Escherichia coli Nissle 1917 secreting functional interleukin 2 targets tumours and enhances the immune response to suppress tumours

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Previous studies have indicated that EcN can accumulate and proliferate selectively in solid tumours in BALB/c mouse models. In this study, EcN was engineered to express human interleukin 2 (hIL-2), which is known to enhance immune responses to tumours by activating a variety of immune cells. IL-2 expressed by EcN was proven to activate PBMCs in vitro. Compared to control EcN, intraperitoneally injected EcN expressing hIL-2 (EcN(hIL-2)) was selectively distributed in the tumour microenvironment and inhibited the growth of CT26 tumours in a tumour-bearing mouse model. Antitumour activity was achieved without toxicity to key normal organs and tissues, such as liver, spleen and kidneys. The antitumour mechanism was associated with the infiltration of inflammatory cells, such as T cells, neutrophils and macrophages. These findings provide evidence that the combination of tumour-targeting EcN bacteria and delivery of the immunostimulatory factor IL-2 can be exploited as a promising tumour immunotherapy. Biotechnology and Bioengineering Environmental Engineering E.coli Nissle 1917 IL-2 Anti-tumor engineering bacteria Targeted cancer therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Immune system function is often inhibited in the microenvironment of solid tumours[ 1-5 ]. Therefore, many attempts are being made to activate immune responses against tumours[ 6 ]. Tumour-targeted therapy is a promising treatment method for many malignant tumours. Colorectal cancer is one of the most common malignant tumors. The global incidence rate of malignant tumors ranks third, and the case fatality rate ranks second. The incidence of colorectal cancer in the world is increasing year by year[ 7 ]. Among the probiotics used in research in recent years, E. coli Nissle 1917 (EcN) is currently the only Gram-negative bacteria in use, and it is also one of the most widely studied probiotics in the world[ 8 ]. EcN is an ideal carrier for vaccines, cytokines and other substances because of its innocuity and versatility in bioengineering. For example, genetically engineered EcN can be used to secrete cystatin, a nematode immunomodulator, in the intestinal tract to treat experimental colitis in mice or pigs[ 9 ]. Jochen et al injected EcN intravenously (i.v.), intraperitoneally (i.p.), or intratumorally (i.t.) into tumor-bearing mice to study its specific targeting property. They found that massive EcN colonized and replicated in tumors and no obvious difference was observed in the CFU/g isolated from organ tissues, regardless of inoculation route[ 10 ]. However, the development of targeted drugs has significantly improved the overall prognosis of patients with colorectal cancer, so we hope to use EcN as a delivery vehicle to treat colorectal cancer. The immunoregulatory cytokine interleukin-2 (IL-2) is a growth and activating factor for a variety of immune cells, including T cells and NK cells[ 11 ]. The cytokine IL-2 is an effective T cell mitogen and activator, which can expand the function of T cells，maintain the proliferation of T lymphovitro for a long time, stimulate T cells to enter the cell division cycle，and increase the immune clearance rate of tumors in the immunosuppressed tumor microenvironment[ 12 ]. Meanwhile, IL-2 can promote the production of NK or T cell-derived cytokines, such as TNF-α, IFN-γ and GM-CSF, which can boost antitumour immunity, and these molecules have synergistic effects[ 13-15 ]. Therefore, we hypothesized that tumour-targeting bacteria expressing the immune-activating cytokine IL-2 may optimally modify the immune microenvironment and improve antitumour effects. In this study, EcN bacteria were engineered to express soluble human IL-2, with the aim of improving the immune function of tumour-bearing mice and inhibiting the growth of tumours. To exploit the role of IL-2 delivered by EcN in tumour tissue, CT26 colon cancer cells were implanted subcutaneously in syngeneic BALB/c mice, and after the tumour was established, EcN(hIL-2) was injected intraperitoneally into the tumour-bearing mouse. Immunohistochemical results showed that EcN(hIL-2) specifically localized in the tumour region and that IL-2 was released in the tumour tissue. Tumour growth in the EcN(hIL-2) group was inhibited approximately 53.91% compared with that in the PBS control group. A myriad of necrotic tumour cells could be observed in the tumour tissue of the EcN (hIL-2)-treated group. To assess the toxicity induced by EcN (hIL-2), we measured the body weight of tumour-bearing mice every two days. These mice were sacrificed after 7 days, and the liver, kidneys and the spleen of the mice in each group were excised and weighed. There was no difference in either body or organ weight between the experimental and control groups. We also investigated the distribution of engineered bacteria in the liver, kidneys and the spleen of tumour-bearing mice using an IVIS spectrum[ 16 , 17 ] and found that the numbers of bacteria in these organs were significantly lower than those in tumours. To further explore the antitumour mechanism of these engineered bacteria, local immune responses induced by EcN(hIL-2) were also detected by H&E staining of tissue sections. Immune cells, such as T lymphocytes, neutrophils and macrophages, infiltrated the tumour microenvironment, but the control groups had insufficient immune cell infiltration. We also examined the cytokines IFN-γ and TGF-β in the serum, as IFN-γ is a known key mediator of IL-2 toxicity and TGF-β is an inhibitory factor. The results showed that EcN(hIL-2) treatment was associated with significantly elevated IFN-γ expression and decreased TGF-β expression. Results Expression analysis of the IL-2 protein in vitro To allow expression of the IL-2 protein in a prokaryotic system, the IL-2 gene was cloned into three different inducible expression vectors (Supplementary Fig. 1 A, 1 B and 1 C). The differential proteins expressed by the three recombinant plasmids in E. coli BL21 (DE3) were verified by Western blotting and mass spectrometry, which proved that the target protein IL-2 was successfully expressed (Supplementary Fig.2 and Table S1). Coomassie Brilliant Blue staining showed that the recombinant protein showed soluble expression under the action of SUMO and IF2 tags (Fig. 1 A). Engineered bacteria need to continuously secrete recombinant proteins inside a tumour, induce immune cell activation, attack tumour cells, and inhibit tumour growth. Therefore, the IL-2 protein can be continuously expressed in hypoxic tumours under the oxygen-dependent promoter of the haemoglobin gene (vhb) of Vitreoscilla and the pelB leader sequence (Fig. 1B and Supplementary Fig. 3). Engineered bacteria were cultured overnight in LB medium, and the recombinant protein was successfully expressed in engineered EcN bacteria, as verified by SDS-PAGE analysis (Fig. 1 C). Western blot analysis indicated that the IL-2 protein was presented in both the cell lysate and medium supernatant of EcN (hIL-2) (Fig. 1 D). To reduce the effect of tags on IL-2 protein activity, a SUMO fusion system with a relatively low molecular weight was selected for subsequent experimental research. SUMO-IL-2 protein can promote the proliferation of PBMCs BL21 (pSmartI-IL 2) bacteria were cultured, and IL-2 protein was collected by Ni-NTA Sefinose (TM) Resin Kit. The IL-2 protein at different concentrations was cocultured with peripheral blood mononuclear cells (PBMCs), and PBS was added to the control group. PBMCs were cocultured with the recombinant protein for 24 h, and then Cell Counting Kit-8 was added to detect the cell survival rate. The results showed that cell proliferation was obviously promoted after the addition of SUMO-IL-2 (Sumo is the solubilizing label on pSmartI), and the proliferation rate of cells also increased as the SUMO-IL-2 protein concentration increased (Fig. 2 A). Additionally, the cell culture medium was centrifuged after the incubation, and the culture supernatant was collected. The concentrations of IFN-γ and TGF-β in the supernatant of the culture medium were detected by enzyme-linked immunosorbent assay (ELISA). The results showed that compared with that in the supernatant of the control group, the concentration of IFN-γ in the supernatant of the experimental group was significantly increased (Fig. 2 B), while the concentration of TGF-β was significantly decreased (Fig. 2 C). EcN specifically colonizes tumour regions in tumour-bearing mice An IVIS can accurately observe the real-time location of bacteria in animals without causing damage to the animals[ 16 , 17 ]. After intraperitoneal injection of 5×10 6 CFU/100 µL EcN(Lux)[ 18 ] into tumour-bearing mice in the experimental group and injection of sterile PBS into tumour-bearing mice in the control group, bacterial colonization in the mice was observed by an IVIS. The results showed that the tumour-bearing mice exhibited a significant fluorescence signal in the tumour area for 5 days after the bacteria were injected, and the fluorescence signal was still observed on the 7th day after injection (Fig. 3 A). The control group did not exhibit a detectable signal. After the mice were euthanized on the 7th day, the tumour, liver, kidneys and spleen of the mice were obtained. IVIS analysis showed that 5 days after EcN(Lux) was intraperitoneally injected into tumour-bearing mice, a strong fluorescence signal was detected in the tumour tissues of the mice, and no fluorescence signal was detected in other organs (Fig. 3 B). These results showed that EcN has excellent targeting to the tumours in CT26 tumour-bearing mice. Bacteria can quickly accumulate in the tumour area and grow and reproduce in the tumour area after intraperitoneal injection into mice, while bacteria in other parts of mice can be removed by the local immune response quickly. Antitumour effect of EcN(hIL-2) To validate the