of glucose (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). To confirm the functionality of P\\u003csub\\u003easpA\\u003c/sub\\u003e as a tumor-specific switch for anticancer agent delivery, we analyzed the P\\u003csub\\u003easpA\\u003c/sub\\u003e region to evaluate promoter activity [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. The promoter was primarily regulated by phosphorylated DcuR, which bound to two regions located at \\u0026minus;\\u0026thinsp;83 to \\u0026minus;\\u0026thinsp;67 and \\u0026minus;\\u0026thinsp;169 to \\u0026minus;\\u0026thinsp;153 upstream of the \\u003cem\\u003easpA\\u003c/em\\u003e transcriptional start site (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB). A gene reporter system was constructed using the unstable GFP variant gfpOVA, which was cloned downstream of P\\u003csub\\u003easpA\\u003c/sub\\u003e in pBR322 to generate pA-gfpOVA. \\u003cem\\u003eE. coli\\u003c/em\\u003e transformed with this plasmid was used to monitor P\\u003csub\\u003easpA\\u003c/sub\\u003e activity. The fluorescence intensity was determined during \\u003cem\\u003ein vitro\\u003c/em\\u003e growth in the aforementioned medium. The fluorescence intensity was \\u0026gt;\\u0026thinsp;4-fold higher in the fumarate medium than in the glucose medium (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC and Supplementary Fig. \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCytotoxic activity of psp-TGFα-PE38 expressed via the aspA promoter\\u003c/h2\\u003e \\u003cp\\u003eNext, we tested whether P\\u003csub\\u003easpA\\u003c/sub\\u003e could drive the expression of anticancer agents. We selected a recombinant immunotoxin composed of transforming growth factor alpha (TGFα) and modified \\u003cem\\u003ePseudomonas\\u003c/em\\u003e exotoxin A (PE38) as a model therapeutic payload, with the psp signal peptide fused in-frame to the N-terminal of TGFα-PE38 to facilitate secretion [\\u003cspan additionalcitationids=\\\"CR39\\\" citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. Therefore, we cloned psp-TGFα-PE38 (TP) under the control of P\\u003csub\\u003easpA\\u003c/sub\\u003e in pBR322 to generate pA-TP (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). Subsequently, we observed whether TP was successfully controlled by P\\u003csub\\u003easpA\\u003c/sub\\u003e under specific nutrient conditions similar to those in TME. TP was efficiently overexpressed with fumarate supplementation compared with glucose supplementation, supporting the functionality of P\\u003csub\\u003easpA\\u003c/sub\\u003e as a switch for the tumor-specific expression of TP (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eTo evaluate the selective cytotoxicity of TP expressed by P\\u003csub\\u003easpA\\u003c/sub\\u003e, MTT assay was performed using the supernatant from \\u003cem\\u003eE. coli\\u003c/em\\u003e cultures carrying pA-TP supplemented with glucose or fumarate. The assay was applied to CT26 and MC38 murine colon cancer cell lines, LLC1 murine lung cancer, and 4T1 murine breast cancer cells, all of which exhibit high levels of epidermal growth factor receptor (EGFR) expression [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e], compared with that in SW620 human colon cancer cells, which express low levels of EGFR [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. The cytotoxic effect increased in proportion to the fumarate supplementation of the supernatant when 0.1 mg of the supernatant fraction was administered to tumor cells for 48 h, except for SW620 cells, which express low levels of EGFR (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). The maximum cytotoxicity of TP was observed in LLC1, a tumor cell line that overexpresses EGFR, showing approximately a 90% increase in cytotoxicity relative to the mock group (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). These results indicate that P\\u003csub\\u003easpA\\u003c/sub\\u003e enables the selective expression of the anticancer agent TP under TME-like conditions while preserving its cytotoxic effect against EGFR-overexpressing tumor cells.