successful expression of the IL-2 molecule carried by EcN in tumour areas, we performed immunohistochemistry on samples from each group of mouse tumours. Mice were euthanized on the 7th day after the third administration, and the tumour tissue was removed, fixed in 4% paraformaldehyde, and then embedded in paraffin. These results showed that a yellow-grey signal appeared in the tumour tissue sections of the EcN (hIL-2) experimental group, while those of the other three groups did not show a positive signal (Fig. 4 A). These results indicate that IL-2 is successfully expressed in the tumour region. The antibody used in immunohistochemistry is Anti-His Tag Rabbit Polyclonal Antibody. To evaluate the antitumour efficacy of EcN (IL-2), we subcutaneously injected CT26 colon cancer cells into the right axillary area of BALB/c mice. The resultant xenograft tumour model was used to study the antitumour effect of EcN (hIL-2). When the tumours in the mice grew to approximately 60 mm 3 , the mice were randomly divided into 4 groups (n = 5, 6, or 7), and the groups were treated respectively by intraperitoneal injection of sterile PBS, EcN, EcN (28a) or EcN (hIL-2). Body weight and tumour volume were measured every two days during the observation period until the animals were sacrificed. The results of the experiment showed that the tumour growth in the EcN (hIL-2) group was significantly inhibited (Fig. 4 B), while the xenograft tumour growth in the other three groups have no difference. The final tumour weight of the EcN (hIL-2) group was also significantly lower than that of the other 3 groups (Fig. 4 C). The tumour volume in the PBS group reached 4.93 ±1.35 cm 3 , but the tumour volume in the EcN (hIL-2) group was significantly smaller, reaching a volume of 2.32 ± 1.43 mm 3 . Tumour volume was calculated according to the formula (Table 1). Tumour growth in the EcN (hIL-2) group was inhibited approximately 53.91% compared to that in the PBS group. Table 1: The comparison of tumor volume, tumor weight of CT26 cancer of the BALB/c mice Group Mean Tumor Volume (cm 3 ) Mean Tumor Weight (g) PBS 4.93 ±1.35 6.41 ± 1.22 EcN 5.53 ± 1.43 6.74 ± 1.35 EcN (28a) 4.62 ± 1.67 6.38 ± 0.91 EcN (IL-2) 2.32 ± 1.43 (53.91%) ** 3.48 ± 1.49 (45.71%) * Notes: *P< 0.05, ** P < 0.01. Tumour histomorphology and safety monitoring of E. coli Nissle 1917 Haematoxylin-eosin staining (H&E staining) is one of the commonly used staining methods for paraffin sections. Haematoxylin dyeing solutions are alkaline, mainly causing chromatin in the nucleus and nucleic acid in the cytoplasm to be stained purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and the extracellular matrix red. Our experimental results showed that the tumour staining results in the PBS group showed normal tumour cell morphology, and no necrotic areas were observed. However, infiltrating inflammatory cells were observed in the EcN experimental group, and the cell morphology was irregular, the phenomenon that inflammatory cells gather in the inflammatory focus. (Fig. 5 A). Additionally, we also performed H&E staining of liver, kidney and spleen tissues from the PBS group and EcN experimental group. The results showed that there was no significant change in histopathological morphology in the liver, kidneys or spleen between the two groups, indicating that EcN had no obvious side effects on the liver, kidneys or spleen in mice (Fig. 5 A). During the experiment, to assess the systemic effects of EcN on the whole body after intraperitoneal injection, we measured mouse body weight every two days. At the end of the experiment, the liver, kidneys and spleen of the mice in each group were excised and weighed, and there were no differences in weight among the four groups (Fig. 5 B). Although the mice exhibited a slight decrease in body weight during treatment, they all approached the same weight by the end of the experiment (Fig. 5 C). All of the above results demonstrate that the toxicity of intraperitoneal administration of EcN to mice is negligible. Examining the tumour microenvironment We explored the mechanism underlying the antitumour immune activity of EcN (hIL-2) in a CT26 colon cancer tumour model. We investigated the immune cell profile in the tumour microenvironment and the changes in cytokines in the blood using the CT26 tumour model. We selected specific antibodies to be combined with antigens on the surface of T lymphocytes, neutrophils and M1 macrophages, and compared the changes in the content of these three cells in the four groups of tumors by immunohistochemistry. Type M1 is a classically activated macrophage (classically activated macrophage), which refers to macrophages that exist in an inflammatory environment, and is affected by gamma interferon, tumor necrosis factor alpha (TNF alpha) and granulocyte macrophage colony stimulating factor (GMCSF) Induction, it can induce an immune response of type I helper T cells (Th1), has the ability to promote inflammation, and plays a very important role in killing bacteria and viruses in the cells. Its characteristics in tumor tissues are mainly manifested as having tumor cell toxicity, effectively presenting antigens and promoting adaptive immune response against tumors. Immunohistochemical results showed that the levels of tumour-infiltrating T lymphocytes and neutrophils were increased in the EcN (hIL-2)-treated group compared with the other three groups (Fig. 6 A, B). The antibody selected for T lymphocytes is CD3, the antibody selected for M1 macrophages is CD11C, and the antibody selected for neutrophils is Ly6G. The results also showed an increase in the level of M1 macrophages in the total macrophage population in the tumour microenvironment after treatment with EcN (hIL-2) (Fig. 6 C). We believe that the yellow signals in the other three groups are the original T lymphocytes, neutrophils and M1 macrophages in the mouse tumor tissues, while the EcN (hIL-2) treatment group has a large area of yellow signals, and the positive signal can be seen more clearly from the 400× partial enlarged image. This can further explain the increase in the number of T lymphocytes, neutrophils and M1 macrophages in tumor-bearing mice after EcN (hIL-2) treatment. Next, we examined the levels of two immune factors in the blood in four groups of mice. The data showed that the IFN-γ level was significantly increased and the TGF-β level was decreased in the blood of mice in the EcN (hIL-2)-treated group compared with that of mice in the other experimental groups (Fig. 6 D, E). Collectively, these data indicate that EcN (hIL-2) treatment improves the immune microenvironment of tumour-bearing mice to some extent, leading to improved survival outcomes after EcN (hIL-2) treatment. Discussion Tumour-targeted therapies and immunotherapy have raised hope for curing many malignant cancers[ 19 ]. Live tumour-targeting bacteria are a distinctive option for tumour therapy. Bacterial vectors can be reprogrammed following simple genetic rules or sophisticated synthetic bioengineering principles to produce and deliver antitumour agents based on clinical needs. Attenuated Salmonella typhimurium , Clostridium novyi, Bifidobacterium and Listeria strains have been tested in animal models and have shown preferential targeting of solid tumours, and several of these strains have advanced to clinical trials[ 20-24 ]. Various therapeutic payloads delivered by these tumour-targeting bacteria have since been developed [ 25-27 ]. However, these strains (except Bifidobacterium ) are all pathogenic bacteria, and systemic toxicity limits their clinical use. Although researchers have been focused on attenuating the virulence of these bacteria, there are many challenges. Escherichia coli Nissle 1917 (EcN) is known to be avirulent and consumed as the probiotic preparation Mutaflor, which is used for the treatment of various intestinal disorders; EcN successfully colonizes the human gut and can survive and proliferate in both hypoxic and oxygenated environments[ 28 , 29 ]. Previous studies have demonstrated that EcN has excellent performance in preferentially localizing to tumours when administered systematically or orally to different tumour-bearing mouse models[ 10 , 18 ]. EcN has also been engineered to deliver various antitumour agents[ 18 , 30 ] , [ 31 ]. IL-2 is a monomeric secreted glycoprotein with a molecular weight of 15 kDa that exerts a wide spectrum of effects on the immune system and plays crucial roles in regulating both immune activation and homeostasis[ 15 ]. It was approved for use in clinical cancer immunotherapy several years ago. However, high-dose IL-2 administration can result in severe systemic toxicity, such as malaise, fever, anasarca, jaundice, renal dysfunction and capillary leak syndrome, in many patients[ 32 , 33 ]. Researchers have attempted to diminish the adverse effects of systemically administered IL-2 by altering the dose, schedule and route of administration[ 34-38 ]. Nevertheless, the toxic effects of IL-2 therapy persist to various degrees[ 39 ]. These shortcomings have made it necessary to create a better method of using IL-2 for tumour therapy. Localized administration delivered by tumour-targeting bacteria is an appropriate option. In an attempt to establish a local delivery system for IL-2 that may diminish or prevent side effects, Denial A and colleagues used attenuated Salmonella typhimurium to produce the human IL-2 protein and significantly reduced the hepatic metastasis of colon cancer through gavage feeding of their engineered bacteria to model mice[ 40 , 41 ]. Based on the current understanding of the tumour microenvironment and recombinant DNA technology, in this study, we addressed three major questions. First, engineered EcN expressing IL-2 can target CT26 in model mice. Second, IL-2 can enhance immune responses in the tumour microenvironment. Last, improved immune responses can disturb tumour tissues and suppress tumour growth. To achieve sustained high levels of IL-2 in the tumour microenvironment while avoiding systemic toxicity, we utilized the oxygen-dependent promoter of the haemoglobin gene (vhb) of Vitreoscilla and the pelB leader sequence to facilitate IL-2 expression in the hypoxic tumour region. These engineered bacteria containing IL-2 are similar to a vaccine against tumours, and this vaccination strategy does not require knowledge of tumour antigens. Therefore, this approach may have advantages over nonimmunogenic approaches. Taken together, our data demonstrate that the tumour-targeted bacteria EcN can express soluble hIL-2, localize in solid tumours and elicit local immune responses that induce tumour suppression while avoiding systemic toxicity. This live vector system is relatively inexpensive and does not require vast laboratory resources to produce antitumour reagents. However, the clinical development of live bacteria as therapeutic agents faces substantial hurdles mainly because of potential infection-associated toxicities, especially when administered systematically. Major efforts should be made to develop proper administration routes that can minimize systemic toxicities. Employing an oral route of administration in a syngeneic, xenograft CT26 colon cancer mouse model and exploring the cellular and immunological mechanisms of how EcN(hIL-2) facilitates the inhibition of tumours are the subjects of our ongoing research. Materials And Methods Animals and cell culture All animal experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Hunan Normal University. Specific pathogen-free (SPF), male BALB/c mice were purchased from the SLRC Laboratory Animal Company (Hunan, China), and used at 6-8 weeks of age. CT26 colon carcinoma lines were maintained by our laboratory, and peripheral blood mononuclear cells (PBMCs) were purchased from Allcells Biotechnology (Shanghai) Co., Ltd. CT26 cell suspensions were seeded in a cell culture dish, and 8 mL RPMI-1640 medium containing 10% FBS was added to resuspend the cells, which were cultured at 37°C in a 5% CO 2 incubator. The cell culture medium was changed every day. PBMCs frozen in liquid nitrogen were thawed and resuspended in RPMI-1640 medium containing 10% FBS at a final concentration of 5×10 6 . The PBMCs were inoculated into 96-well plates, and different concentrations of recombinant protein were added. After 48h of incubation at 37°C in a 5% CO 2 incubator, adding 10 μL of CCK-8 solution to each well, place it in an incubator for 2 hours, and then use Microplate Reader to measure the absorbance of the cells in each well at 460mm to calculate cell viability. CT26 cells are stored in this laboratory. CCK-8 is an upgraded product of MTT (Methylthiazolyldiphenyl-tetrazolium bromide), and its working principle is that it can be reduced by dehydrogenase in mitochondria in the presence of electronic coupling reagent to produce highly water-soluble orange-yellow formazan. The color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. OD value was measured at 450nm by microplate reader, which indirectly reflected the number of living cells. Gene cloning and soluble expression of IL-2 E. coli bacteria (pINCY-IL-2) were a gift from Li Qing in Wuhan. The primers used in the study are listed in Supplementary Table 2. Prepare competent cells by CaCl 2 method. IL-2 was cloned into the BamH I and Hind III restriction sites of pET- 28a, pSmart-I (a small ubiquitin-related modifier-SUMO fusion expression system) and pSmart II (an initiation factor-IF2 protein structure domain I fusion expression system). The three plasmids (pET-28a-IL 2, pSmart-I-IL 2, and pSmart-II-IL 2) were transformed into E. coli BL21(DE3) cells, cultured at 37 °C and 220 rpm overnight, and transferred into 2% LB medium supplemented with 50 μg/ml kanamycin. Until the OD600 value reached 0.4–0.6, IPTG (final concentration of 0.4-0.5 μg/mL) was added, and incubation was performed for 120 min at 30°C. The bacteria-inducing culture solution was placed in an EP (Eppendorf) tube and centrifuged at 9000 rpm for 3 min to collect the cells. After washing twice and resuspension in a mixture of 50 mM NaH 2 PO 4 and 300 mM NaCl at pH 8.0, the cells were lysed by sonication, and the supernatants and pellets were analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) after centrifugation. The recombinant protein was cut out of the gel with a scalpel and identified by LTQ XL mass spectrometry (Thermo Fisher) after proteolysis. Construction of EcN expression strains The Vitreoscilla haemoglobin gene promoter Pvhb was amplified from pET-28a-Pvhb-pelB-asp (Lab Store). In addition, the Sumo–IL 2 fragment was amplified from pSmart-I-IL 2 (constructed in this study). Pvhb-pelB-SUMO-IL 2 was obtained by overlap extension PCR and inserted into pET-28a after digestion by Apa I and Hin d III. The sequenced vector was transformed into EcN, which was then named EcN (hIL-2). Tumour inoculation and animal studies Animals were quarantined for 1 week prior to their use in the study. For the colorectal tumour model, 1×10 5 CT26 cancer cells suspended in 100 µL PBS were injected subcutaneously into the right axillary region of BALB/c mice. After the tumour volume reached approximately 60 mm 3 , the mice were randomly divided into 4 groups (5-7 mice per group). EcN, EcN (28a), and EcN (IL 2) were activated overnight, and EcN(28a) refers to the EcN strain containing pET28a plasmid. For intraperitoneal injection of sterile PBS, EcN, EcN (28a) or EcN (IL 2) into the mice, the number of bacteria injected was 5×10 6 colony forming units (CFU)/100 μL, and the injections were performed once every 7 days, for a total of 3 injections. Then, the mice were sacrificed and analysed on the fourteenth day, and the tumour, liver, kidney, and spleen weights of the mice were measured after the end of the experiment. Tumour volumes were determined by measuring two perpendicular diameters with a calliper according to the formula volume = (a×b 2 )/2, where a is the largest dimension and b is the smallest dimension of the tumour. Body weight and tumour volume were measured every 2 days over the whole experiment. The antitumour activities of the treatments were evaluated by monitoring tumour growth inhibition. The tumour suppression percentage was calculated by the following computational formula: (control group-treatment group) / control group ×100% (with tumour volume or tumour weight used for calculations). Non-invasive in vivo imaging The bacterial distribution was monitored in injected mice. After 10 days of modelling in tumour-bearing mice, when the tumours of the mice had grown to approximately 100 mm 3 , the mice were intraperitoneally (i.p.) injected with 5×10 6 CFU/100 μL EcN (Lux) (the plasmid is preserved in our laboratory) to observe the colonization of living animals by the bacteria at different time points using an in vivo imaging system (IVIS; Calipers). The mice were anaesthetized with 2% isoflurane by using an XGI-8 gas system (Calipers). The mice were sacrificed 1 day or 7 days after the injection of EcN (Lux), and the tumour, liver, kidneys and spleen of the mice were dissected. The distribution of bacteria in each tissue was observed by IVIS. Histological morphology and the tumour microenvironment Tumours isolated from mice after sacrifice were placed in 4% paraformaldehyde overnight and then embedded in paraffin. Then, the tumours were prepared for haematoxylin and eosin staining and immunohistochemical staining assays in accordance with standard laboratory procedures. Using the principle of specific binding of antigen and antibody, the color reagent (fluorescein, enzyme, metal ion, isotope) of the labeled antibody is developed by chemical reaction to determine the antigen (polypeptide and protein) in the tissue cell, and carries out localization, qualitative and quantitative research on them, which is called immunohistochemistry. The antibody used in immunohistochemistry is Anti-His Tag Rabbit Polyclonal Antibody. The expression of IL-2 and changes in several important immune factors within the tumour microenvironment were analysed by immunohistochemical staining. The liver, spleen, and kidneys of the mice in the PBS group and EcN groups were also prepared for H&E staining to determine whether EcN has noticeable toxicity to mice. After the cell experiment and the mouse experiment, the cell culture supernatant and mouse serum were separately collected, and γ-interferon (IFN-γ) and transforming growth factor-β (TGF-β) levels were measured by ELISA. Statistics All data are expressed as the mean ± standard deviation and were analysed using IBM SPSS statistics 21.0 software. Statistical analyses were performed using an unpaired Student’s t-test. Differences with P values less than 0.05 were considered to be statistically significant, whereas P < 0.01 was considered to be very significant. Abbreviations bp Base pair CCK-8 Cell Counting Kit-8 OD Opitical Density LD linear dichroism PBS Phosphate-buffered saline PCR Polymerase chain reaction LC-MS/MS Liquid chromatography-tandem mass spectrometry EDTA Ethylene diamine tetraacetic acid SDS-PAGE SDS-poly acrylamidegel electrophoresis Tris Tris (hydroxymethyl) aminomethane kDa kiloDalton H&E Hematoxylin-eosin stainin IHC Immunohistochemistry E.coli Escherichia coli EcN E.coli Nissle 1917 IL-2 Interleukin-2 ELISA enzyme linked immunosorbent assay Declarations Acknowledgements Not applicable. Funding This work was supported by the National Natural Science Foundation of China (31770106),the National Basic Research Program (973) of China (2012CB722301), the International Cooperation Project (0102011DFA32610) and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486). Author Contributions: Conceived the research idea: BHL, LQX, HJY. Designed the experiments: BHL, HJY, LQX, YJS. Methodology: BHL, FL, XZD, HCH. Performed the experiments: BHL, HJY, HCH. Discussed the results: BHL, HJY, LQX. Analyzed the data: BHL, HJY, SBH. Wrote the manuscript: BHL,HJY. Discussed and revised the manuscript: BHL, HJY, HCH, YJS, XZD, LQX. All authors read and approved the final manuscript. Availability of data and materials All data generated or analysed during this study are included in thispublished article and its supplementary information files. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Conflict of interest The authors declare that they have no conflict of interest. Author details 1 Hunan Provincial Key Laboratory of Microbial Molecular Biology, State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha 410081, People’s Republic of China.. 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Rosenberg SA, Spiess PJ, Kleiner DE: Antitumor effects in mice of the intravenous injection of attenuated Salmonella typhimurium. Journal of immunotherapy. 2002, 25(3):218-225. Roberts NJ, Zhang L, Janku F, Collins A, Bai RY, Staedtke V et al : Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses. Science translational medicine. 2014, 6(249):249ra111. Yazawa K, Fujimori, M., Amano, J., Kano, Y., Taniguchi,S.: Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors. Cancer Gene Ther. 2000.7:269–274. Brockstedt DG, Giedlin MA, Leong ML, Bahjat KS, Gao Y, Luckett W et al : Listeria-based cancer vaccines that segregate immunogenicity from toxicity. Proceedings of the National Academy of Sciences of the United States of America. 2004, 101(38):13832-13837. Toso JF, Gill VJ, Hwu P, Marincola FM, Restifo NP, Schwartzentruber DJ et al : Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2002, 20(1):142-152. Low KB, Ittensohn M, Le T, Platt J, Sodi S, Amoss M et al : Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nature biotechnology. 1999, 17(1):37-41. Zhao M, Yang M, Li XM, Jiang P, Baranov E, Li S et al : Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proceedings of the National Academy of Sciences of the United States of America. 2005, 102(3):755-760. Loeffler M, Le'Negrate G, Krajewska M, Reed JC: Attenuated Salmonella engineered to produce human cytokine LIGHT inhibit tumor growth. Proceedings of the National Academy of Sciences of the United States of America. 2007, 104(31):12879-12883. Rembacken BJ, Snelling AM, Hawkey PM, Chalmers DM, Axon AT: Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet. 1999, 354(9179):635-639. Zhang Y, Ji W, He L, Chen Y, Ding X, Sun Y et al : E. coli Nissle 1917-Derived Minicells for Targeted Delivery of Chemotherapeutic Drug to Hypoxic Regions for Cancer Therapy. Theranostics. 2018, 8(6):1690-1705. He L, Yang H, Liu F, Chen Y, Tang J, Liu Z: Escherichia coli Nissle 1917 engineered to express Tum-5 can restrain murine melanoma growth. Oncotarget. 2017, 8(49):85772-85782. He L, Yang H, Tang J, Liu Z, Chen Y, Lu B et al : Intestinal probiotics E. coli Nissle 1917 as a targeted vehicle for delivery of p53 and Tum-5 to solid tumors for cancer therapy. Journal of biological engineering. 2019, 13:58. Klapper JA, Downey SG, Smith FO, Yang JC, Hughes MS, Kammula US et al : High-dose interleukin-2 for the treatment of metastatic renal cell carcinoma : a retrospective analysis of response and survival in patients treated in the surgery branch at the National Cancer Institute between 1986 and 2006. Cancer. 2008, 113(2):293-301. Rosalia RA, Arenas-Ramirez N, Bouchaud G, Raeber ME, Boyman O: Use of enhanced interleukin-2 formulations for improved immunotherapy against cancer. Current Opinion in Chemical Biology. 2014:23:39-46. Hu P, Mizokami M, Ruoff G, Khawli LA, Epstein AL: Generation of low-toxicity interleukin-2 fusion proteins devoid of vasopermeability activity. Blood. 2003, 101(12):4853-4861. Vazquez-Lombardi R, Loetsch C, Zinkl D, Jackson J, Schofield P, Deenick EK et al : Potent antitumour activity of interleukin-2-Fc fusion proteins requires Fc-mediated depletion of regulatory T-cells. Nature communications. 2017, 8:15373. Levin AM, Bates DL, Ring AM, Krieg C, Lin JT, Su L et al : Exploiting a natural conformational switch to engineer an interleukin-2 'superkine'. Nature. 2012, 484(7395):529-533. Lazear E, Ghasemi R, Hein SM, Westwick J, Watkins D, Fremont DH et al : Targeting of IL-2 to cytotoxic lymphocytes as an improved method of cytokine-driven immunotherapy. Oncoimmunology. 2017, 6(2):e1265721. Chen X, Ai X, Wu C, Wang H, Zeng G, Yang P et al : A novel human IL-2 mutein with minimal systemic toxicity exerts greater antitumor efficacy than wild-type IL-2. Cell death & disease. 2018, 9(10):989. Smith KA: Lowest dose interleukin-2 immunotherapy. Blood. 1993, 81(6):1414-1423. Saltzman DA, Katsanis E, Heise CP, Hasz DE, Kelly SM, Curtiss R, 3rd et al : Patterns of hepatic and splenic colonization by an attenuated strain of Salmonella typhimurium containing the gene for human interleukin-2: a novel anti-tumor agent. Cancer biotherapy & radiopharmaceuticals. 1997, 12(1):37-45. Sorenson BS, Banton KL, Frykman NL, Leonard AS, Saltzman DA: Attenuated Salmonella typhimurium with IL-2 gene reduces pulmonary metastases in murine osteosarcoma. Clinical orthopaedics and related research. 2008, 466(6):1285-1291. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Posted Version 4 posted You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-27294","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":5113747,"identity":"6d530778-e4e7-4018-8b51-71d08e5b1dae","order_by":0,"name":"Binghua Lu","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Binghua","middleName":"","lastName":"Lu","suffix":""},{"id":5113748,"identity":"8d10a393-0085-473a-af44-dbabf488e5fc","order_by":1,"name":"Huijun Yang","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huijun","middleName":"","lastName":"Yang","suffix":""},{"id":5113749,"identity":"45b9c417-05ec-4af9-8236-4253dd3fc2bc","order_by":2,"name":"Fei Liu","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Liu","suffix":""},{"id":5113750,"identity":"d3ebde4b-5445-4d93-9f46-ea594a5709b3","order_by":3,"name":"Yunjun Sun","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunjun","middleName":"","lastName":"Sun","suffix":""},{"id":5113751,"identity":"8ae7ebd0-4320-4e72-b063-0514da7101d2","order_by":4,"name":"Haocheng He","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haocheng","middleName":"","lastName":"He","suffix":""},{"id":5113752,"identity":"559abac5-085b-452f-979e-7a45d3ca493f","order_by":5,"name":"Xuezhi Ding","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuezhi","middleName":"","lastName":"Ding","suffix":""},{"id":5113753,"identity":"e88006d0-39fb-45b4-b209-8ade1f82cfe4","order_by":6,"name":"Shengbiao Hu","email":"","orcid":"","institution":"Hunan Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shengbiao","middleName":"","lastName":"Hu","suffix":""},{"id":5113754,"identity":"6c56068a-e544-49ed-9ce6-a5ed5111b1e0","order_by":7,"name":"liqiu xia","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYDACZgbGAwwVB6A8NuK0MBxgOEOSFiA4wNhGihaD48wPDryddydxu3SPAcOHssMM/LMb8GuRbGYzODh327PEnXPOGDDOOHeYQeLOAfxa+JkZDA7zbjucuOFGjgEzb9thBgOJBPxa2JjZPxzmnQPV8pcYLfzMPEBbGqBaGInRItnMU3BwzrHDxhtupBUc7DmXziNxg4AWg/PHNz54U3NYdsON5I0PfpRZy/HPIKAFDHig9AEkNpFaRsEoGAWjYBRgBQAqXUjPT9mpRAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8443-2916","institution":"Hunan Normal University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"liqiu","middleName":"","lastName":"xia","suffix":""}],"badges":[],"createdAt":"2020-05-06 23:21:34","currentVersionCode":4,"declarations":"","doi":"10.21203/rs.3.rs-27294/v4","doiUrl":"https://doi.org/10.21203/rs.3.rs-27294/v4","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":3927823,"identity":"1a8cf05d-20e5-4676-b9bc-6dc73694eb5c","added_by":"auto","created_at":"2020-12-01 18:06:47","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52159,"visible":true,"origin":"","legend":"Characterization of IL-2 expression in vitro. (A) Coomassie Brilliant Blue staining of an SDS–PAGE gel showing IL-2 expression in E. coli BL21 (DE3). (B) Map of the recombination plasmid containing the IL-2 gene. (C) SDS–PAGE analysis of IL-2 expression in EcN, EcN (28a), and EcN (hIL-2). (D) Western blot analysis of IL-2 expression in cell lysates (supernatants) or culture supernatants of EcN, EcN (28a), and EcN (hIL-2); In the figure Sup stands for supernatants.","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27294/v4/df565815855fad6b8bf72b74.jpg"},{"id":3927825,"identity":"5960f7f3-90d1-4be4-aaeb-e6a8bdc2716a","added_by":"auto","created_at":"2020-12-01 18:06:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":61854,"visible":true,"origin":"","legend":"SUMO-IL-2 protein promotes the proliferation of PBMCs. The SUMO-IL-2 protein was divided into 5 different concentrations and incubated with PBMCs for 24 h before cell viability was detected. (A) A CCK-8 kit was used to detect cell viability after the incubation (PI: proliferation index; C: concentration). ELISA was used to detect the concentrations of IFN-γ (B) and TGF-β (B) in the culture supernatant. Different letters indicate significant differences (P \u003c0.05); the same letters indicate no significant difference.","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27294/v4/8f00ad6a6495f613f6e287f0.jpg"},{"id":3927826,"identity":"27d74f6e-65fc-4226-9dff-270e3396dfbc","added_by":"auto","created_at":"2020-12-01 18:06:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39339,"visible":true,"origin":"","legend":"Colonization of a CT26 tumour model mice with EcN. CT26 cancer cells were inoculated subcutaneously in the right axilla of BALB/c mice, and 7 days later, the mice were i.p. injected with 5×106 CFU/100 μL EcN (Lux). (A) An IVIS was used to detect the distribution of EcN in tumour-bearing mice at different time points. (B) Liver, kidneys and spleen of mice in the two groups of mice were isolated and observed using the IVIS.","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27294/v4/cff98c9f124b10a605b181c2.jpg"},{"id":3927827,"identity":"dfe356d4-6ad3-4617-bb0b-153838d60faf","added_by":"auto","created_at":"2020-12-01 18:06:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75138,"visible":true,"origin":"","legend":"Stable expression of EcN (hIL-2) in tumour regions and its inhibition of CT26 colon tumours. (A) Successful expression of IL-2 in tumours