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eTumor-specific TP expression via P\\u003c/b\\u003e \\u003csub\\u003e \\u003cb\\u003easpA\\u003c/b\\u003e \\u003c/sub\\u003e \\u003cb\\u003esuppressed tumor growth.\\u003c/b\\u003e\\u003c/p\\u003e \\u003cp\\u003eThe role of P\\u003csub\\u003easpA\\u003c/sub\\u003e as a metabolically responsive promoter that enables tumor-specific expression of the immunotoxin TP was initially validated \\u003cem\\u003ein vitro\\u003c/em\\u003e. Subsequently, we evaluated its ability to drive selective TP expression in the TME and minimize off-target expression in normal tissues \\u003cem\\u003ein vivo\\u003c/em\\u003e. To this end, CT26 tumor-bearing mice were intravenously administered \\u003cem\\u003eE. coli\\u003c/em\\u003e carrying pA-TP. TP expression was confirmed in the excised tumor tissue over the indicated days post-injection (dpi), with no significant expression detected in the liver, as shown using western blotting (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). The results revealed that TP is selectively expressed within the TME under the control of P\\u003csub\\u003easpA\\u003c/sub\\u003e. Furthermore, tumor volumes were used to evaluate the anti-tumor efficacy of this system. Mice treated with \\u003cem\\u003eE. coli\\u003c/em\\u003e carrying pA-TP exhibited a 2.8-fold reduction in tumor growth compared with the mock treatment and a 2.1-fold reduction compared with wild-type \\u003cem\\u003eE. coli\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB, with images of all tumors presented in Supplementary Fig. S2). Consistent with the tumor suppression results, survival rate analysis indicated that treatment with \\u003cem\\u003eE. coli\\u003c/em\\u003e carrying pA-TP prolonged the lifespan of tumor-bearing mice by up to 35 days (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC). Although \\u003cem\\u003eE. coli\\u003c/em\\u003e carrying pA-TP showed precise efficacy in delivering anticancer agents, the associated side effects of the bacteria should be carefully monitored. The body weight loss in the treated mice was \\u0026lt;\\u0026thinsp;6%, with a noticeable recovery after 4 dpi (Supplementary Fig. S3A). The bacterial loads in normal tissues were over 100,000-fold lower than those in tumor tissues after 3 days (Supplementary Fig. S3B). Correspondingly, serum IL-6 levels recovered significantly after 3 dpi compared with those observed after 1 dpi, indicating a reduction in systemic inflammatory responses (Supplementary Fig. S3C).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eEffective cancer therapy is limited by poor tumor selectivity and systemic toxicity. Bacterial cancer therapy has become a modern strategy that harnesses engineered microbes to colonize the TME and deliver therapeutic payload locally [\\u003cspan additionalcitationids=\\\"CR6\\\" citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. However, studies specifically exploiting the metabolic features of the TME to achieve tumor-selective expression of anticancer agents are limited [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. In this study, we successfully developed and validated a TME-responsive gene expression system in \\u003cem\\u003eE. coli\\u003c/em\\u003e by leveraging the distinctive metabolic characteristics of the TME as intrinsic cues for therapeutic gene expression.\\u003c/p\\u003e \\u003cp\\u003eWe began by systematically characterizing the nutrient heterogeneity within the TME of CT26 tumor-bearing mice. Consistent with previous reports [\\u003cspan additionalcitationids=\\\"CR12 CR13\\\" citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e], our metabolic analysis unequivocally showed significant glucose depletion and fumarate accumulation in tumor tissues compared with that in normal liver tissues (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA and B). This distinct metabolic signature provided the foundational rationale for exploiting TME-specific cues. Based on these findings, we investigated the DcuSR regulon as a promising metabolic-responsive system (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC) [\\u003cspan additionalcitationids=\\\"CR26 CR27\\\" citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. Crucially, the aspartase-encoding \\u003cem\\u003easpA\\u003c/em\\u003e gene exhibited the most pronounced tumor-specific upregulation (65-fold, Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD), positioning its promoter, P\\u003csub\\u003easpA\\u003c/sub\\u003e, as an ideal candidate for a tumor-specific, auto-switch.\\u003c/p\\u003e \\u003cp\\u003eOur \\u003cem\\u003ein vitro\\u003c/em\\u003e experiments confirmed robust activation of P\\u003csub\\u003easpA\\u003c/sub\\u003e under tumor-like metabolic conditions and minimal activity under\\u003c/p\\u003e \\u003cp\\u003enormal metabolic conditions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). To translate this into a therapeutic application, we engineered pA-TP, encoding the recombinant immunotoxin TP under P\\u003csub\\u003easpA\\u003c/sub\\u003e control (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). TP comprises two functional domains designed for selective cytotoxicity: a TGFα moiety that binds the epidermal growth factor receptor (EGFR), which is frequently overexpressed in malignant tumors, and a truncated Pseudomonas exotoxin A (PE38) domain, which inhibits protein synthesis upon internalization, leading to cell death. Through this design, TP combines receptor-ligand targeting specificity with the potent cytotoxicity of bacterial toxins. Despite the therapeutic promise of immunotoxins, their systemic administration often results in off-target toxicity and limited efficacy, highlighting the need for localized delivery strategies. In this context, the tumor-responsive P\\u003csub\\u003easpA\\u003c/sub\\u003e system offers a strategy to localize TP expression within the tumor microenvironments.\\u003c/p\\u003e \\u003cp\\u003e \\u003cem\\u003eIn vitro\\u003c/em\\u003e assays showed that TP expression was precisely induced under tumor-like conditions, with minimal expression in normal metabolic environments (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). Furthermore, the secreted TP retained its EGFR-TGFα-mediated selective cytotoxicity against EGFR-overexpressing cancer cells (Figs.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC and D), confirming its functional integrity. The therapeutic relevance of the P\\u003csub\\u003easpA\\u003c/sub\\u003e system was further validated \\u003cem\\u003ein vivo\\u003c/em\\u003e. Intravenous administration of \\u003cem\\u003eE. coli\\u003c/em\\u003e carrying pA-TP into CT26 tumor-bearing mice resulted in selective TP expression within tumors (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). This selective expression led to remarkable tumor regression and a substantial prolongation of survival in treated mice (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB and C). These compelling \\u003cem\\u003ein vivo\\u003c/em\\u003e results confirm that the P\\u003csub\\u003easpA\\u003c/sub\\u003e system effectively leverages TME-specific metabolic conditions to enable the selective expression of therapeutic proteins. Our approach enhanced on-target accuracy while significantly reducing the risk of off-target toxicity, thereby improving the overall efficacy of bacteria-mediated anti-cancer therapy.\\u003c/p\\u003e \\u003cp\\u003eDespite these promising outcomes, complete tumor elimination was not achieved, likely because of the inherent challenge of stable plasmid maintenance in bacteria \\u003cem\\u003ein vivo\\u003c/em\\u003e (Supplementary Fig. S4). This limitation, which can lead to a loss of therapeutic gene expression over time, represents a common hurdle in bacteria-mediated therapies. Future strategies to overcome this critical issue include employing essential gene complementation systems, such as \\u003cem\\u003easd\\u003c/em\\u003e and \\u003cem\\u003eglmS\\u003c/em\\u003e, which can significantly promote stable plasmid inheritance and thus enhance the long-term therapeutic potential of the P\\u003csub\\u003easpA\\u003c/sub\\u003e system. With the ongoing advancements in synthetic biology and genetic engineering, we anticipate that the P\\u003csub\\u003easpA\\u003c/sub\\u003e system will evolve into a versatile and highly reliable platform. Its auto-inducible, tumor-specific nature makes it particularly promising for integration with other therapeutic strategies, potentially paving the way for more effective and precisely targeted cancer treatments.