was verified by IHC (200×). The red arrow refers to positive staining (400×). The therapeutic effect of EcN (hIL-2) on CT26 tumour-bearing mice was examined. (B) PBS, EcN, EcN (28a) or EcN (hIL-2) was injected i.p. into mice (bacterial injections contained 5×106 CFU/100 μL), and tumour volumes (cm 3) were estimated using external callipers (values are expressed as the mean ± standard deviation [SD]). (C) Tumours in the EcN (hIL-2)-treated group were significantly smaller than those in the other three groups. After the mice were sacrificed, their tumours were dissected and weighed; * P \u003c0.05, ** P \u003c0.01.","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27294/v4/410c6a248f17d16aaf912b98.jpg"},{"id":3927828,"identity":"e39297e7-73c0-48f5-9e48-1960aff7a4a4","added_by":"auto","created_at":"2020-12-01 18:06:48","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101957,"visible":true,"origin":"","legend":"H\u0026E staining of tumour tissue sections and the effect of EcN on the liver, kidneys, spleen and body weight of mice. (A) H\u0026E staining (200×) of tumour tissue sections was used to observe an invasive inflammatory phenomenon in tumour tissue. H\u0026E staining (200×) of the liver, kidneys and spleen was used to observe the pathological morphology of the biopsies. (B) After mice in the four groups were sacrificed, the liver, kidneys and spleen of the mice were weighed (shown as the mean and SD). (C) Mouse body weights were recorded every two days during the experiment. No significant differences were observed among the four groups of mice.","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27294/v4/fe1d735019b1764ddd8ebe95.jpg"},{"id":3927829,"identity":"4f134c9b-2dd3-4c0c-a2f7-24149765ed1d","added_by":"auto","created_at":"2020-12-01 18:06:48","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":115908,"visible":true,"origin":"","legend":"Immune status change in the tumour microenvironment post-bacterial treatments. In total, 1×105 CT26 cancer cells were inoculated subcutaneously into the right axilla of BALB/c mice, which were then treated with PBS, EcN, EcN (28a) or EcN (hIL-2) at 7 days post-tumour inoculation. At the end of the experiment, the mice were sacrificed, and tumours were collected to determine T lymphocyte (A), neutrophil (B) and M1 macrophage (C) levels by immunohistochemistry. The red arrow refers to positive staining (400×). Blood was collected from mice, and the tumour microenvironment was assessed by measuring INF-γ and TGF-β levels in the serum by ELISA (D, E); * P \u003c 0.05.","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-27294/v4/b705a27b3d44e77af0b94a08.jpg"},{"id":15671370,"identity":"60890fa8-56e4-4d19-ac2c-968585c1ac34","added_by":"auto","created_at":"2021-11-18 14:05:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":647440,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-27294/v4/2ae77843-017f-44e4-a979-23e7df639156.pdf"},{"id":3927824,"identity":"c11a3774-b285-482b-8dd9-5289d1a05a83","added_by":"auto","created_at":"2020-12-01 18:06:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4109579,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-27294/v4/9f788565900828f10f3c72af.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eEscherichia coli \u003c/em\u003eNissle 1917 secreting functional interleukin 2 targets tumours and enhances the immune response to suppress tumours\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImmune system function is often inhibited in the microenvironment of solid tumours[\u003ca href=\"#_ENREF_1\"\u003e1-5\u003c/a\u003e]. Therefore, many attempts are being made to activate immune responses against tumours[\u003ca href=\"#_ENREF_6\"\u003e6\u003c/a\u003e]. Tumour-targeted therapy is a promising treatment method for many malignant tumours. Colorectal cancer is one of the most common malignant tumors. The global incidence rate of malignant tumors ranks third, and the case fatality rate ranks second. The incidence of colorectal cancer in the world is increasing year by year[\u003ca href=\"#_ENREF_7\"\u003e7\u003c/a\u003e]. Among the probiotics used in research in recent years, \u003cem\u003eE. coli Nissle\u003c/em\u003e 1917 (EcN) is currently the only Gram-negative bacteria in use, and it is also one of the most widely studied probiotics in the world[\u003ca href=\"#_ENREF_8\"\u003e8\u003c/a\u003e]. EcN is an ideal carrier for vaccines, cytokines and other substances because of its innocuity and versatility in bioengineering. For example, genetically engineered EcN can be used to secrete cystatin, a nematode immunomodulator, in the intestinal tract to treat experimental colitis in mice or pigs[\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e]. Jochen et al injected EcN intravenously (i.v.), intraperitoneally (i.p.), or intratumorally (i.t.) into tumor-bearing mice to study its specific targeting property. They found that massive EcN colonized and replicated in tumors and no obvious difference was observed in the CFU/g isolated from organ tissues, regardless of inoculation route[\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e]. However, the development of targeted drugs has significantly improved the overall prognosis of patients with colorectal cancer, so we hope to use EcN as a delivery vehicle to treat colorectal cancer.\u003c/p\u003e\n\u003cp\u003eThe immunoregulatory cytokine interleukin-2 (IL-2) is a growth and activating factor for a variety of immune cells, including T cells and NK cells[\u003ca href=\"#_ENREF_11\"\u003e11\u003c/a\u003e]. The cytokine IL-2 is an effective T cell mitogen and activator, which can expand the function of T cells，maintain the proliferation of T lymphovitro for a long time, stimulate T cells to enter the cell division cycle，and increase the immune clearance rate of tumors in the immunosuppressed tumor microenvironment[\u003ca href=\"#_ENREF_12\"\u003e12\u003c/a\u003e]. Meanwhile, IL-2 can promote the production of NK or T cell-derived cytokines, such as TNF-\u0026alpha;, IFN-\u0026gamma; and GM-CSF, which can boost antitumour immunity, and these molecules have synergistic effects[\u003ca href=\"#_ENREF_13\"\u003e13-15\u003c/a\u003e]. Therefore, we hypothesized that tumour-targeting bacteria expressing the immune-activating cytokine IL-2 may optimally modify the immune microenvironment and improve antitumour effects.\u003c/p\u003e\n\u003cp\u003eIn this study, EcN bacteria were engineered to express soluble human IL-2, with the aim of improving the immune function of tumour-bearing mice and inhibiting the growth of tumours. To exploit the role of IL-2 delivered by EcN in tumour tissue, CT26 colon cancer cells were implanted subcutaneously in syngeneic BALB/c mice, and after the tumour was established, EcN(hIL-2) was injected intraperitoneally into the tumour-bearing mouse. Immunohistochemical results showed that EcN(hIL-2) specifically localized in the tumour region and that IL-2 was released in the tumour tissue. Tumour growth in the EcN(hIL-2) group was inhibited approximately 53.91% compared with that in the PBS control group. A myriad of necrotic tumour cells could be observed in the tumour tissue of the EcN (hIL-2)-treated group. To assess the toxicity induced by EcN (hIL-2), we measured the body weight of tumour-bearing mice every two days. These mice were sacrificed after 7 days, and the liver, kidneys and the spleen of the mice in each group were excised and weighed. There was no difference in either body or organ weight between the experimental and control groups. We also investigated the distribution of engineered bacteria in the liver, kidneys and the spleen of tumour-bearing mice using an IVIS spectrum[\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e, \u003ca href=\"#_ENREF_17\"\u003e17\u003c/a\u003e] and found that the numbers of bacteria in these organs were significantly lower than those in tumours.\u003c/p\u003e\n\u003cp\u003eTo further explore the antitumour mechanism of these engineered bacteria, local immune responses induced by EcN(hIL-2) were also detected by H\u0026amp;E staining of tissue sections. Immune cells, such as T lymphocytes, neutrophils and macrophages, infiltrated the tumour microenvironment, but the control groups had insufficient immune cell infiltration. We also examined the cytokines IFN-\u0026gamma; and TGF-\u0026beta; in the serum, as IFN-\u0026gamma; is a known key mediator of IL-2 toxicity and TGF-\u0026beta; is an inhibitory factor. The results showed that EcN(hIL-2) treatment was associated with significantly elevated IFN-\u0026gamma; expression and decreased TGF-\u0026beta; expression.