\\u003c/p\\u003e\"},{\"header\":\"Conclusion\",\"content\":\"\\u003cp\\u003eWe developed a tumor-responsive gene expression system based on the \\u003cem\\u003easpA\\u003c/em\\u003e promoter that enables tumor-selective expression of anticancer agents by exploiting metabolic features of the TME. Our findings demonstrate that TME-associated nutrient heterogeneity, particularly glucose depletion and fumarate accumulation, can be harnessed as intrinsic regulatory cues for controlling therapeutic gene expression in bacteria (Fig.\\u0026nbsp;5). This strategy addresses a critical limitation in bacterial cancer therapy by enabling spatially restricted expression of therapeutic payloads, thereby improving targeting precision and reducing the risk of off-target toxicity. Importantly, the P\\u003csub\\u003easpA\\u003c/sub\\u003e \\u0026ndash; based system represents a modular and adaptable platform that can be extended to a wide range of therapeutic protein beyond immunotoxins. Overall, our study provides a new framework for leveraging endogenous tumor-associated signals to achieve precise and context-dependent control of therapeutic gene expression in bacterial cancer therapy.\\u003c/p\\u003e\"},{\"header\":\"Abbreviations\",\"content\":\"\\u003cp\\u003eANOVA, analysis of variance; dpi, days post-injection; EGFR, epidermal growth factor receptor; PBS, phosphate-buffered saline; qPCR, quantitative polymerase chain reaction; TGF\\u0026alpha;, transforming growth factor alpha; TME, tumor microenvironment.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll animal procedures were approved by the Institutional Animal Care and Use Committee of Kangwon National University (approval numbers KW-240326-3).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication:\\u0026nbsp;\\u003c/strong\\u003eNot applicable\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe raw data supporting the conclusions of this article will be made available by the authors upon reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the National Research Foundation (NRF) of Korea grants funded, Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry by the Korean government and the Regional Innovation System and Education program through the Gangwon RISE Center, [grant number NRF-RS-2023-00210053, RS-2025-02263715 and 2025-RISE-10-002 to Daejin Lim]; the NRF of Korea [grant numbers RS-2026-25487152 to Miryoung Song]; and the Hankuk University of Foreign Studies Research Fund of 2026 [to Miryoung Song].\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors' contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eConceptualization, D.L.; Data curation, D.L., M.S. and D.L.; Formal analysis, D.L.; Investigation, D.L, J.K., and S.H.; Methodology, D.L.; Project administration, D.L., and D.L.; Validation, D.L., J.K., and S.H.; Visualization, D.L.; Writing – original draft preparation, D.L.; Supervision, M.S., and D.L.; Writing – review \\u0026amp; editing, M.S., and D.L.; Funding acquisition, D.L.; Project administration, D.L; Resources, D.L. All authors have read and agreed to the published version of the manuscript.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eForbes NS. Engineering the perfect (bacterial) cancer therapy. Nature Reviews Cancer [Internet]. 2010;10:785\\u0026ndash;94. https://doi.org/10.1038/nrc2934\\u003c/li\\u003e\\n\\u003cli\\u003eGurbatri CR, Arpaia N, Danino T. Engineering bacteria as interactive cancer therapies. Science. 2022;378:858\\u0026ndash;64. https://doi.org/10.1126/science.add9667\\u003c/li\\u003e\\n\\u003cli\\u003eKucerova P, Cervinkova M. Spontaneous regression of tumour and the role of microbial infection--possibilities for cancer treatment. Anticancer Drugs. 2016;27:269\\u0026ndash;77. https://doi.org/10.1097/CAD.0000000000000337\\u003c/li\\u003e\\n\\u003cli\\u003eAshu EE, Xu J, Yuan Z-C. Bacteria in Cancer Therapeutics: A Framework for Effective Therapeutic Bacterial Screening and Identification. J Cancer. 2019;10:1781\\u0026ndash;93. https://doi.org/10.7150/jca.31699\\u003c/li\\u003e\\n\\u003cli\\u003eZhou S, Gravekamp C, Bermudes D, Liu K. Tumour-targeting bacteria engineered to fight cancer. Nat Rev Cancer. 2018;18:727\\u0026ndash;43. https://doi.org/10.1038/s41568-018-0070-z\\u003c/li\\u003e\\n\\u003cli\\u003eLim D, Jung WC, Jeong J-H, Song M. Targeted Delivery of the Mitochondrial Target Domain of Noxa to Tumor Tissue via Synthetic Secretion System in E. coli. Front Bioeng Biotechnol. 2020;8:840. https://doi.org/10.3389/fbioe.2020.00840\\u003c/li\\u003e\\n\\u003cli\\u003eMai P-T, Lim D, So E, Kim HY, Duysak T, Tran T-Q, et al. Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria. Cancers (Basel). 2023;15:1486. https://doi.org/10.3390/cancers15051486\\u003c/li\\u003e\\n\\u003cli\\u003eJiang S-N, Phan TX, Nam T-K, Nguyen VH, Kim H-S, Bom H-S, et al. 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The Complex Interplay between Antioxidants and ROS in Cancer. Trends Cell Biol. 2020;30:440\\u0026ndash;51. https://doi.org/10.1016/j.tcb.2020.03.002\\u003c/li\\u003e\\n\\u003cli\\u003eLiu JY, Wellen KE. Advances into understanding metabolites as signaling molecules in cancer progression. Curr Opin Cell Biol. 2020;63:144\\u0026ndash;53. https://doi.org/10.1016/j.ceb.2020.01.013\\u003c/li\\u003e\\n\\u003cli\\u003eThompson CB. Metabolic enzymes as oncogenes or tumor suppressors. N Engl J Med. 2009;360:813\\u0026ndash;5. https://doi.org/10.1056/NEJMe0810213\\u003c/li\\u003e\\n\\u003cli\\u003eDando I, Pozza ED, Ambrosini G, Torrens-Mas M, Butera G, Mullappilly N, et al. Oncometabolites in cancer aggressiveness and tumour repopulation. Biol Rev Camb Philos Soc. 2019;94:1530\\u0026ndash;46. https://doi.org/10.1111/brv.12513\\u003c/li\\u003e\\n\\u003cli\\u003eLinehan WM, Schmidt LS, Crooks DR, Wei D, Srinivasan R, Lang M, et al. The Metabolic Basis of Kidney Cancer. Cancer Discov. 2019;9:1006\\u0026ndash;21. https://doi.org/10.1158/2159-8290.CD-18-1354\\u003c/li\\u003e\\n\\u003cli\\u003eCheng J, Yan J, Liu Y, Shi J, Wang H, Zhou H, et al. Cancer-cell-derived fumarate suppresses the anti-tumor capacity of CD8+ T cells in the tumor microenvironment. Cell Metab. 2023;35:961-978.e10. https://doi.org/10.1016/j.cmet.2023.04.017\\u003c/li\\u003e\\n\\u003cli\\u003eChang C-H, Qiu J, O\\u0026rsquo;Sullivan D, Buck MD, Noguchi T, Curtis JD, et al. Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression. Cell. 2015;162:1229\\u0026ndash;41. https://doi.org/10.1016/j.cell.2015.08.016\\u003c/li\\u003e\\n\\u003cli\\u003eDePeaux K, Delgoffe GM. Metabolic barriers to cancer immunotherapy. Nature Reviews Immunology [Internet]. 2021;21:785\\u0026ndash;97. https://doi.org/10.1038/s41577-021-00541-y\\u003c/li\\u003e\\n\\u003cli\\u003eDawan J, Ahn J. Bacterial Stress Responses as Potential Targets in Overcoming Antibiotic Resistance. Microorganisms. 2022;10:1385. https://doi.org/10.3390/microorganisms10071385\\u003c/li\\u003e\\n\\u003cli\\u003eUnden G, Strecker A, Kleefeld A, Kim OB. C4-Dicarboxylate Utilization in Aerobic and Anaerobic Growth. EcoSal Plus. 2016;7. https://doi.org/10.1128/ecosalplus.ESP-0021-2015\\u003c/li\\u003e\\n\\u003cli\\u003eJanausch IG, Garcia-Moreno I, Unden G. Function of DcuS from Escherichia coli as a fumarate-stimulated histidine protein kinase in vitro. J Biol Chem. 2002;277:39809\\u0026ndash;14. https://doi.org/10.1074/jbc.M204482200\\u003c/li\\u003e\\n\\u003cli\\u003eGolby P, Davies S, Kelly DJ, Guest JR, Andrews SC. Identification and characterization of a two-component sensor-kinase and response-regulator system (DcuS-DcuR) controlling gene expression in response to C4-dicarboxylates in Escherichia coli. J Bacteriol. 1999;181:1238\\u0026ndash;48. https://doi.org/10.1128/JB.181.4.1238-1248.1999\\u003c/li\\u003e\\n\\u003cli\\u003eZientz E, Bongaerts J, Unden G. 