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eExpression analysis of the IL-2 protein in vitro\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo allow expression of the IL-2 protein in a prokaryotic system, the IL-2 gene was cloned into three different inducible expression vectors (Supplementary Fig. 1 A, 1 B and 1 C). The differential proteins expressed by the three recombinant plasmids in\u003cem\u003e E. coli\u003c/em\u003e BL21 (DE3) were verified by Western blotting and mass spectrometry, which proved that the target protein IL-2 was successfully expressed (Supplementary Fig.2 and Table S1). Coomassie Brilliant Blue staining showed that the recombinant protein showed soluble expression under the action of SUMO and IF2 tags (Fig. 1 A). Engineered bacteria need to continuously secrete recombinant proteins inside a tumour, induce immune cell activation, attack tumour cells, and inhibit tumour growth. Therefore, the IL-2 protein can be continuously expressed in hypoxic tumours under the oxygen-dependent promoter of the haemoglobin gene (vhb) of Vitreoscilla and the pelB leader sequence (Fig. 1B and Supplementary Fig. 3). Engineered bacteria were cultured overnight in LB medium, and the recombinant protein was successfully expressed in engineered EcN bacteria, as verified by SDS-PAGE analysis (Fig. 1 C). Western blot analysis indicated that the IL-2 protein was presented in both the cell lysate and medium supernatant of EcN (hIL-2) (Fig. 1 D). To reduce the effect of tags on IL-2 protein activity, a SUMO fusion system with a relatively low molecular weight was selected for subsequent experimental research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSUMO-IL-2 protein can promote the proliferation of \u003c/strong\u003e\u003cstrong\u003ePBMCs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBL21 (pSmartI-IL 2) bacteria were cultured, and IL-2 protein was collected by Ni-NTA Sefinose (TM) Resin Kit. The IL-2 protein at different concentrations was cocultured with peripheral blood mononuclear cells (PBMCs), and PBS was added to the control group. PBMCs were cocultured with the recombinant protein for 24 h, and then Cell Counting Kit-8 was added to detect the cell survival rate. The results showed that cell proliferation was obviously promoted after the addition of SUMO-IL-2 (Sumo is the solubilizing label on pSmartI), and the proliferation rate of cells also increased as the SUMO-IL-2 protein concentration increased (Fig. 2 A). Additionally, the cell culture medium was centrifuged after the incubation, and the culture supernatant was collected. The concentrations of IFN-\u0026gamma; and TGF-\u0026beta; in the supernatant of the culture medium were detected by enzyme-linked immunosorbent assay (ELISA). The results showed that compared with that in the supernatant of the control group, the concentration of IFN-\u0026gamma; in the supernatant of the experimental group was significantly increased (Fig. 2 B), while the concentration of TGF-\u0026beta; was significantly decreased (Fig. 2 C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEcN specifically colonizes tumour regions in tumour-bearing mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn IVIS can accurately observe the real-time location of bacteria in animals without causing damage to the animals[\u003ca href=\"#_ENREF_16\"\u003e16\u003c/a\u003e, \u003ca href=\"#_ENREF_17\"\u003e17\u003c/a\u003e]. After intraperitoneal injection of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/100 \u0026micro;L EcN(Lux)[\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e] into tumour-bearing mice in the experimental group and injection of sterile PBS into tumour-bearing mice in the control group, bacterial colonization in the mice was observed by an IVIS. The results showed that the tumour-bearing mice exhibited a significant fluorescence signal in the tumour area for 5 days after the bacteria were injected, and the fluorescence signal was still observed on the 7th day after injection (Fig. 3 A). The control group did not exhibit a detectable signal. After the mice were euthanized on the 7th day, the tumour, liver, kidneys and spleen of the mice were obtained. IVIS analysis showed that 5 days after EcN(Lux) was intraperitoneally injected into tumour-bearing mice, a strong fluorescence signal was detected in the tumour tissues of the mice, and no fluorescence signal was detected in other organs (Fig. 3 B). These results showed that EcN has excellent targeting to the tumours in CT26 tumour-bearing mice. Bacteria can quickly accumulate in the tumour area and grow and reproduce in the tumour area after intraperitoneal injection into mice, while bacteria in other parts of mice can be removed by the local immune response quickly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntitumour effect of EcN(hIL-2)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the successful expression of the IL-2 molecule carried by EcN in tumour areas, we performed immunohistochemistry on samples from each group of mouse tumours. Mice were euthanized on the 7th day after the third administration, and the tumour tissue was removed, fixed in 4% paraformaldehyde, and then embedded in paraffin. These results showed that a yellow-grey signal appeared in the tumour tissue sections of the EcN (hIL-2) experimental group, while those of the other three groups did not show a positive signal (Fig. 4 A). These results indicate that IL-2 is successfully expressed in the tumour region. The antibody used in immunohistochemistry is Anti-His Tag Rabbit Polyclonal Antibody.\u003c/p\u003e\n\u003cp\u003eTo evaluate the antitumour efficacy of EcN (IL-2), we subcutaneously injected CT26 colon cancer cells into the right axillary area of BALB/c mice. The resultant xenograft tumour model was used to study the antitumour effect of EcN (hIL-2). When the tumours in the mice grew to approximately 60 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly divided into 4 groups (n = 5, 6, or 7), and the groups were treated respectively by intraperitoneal injection of sterile PBS, EcN, EcN (28a) or EcN (hIL-2). Body weight and tumour volume were measured every two days during the observation period until the animals were sacrificed. The results of the experiment showed that the tumour growth in the EcN (hIL-2) group was significantly inhibited (Fig. 4 B), while the xenograft tumour growth in the other three groups have no difference. The final tumour weight of the EcN (hIL-2) group was also significantly lower than that of the other 3 groups (Fig. 4 C). The tumour volume in the PBS group reached 4.93 \u0026plusmn;1.35 cm\u003csup\u003e3\u003c/sup\u003e, but the tumour volume in the EcN (hIL-2) group was significantly smaller, reaching a volume of 2.32 \u0026plusmn; 1.43 mm\u003csup\u003e3\u003c/sup\u003e. Tumour volume was calculated according to the formula (Table 1). Tumour growth in the EcN (hIL-2) group was inhibited approximately 53.91% compared to that in the PBS group.\u003c/p\u003e\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:0in;text-align:center;text-indent:0in;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003eTable 1: The comparison of tumor volume, tumor weight of CT26 cancer of the\u0026nbsp;\u003c/span\u003e\u003cspan style=\"font-size:15px;\"\u003eBALB/c\u003c/span\u003e\u003cspan style=\"font-size:15px;\"\u003e\u0026nbsp;mice\u003c/span\u003e\u003c/p\u003e\n\u003cdiv align=\"center\" style='margin:0in;text-align:justify;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\n \u003ctable style=\"width: 4.1e+2pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:113.4pt;border-top:solid black 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:22.6pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003eGroup\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:2.25in;border-top:solid black 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:22.6pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003eMean Tumor Volume (cm\u003csup\u003e3\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:138.55pt;border-top:solid black 1.5pt;border-left:none;border-bottom:solid black 1.0pt;border-right:none;padding:0in 5.4pt 0in 5.4pt;height:22.6pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003eMean Tumor Weight (g)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:113.4pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:11.15pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003ePBS\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:2.25in;border:none;padding:0in 5.4pt 0in 5.4pt;height:11.15pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4.93 \u0026plusmn;1.35\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:138.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:11.15pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003e6.41 \u0026plusmn; 1.22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:113.4pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:16.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003eEcN\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:2.25in;border:none;padding:0in 5.4pt 0in 5.4pt;height:16.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003e5.53 \u0026plusmn; 1.43\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:138.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:16.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003e6.74 \u0026plusmn; 1.35\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:113.4pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:16.7pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003eEcN (28a)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:2.25in;border:none;padding:0in 5.4pt 0in 5.4pt;height:16.7pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003e4.62 \u0026plusmn; 1.67\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:138.55pt;border:none;padding:0in 5.4pt 0in 5.4pt;height:16.7pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003e6.38 \u0026plusmn; 0.91\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:113.4pt;border:none;border-bottom:solid black 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:16.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003eEcN (IL-2)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:2.25in;border:none;border-bottom:solid black 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:16.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003e2.32 \u0026plusmn; 1.43 (53.91%)\u003csup\u003e**\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:138.55pt;border:none;border-bottom:solid black 1.5pt;padding:0in 5.4pt 0in 5.4pt;height:16.5pt;\"\u003e\n \u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003e3.48 \u0026plusmn; 1.49 (45.71%)\u003csup\u003e*\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp style='margin-top:0in;margin-right:0in;margin-bottom:0in;margin-left:21.0pt;text-align:left;text-indent:22.0pt;font-size:14px;font-family:\"Calibri\",sans-serif;'\u003e\u003cspan style=\"font-size:15px;\"\u003eNotes: *P\u0026lt; 0.05, ** P \u0026lt; 0.01.