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Fumarate dependent protein composition under aerobic and anaerobic growth conditions in Escherichia coli. J Proteomics. 2020;212:103583. https://doi.org/10.1016/j.jprot.2019.103583\\u003c/li\\u003e\\n\\u003cli\\u003eMeng F, Luo X, Li C, Wang G. LncRNA LINC00525 activates HIF-1\\u0026alpha; through miR-338-3p / UBE2Q1 / \\u0026beta;-catenin axis to regulate the Warburg effect in colorectal cancer. Bioengineered. 2022;13:2554\\u0026ndash;67. https://doi.org/10.1080/21655979.2021.2018538\\u003c/li\\u003e\\n\\u003cli\\u003eAggarwal RK, Luchtel RA, Machha V, Tischer A, Zou Y, Pradhan K, et al. Functional succinate dehydrogenase deficiency is a common adverse feature of clear cell renal cancer. Proc Natl Acad Sci U S A. 2021;118:e2106947118. https://doi.org/10.1073/pnas.2106947118\\u003c/li\\u003e\\n\\u003cli\\u003eGencheva S, Dersch S, Surmann K, Wernet M, Antelo L, Hammer E, et al. Tight Complex Formation of the Fumarate Sensing DcuS-DcuR Two-Component System at the Membrane and Target Promoter Search by Free DcuR Diffusion. mSphere. 2022;7:e0023522. https://doi.org/10.1128/msphere.00235-22\\u003c/li\\u003e\\n\\u003cli\\u003eLim D, Kim KS, Kim H, Ko K-C, Song JJ, Choi JH, et al. Anti-tumor activity of an immunotoxin (TGF\\u0026alpha;-PE38) delivered by attenuated Salmonella typhimurium. Oncotarget. 2017;8:37550\\u0026ndash;60. https://doi.org/10.18632/oncotarget.17197\\u003c/li\\u003e\\n\\u003cli\\u003eGrandis JR, Tweardy DJ. Elevated levels of transforming growth factor alpha and epidermal growth factor receptor messenger RNA are early markers of carcinogenesis in head and neck cancer. Cancer Res. 1993;53:3579\\u0026ndash;84. \\u003c/li\\u003e\\n\\u003cli\\u003eWeldon JE, Pastan I. A guide to taming a toxin--recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer. FEBS J. 2011;278:4683\\u0026ndash;700. https://doi.org/10.1111/j.1742-4658.2011.08182.x\\u003c/li\\u003e\\n\\u003cli\\u003eTurker NS, Heidari P, Kucherlapati R, Kucherlapati M, Mahmood U. An EGFR targeted PET imaging probe for the detection of colonic adenocarcinomas in the setting of colitis. Theranostics. 2014;4:893\\u0026ndash;903. https://doi.org/10.7150/thno.9425\\u003c/li\\u003e\\n\\u003cli\\u003eDykxhoorn DM, Wu Y, Xie H, Yu F, Lal A, Petrocca F, et al. miR-200 enhances mouse breast cancer cell colonization to form distant metastases. PLoS One. 2009;4:e7181. https://doi.org/10.1371/journal.pone.0007181\\u003c/li\\u003e\\n\\u003cli\\u003eBalin-Gauthier D, Delord J-P, Rochaix P, Mallard V, Thomas F, Hennebelle I, et al. In vivo and in vitro antitumor activity of oxaliplatin in combination with cetuximab in human colorectal tumor cell lines expressing different level of EGFR. Cancer Chemother Pharmacol. 2006;57:709\\u0026ndash;18. https://doi.org/10.1007/s00280-005-0123-3\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-biological-engineering\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jbie\",\"sideBox\":\"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)\",\"snPcode\":\"13036\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13036/3\",\"title\":\"Journal of Biological Engineering\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"E. coli, Fumarate respiration, aspA promoter, Tumor-specific expression, Tumor-microenvironment, Nutrient heterogeneity\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-9515764/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-9515764/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eBacterial 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 \\u003cem\\u003eEscherichia coli\\u003c/em\\u003e (\\u003cem\\u003eE. coli\\u003c/em\\u003e) 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\\u0026thinsp;\\u0026gt;\\u0026thinsp;3-fold lower glucose and \\u0026gt;\\u0026thinsp;5-fold higher fumarate levels than those in the liver. In response to this nutrient heterogeneity, \\u003cem\\u003eE. coli\\u003c/em\\u003e upregulated gene involved in fumarate respiration including \\u003cem\\u003efrdA\\u003c/em\\u003e, \\u003cem\\u003edctA\\u003c/em\\u003e, \\u003cem\\u003edcuB\\u003c/em\\u003e, and \\u003cem\\u003easpA\\u003c/em\\u003e in the DcuSR regulon. \\u003cem\\u003easpA\\u003c/em\\u003e was most upregulated (65-fold) in tumors, suggesting that its promoter exhibits the strongest activity under TME. We used the \\u003cem\\u003easpA\\u003c/em\\u003e promoter (P\\u003csub\\u003easpA\\u003c/sub\\u003e) as a tumor-specific switch to control of TP expression, targeting secreted functional TP and induced cytotoxicity in vitro. TP was specifically expressed by P\\u003csub\\u003easpA\\u003c/sub\\u003e in tumors, reducing tumor size by 2.8-fold and extending survival by 35 days in vivo. The results suggest that the P\\u003csub\\u003easpA\\u003c/sub\\u003e as a tumor-specific expression system.\\u003c/p\\u003e\",\"manuscriptTitle\":\"aspA promoter as a tumor microenvironment-responsive autoswitch for anti- cancer agent expression in Escherichia coli\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-05-11 15:21:14\",\"doi\":\"10.21203/rs.3.rs-9515764/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-13T02:05:09+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-07T04:53:59+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"297038449599332015236046075404188742142\",\"date\":\"2026-05-04T04:29:52+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"72317077421308755932895909411483651075\",\"date\":\"2026-05-04T00:26:46+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"47616570607822784769979935834137916474\",\"date\":\"2026-05-03T03:04:58+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"206893786448789067832909684174470602918\",\"date\":\"2026-05-02T00:54:29+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"115867720658860117558737552562595326530\",\"date\":\"2026-05-01T23:26:14+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2026-05-01T16:56:35+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2026-04-28T11:32:29+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2026-04-28T11:31:38+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Biological Engineering\",\"date\":\"2026-04-24T09:54:20+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-biological-engineering\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jbie\",\"sideBox\":\"Learn more about [Journal of Biological Engineering](http://jbioleng.biomedcentral.com/)\",\"snPcode\":\"13036\",\"submissionUrl\":\"https://submission.nature.com/new-submission/13036/3\",\"title\":\"Journal of Biological Engineering\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"0b37ecea-da79-448e-95a9-8a48a0480232\",\"owner\":[],\"postedDate\":\"May 11th, 2026\",\"published\":true,\"recentEditorialEvents\":[{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-13T02:05:09+00:00\",\"index\":30,\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2026-05-07T04:53:59+00:00\",\"index\":27,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"297038449599332015236046075404188742142\",\"date\":\"2026-05-04T04:29:52+00:00\",\"index\":24,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"72317077421308755932895909411483651075\",\"date\":\"2026-05-04T00:26:46+00:00\",\"index\":23,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"47616570607822784769979935834137916474\",\"date\":\"2026-05-03T03:04:58+00:00\",\"index\":21,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"206893786448789067832909684174470602918\",\"date\":\"2026-05-02T00:54:29+00:00\",\"index\":20,\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"115867720658860117558737552562595326530\",\"date\":\"2026-05-01T23:26:14+00:00\",\"index\":19,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"14\",\"date\":\"2026-05-01T16:56:35+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"under-review\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-05-11T15:21:14+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-05-11 15:21:14\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-9515764\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-9515764\",\"identity\":\"rs-9515764\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}