\u003c/span\u003e\u003c/p\u003e\u003cbr\u003e\u003cp\u003e\u003cstrong\u003eTumour histomorphology and safety monitoring of \u003cem\u003eE. coli\u003c/em\u003e Nissle 1917\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHaematoxylin-eosin staining (H\u0026amp;E staining) is one of the commonly used staining methods for paraffin sections. Haematoxylin dyeing solutions are alkaline, mainly causing chromatin in the nucleus and nucleic acid in the cytoplasm to be stained purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and the extracellular matrix red. Our experimental results showed that the tumour staining results in the PBS group showed normal tumour cell morphology, and no necrotic areas were observed. However, infiltrating inflammatory cells were observed in the EcN experimental group, and the cell morphology was irregular, the phenomenon that inflammatory cells gather in the inflammatory focus. (Fig. 5 A).\u003c/p\u003e\n\u003cp\u003eAdditionally, we also performed H\u0026amp;E staining of liver, kidney and spleen tissues from the PBS group and EcN experimental group. The results showed that there was no significant change in histopathological morphology in the liver, kidneys or spleen between the two groups, indicating that EcN had no obvious side effects on the liver, kidneys or spleen in mice (Fig. 5 A). During the experiment, to assess the systemic effects of EcN on the whole body after intraperitoneal injection, we measured mouse body weight every two days. At the end of the experiment, the liver, kidneys and spleen of the mice in each group were excised and weighed, and there were no differences in weight among the four groups (Fig. 5 B). Although the mice exhibited a slight decrease in body weight during treatment, they all approached the same weight by the end of the experiment (Fig. 5 C). All of the above results demonstrate that the toxicity of intraperitoneal administration of EcN to mice is negligible.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExamining the tumour microenvironment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe explored the mechanism underlying the antitumour immune activity of EcN (hIL-2) in a CT26 colon cancer tumour model. We investigated the immune cell profile in the tumour microenvironment and the changes in cytokines in the blood using the CT26 tumour model. We selected specific antibodies to be combined with antigens on the surface of T lymphocytes, neutrophils and M1 macrophages, and compared the changes in the content of these three cells in the four groups of tumors by immunohistochemistry. Type M1 is a classically activated macrophage (classically activated macrophage), which refers to macrophages that exist in an inflammatory environment, and is affected by gamma interferon, tumor necrosis factor alpha (TNF alpha) and granulocyte macrophage colony stimulating factor (GMCSF) Induction, it can induce an immune response of type I helper T cells (Th1), has the ability to promote inflammation, and plays a very important role in killing bacteria and viruses in the cells. Its characteristics in tumor tissues are mainly manifested as having tumor cell toxicity, effectively presenting antigens and promoting adaptive immune response against tumors. Immunohistochemical results showed that the levels of tumour-infiltrating T lymphocytes and neutrophils were increased in the EcN (hIL-2)-treated group compared with the other three groups (Fig. 6 A, B). The antibody selected for T lymphocytes is CD3, the antibody selected for M1 macrophages is CD11C, and the antibody selected for neutrophils is Ly6G. The results also showed an increase in the level of M1 macrophages in the total macrophage population in the tumour microenvironment after treatment with EcN (hIL-2) (Fig. 6 C). We believe that the yellow signals in the other three groups are the original T lymphocytes, neutrophils and M1 macrophages in the mouse tumor tissues, while the EcN (hIL-2) treatment group has a large area of yellow signals, and the positive signal can be seen more clearly from the 400\u0026times; partial enlarged image. \u0026nbsp;This can further explain the increase in the number of T lymphocytes, neutrophils and M1 macrophages in tumor-bearing mice after EcN (hIL-2) treatment. Next, we examined the levels of two immune factors in the blood in four groups of mice. The data showed that the IFN-\u0026gamma; level was significantly increased and the TGF-\u0026beta; level was decreased in the blood of mice in the EcN (hIL-2)-treated group compared with that of mice in the other experimental groups (Fig. 6 D, E). Collectively, these data indicate that EcN (hIL-2) treatment improves the immune microenvironment of tumour-bearing mice to some extent, leading to improved survival outcomes after EcN (hIL-2) treatment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTumour-targeted therapies and immunotherapy have raised hope for curing many malignant cancers[\u003ca href=\"#_ENREF_19\"\u003e19\u003c/a\u003e]. Live tumour-targeting bacteria are a distinctive option for tumour therapy. Bacterial vectors can be reprogrammed following simple genetic rules or sophisticated synthetic bioengineering principles to produce and deliver antitumour agents based on clinical needs. Attenuated \u003cem\u003eSalmonella typhimurium\u003c/em\u003e\u003cem\u003e, Clostridium \u003c/em\u003e\u003cem\u003enovyi, Bifidobacterium \u003c/em\u003eand \u003cem\u003eListeria\u003c/em\u003e strains have been tested in animal models and have shown preferential targeting of solid tumours, and several of these strains have advanced to clinical trials[\u003ca href=\"#_ENREF_20\"\u003e20-24\u003c/a\u003e]. Various therapeutic payloads delivered by these tumour-targeting bacteria have since been developed [\u003ca href=\"#_ENREF_25\"\u003e25-27\u003c/a\u003e]. However, these strains (except \u003cem\u003eBifidobacterium\u003c/em\u003e) are all pathogenic bacteria, and systemic toxicity limits their clinical use. Although researchers have been focused on attenuating the virulence of these bacteria, there are many challenges.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u0026nbsp;Nissle\u003cem\u003e\u0026nbsp;1917 \u003c/em\u003e(EcN) is known to be avirulent and consumed as the probiotic preparation Mutaflor, which is used for the treatment of various intestinal disorders; EcN successfully colonizes the human gut and can survive and proliferate in both hypoxic and oxygenated environments[\u003ca href=\"#_ENREF_28\"\u003e28\u003c/a\u003e, \u003ca href=\"#_ENREF_29\"\u003e29\u003c/a\u003e]. Previous studies have demonstrated that EcN has excellent performance in preferentially localizing to tumours when administered systematically or orally to different tumour-bearing mouse models[\u003ca href=\"#_ENREF_10\"\u003e10\u003c/a\u003e, \u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e]. EcN has also been engineered to deliver various antitumour agents[\u003ca href=\"#_ENREF_18\"\u003e18\u003c/a\u003e, \u003ca href=\"#_ENREF_30\"\u003e30\u003c/a\u003e]\u003csup\u003e,\u003c/sup\u003e[\u003ca href=\"#_ENREF_31\"\u003e31\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eIL-2 is a monomeric secreted glycoprotein with a molecular weight of 15 kDa that exerts a wide spectrum of effects on the immune system and plays crucial roles in regulating both immune activation and homeostasis[\u003ca href=\"#_ENREF_15\"\u003e15\u003c/a\u003e]. It was approved for use in clinical cancer immunotherapy several years ago. However, high-dose IL-2 administration can result in severe systemic toxicity, such as malaise, fever, anasarca, jaundice, renal dysfunction and capillary leak syndrome, in many patients[\u003ca href=\"#_ENREF_32\"\u003e32\u003c/a\u003e, \u003ca href=\"#_ENREF_33\"\u003e33\u003c/a\u003e]. Researchers have attempted to diminish the adverse effects of systemically administered IL-2 by altering the dose, schedule and route of administration[\u003ca href=\"#_ENREF_34\"\u003e34-38\u003c/a\u003e]. Nevertheless, the toxic effects of IL-2 therapy persist to various degrees[\u003ca href=\"#_ENREF_39\"\u003e39\u003c/a\u003e]. These shortcomings have made it necessary to create a better method of using IL-2 for tumour therapy. Localized administration delivered by tumour-targeting bacteria is an appropriate option. In an attempt to establish a local delivery system for IL-2 that may diminish or prevent side effects, Denial A and colleagues used attenuated \u003cem\u003eSalmonella typhimurium\u003c/em\u003e to produce the human IL-2 protein and significantly reduced the hepatic metastasis of colon cancer through gavage feeding of their engineered bacteria to model mice[\u003ca href=\"#_ENREF_40\"\u003e40\u003c/a\u003e, \u003ca href=\"#_ENREF_41\"\u003e41\u003c/a\u003e].\u003c/p\u003e\n\u003cp\u003eBased on the current understanding of the tumour microenvironment and recombinant DNA technology, in this study, we addressed three major questions. First, engineered EcN expressing IL-2 can target CT26 in model mice. Second, IL-2 can enhance immune responses in the tumour microenvironment. Last, improved immune responses can disturb tumour tissues and suppress tumour growth. To achieve sustained high levels of IL-2 in the tumour microenvironment while avoiding systemic toxicity, we utilized the oxygen-dependent promoter of the haemoglobin gene (vhb) of \u003cem\u003eVitreoscilla\u003c/em\u003e and the pelB leader sequence to facilitate IL-2 expression in the hypoxic tumour region. These engineered bacteria containing IL-2 are similar to a vaccine against tumours, and this vaccination strategy does not require knowledge of tumour antigens. Therefore, this approach may have advantages over nonimmunogenic approaches.\u003c/p\u003e\n\u003cp\u003eTaken together, our data demonstrate that the tumour-targeted bacteria EcN can express soluble hIL-2, localize in solid tumours and elicit local immune responses that induce tumour suppression while avoiding systemic toxicity. This live vector system is relatively inexpensive and does not require vast laboratory resources to produce antitumour reagents. However, the clinical development of live bacteria as therapeutic agents faces substantial hurdles mainly because of potential infection-associated toxicities, especially when administered systematically. Major efforts should be made to develop proper administration routes that can minimize systemic toxicities. Employing an oral route of administration in a syngeneic, xenograft CT26 colon cancer mouse model and exploring the cellular and immunological mechanisms of how EcN(hIL-2) facilitates the inhibition of tumours are the subjects of our ongoing research.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals and cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Hunan Normal University. Specific pathogen-free (SPF), male BALB/c mice were purchased from the SLRC Laboratory Animal Company (Hunan, China), and used at 6-8 weeks of age. CT26 colon carcinoma lines were maintained by our laboratory, and peripheral blood mononuclear cells (PBMCs) were purchased from Allcells Biotechnology (Shanghai) Co., Ltd.\u003c/p\u003e\n\u003cp\u003eCT26 cell suspensions were seeded in a cell culture dish, and 8 mL RPMI-1640 medium containing 10% FBS was added to resuspend the cells, which were cultured at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. The cell culture medium was changed every day. PBMCs frozen in liquid nitrogen were thawed and resuspended in RPMI-1640 medium containing 10% FBS at a final concentration of 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e. The PBMCs were inoculated into 96-well plates, and different concentrations of recombinant protein were added. After 48h of incubation at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator, adding 10 \u0026mu;L of CCK-8 solution to each well, place it in an incubator for 2 hours, and then use Microplate Reader to measure the absorbance of the cells in each well at 460mm to calculate cell viability. CT26 cells are stored in this laboratory. CCK-8 is an upgraded product of MTT (Methylthiazolyldiphenyl-tetrazolium bromide), and its working principle is that it can be reduced by dehydrogenase in mitochondria in the presence of electronic coupling reagent to produce highly water-soluble orange-yellow formazan. The color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. OD value was measured at 450nm by microplate reader, which indirectly reflected the number of living cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene cloning and soluble expression of IL-2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e bacteria (pINCY-IL-2) were a gift from Li Qing in Wuhan. The primers used in the study are listed in Supplementary Table 2. Prepare competent cells by CaCl\u003csub\u003e2\u003c/sub\u003e method. IL-2 was cloned into the BamH I and Hind III restriction sites of pET- 28a, pSmart-I (a small ubiquitin-related modifier-SUMO fusion expression system) and pSmart II (an initiation factor-IF2 protein structure domain I fusion expression system). The three plasmids (pET-28a-IL 2, pSmart-I-IL 2, and pSmart-II-IL 2) were transformed into \u003cem\u003eE. coli\u003c/em\u003e BL21(DE3) cells, cultured at 37 \u0026deg;C and 220 rpm overnight, and transferred into 2% LB medium supplemented with 50 \u0026mu;g/ml kanamycin. Until the OD600 value reached 0.4\u0026ndash;0.6, IPTG (final concentration of 0.4-0.5 \u0026mu;g/mL) was added, and incubation was performed for 120 min at 30\u0026deg;C. The bacteria-inducing culture solution was placed in an EP (Eppendorf) tube and centrifuged at 9000 rpm for 3 min to collect the cells. After washing twice and resuspension in a mixture of 50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and 300 mM NaCl at pH 8.0, the cells were lysed by sonication, and the supernatants and pellets were analysed by sodium dodecyl sulfate\u0026ndash;polyacrylamide gel electrophoresis (SDS\u0026ndash;PAGE) after centrifugation. The recombinant protein was cut out of the gel with a scalpel and identified by LTQ XL mass spectrometry (Thermo Fisher) after proteolysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of EcN expression strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Vitreoscilla haemoglobin gene promoter Pvhb was amplified from pET-28a-Pvhb-pelB-asp (Lab Store). In addition, the Sumo\u0026ndash;IL 2 fragment was amplified from pSmart-I-IL 2 (constructed in this study). Pvhb-pelB-SUMO-IL 2 was obtained by overlap extension PCR and inserted into pET-28a after digestion by \u003cem\u003eApa\u003c/em\u003e I and \u003cem\u003eHin\u003c/em\u003ed III. The sequenced vector was transformed into EcN, which was then named EcN (hIL-2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTumour inoculation and animal studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were quarantined for 1 week prior to their use in the study. For the colorectal tumour model, 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e CT26 cancer cells suspended in 100 \u0026micro;L PBS were injected subcutaneously into the right axillary region of BALB/c mice.\u003c/p\u003e\n\u003cp\u003eAfter the tumour volume reached approximately 60 mm\u003csup\u003e3\u003c/sup\u003e, the mice were randomly divided into 4 groups (5-7 mice per group). EcN, EcN (28a), and EcN (IL 2) were activated overnight, and EcN(28a) refers to the EcN strain containing pET28a plasmid. For intraperitoneal injection of sterile PBS, EcN, EcN (28a) or EcN (IL 2) into the mice, the number of bacteria injected was 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e colony forming units (CFU)/100 \u0026mu;L, and the injections were performed once every 7 days, for a total of 3 injections. Then, the mice were sacrificed and analysed on the fourteenth day, and the tumour, liver, kidney, and spleen weights of the mice were measured after the end of the experiment. Tumour volumes were determined by measuring two perpendicular diameters with a calliper according to the formula volume = (a\u0026times;b\u003csup\u003e2\u003c/sup\u003e)/2, where a is the largest dimension and b is the smallest dimension of the tumour. Body weight and tumour volume were measured every 2 days over the whole experiment. The antitumour activities of the treatments were evaluated by monitoring tumour growth inhibition. The tumour suppression percentage was calculated by the following computational formula: (control group-treatment group) / control group \u0026times;100% (with tumour volume or tumour weight used for calculations).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNon-invasive in vivo imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe bacterial distribution was monitored in injected mice. After 10 days of modelling in tumour-bearing mice, when the tumours of the mice had grown to approximately 100 mm\u003csup\u003e3\u003c/sup\u003e, the mice were intraperitoneally (i.p.) injected with 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e CFU/100 \u0026mu;L EcN (Lux) (the plasmid is preserved in our laboratory) to observe the colonization of living animals by the bacteria at different time points using an in vivo imaging system (IVIS; Calipers). The mice were anaesthetized with 2% isoflurane by using an XGI-8 gas system (Calipers). The mice were sacrificed 1 day or 7 days after the injection of EcN (Lux), and the tumour, liver, kidneys and spleen of the mice were dissected. The distribution of bacteria in each tissue was observed by IVIS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological morphology and the tumour microenvironment \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTumours isolated from mice after sacrifice were placed in 4% paraformaldehyde overnight and then embedded in paraffin. Then, the tumours were prepared for haematoxylin and eosin staining and immunohistochemical staining assays in accordance with standard laboratory procedures. Using the principle of specific binding of antigen and antibody, the color reagent (fluorescein, enzyme, metal ion, isotope) of the labeled antibody is developed by chemical reaction to determine the antigen (polypeptide and protein) in the tissue cell, and carries out localization, qualitative and quantitative research on them, which is called immunohistochemistry. The antibody used in immunohistochemistry is Anti-His Tag Rabbit Polyclonal Antibody.\u003c/p\u003e\n\u003cp\u003eThe expression of IL-2 and changes in several important immune factors within the tumour microenvironment were analysed by immunohistochemical staining. The liver, spleen, and kidneys of the mice in the PBS group and EcN groups were also prepared for H\u0026amp;E staining to determine whether EcN has noticeable toxicity to mice.\u003c/p\u003e\n\u003cp\u003eAfter the cell experiment and the mouse experiment, the cell culture supernatant and mouse serum were separately collected, and \u0026gamma;-interferon (IFN-\u0026gamma;) and transforming growth factor-\u0026beta; (TGF-\u0026beta;) levels were measured by ELISA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are expressed as the mean \u0026plusmn; standard deviation and were analysed using IBM SPSS statistics 21.0 software. Statistical analyses were performed using an unpaired Student\u0026rsquo;s t-test. Differences with P values less than 0.05 were considered to be statistically significant, whereas P \u0026lt; 0.01 was considered to be very significant.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003ebp\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eBase pair\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eCCK-8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eCell Counting Kit-8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eOD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eOpitical Density\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eLD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003elinear dichroism\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003ePBS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003ePhosphate-buffered saline\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003ePCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003ePolymerase chain reaction\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eLC-MS/MS\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eLiquid chromatography-tandem mass spectrometry\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eEDTA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eEthylene diamine tetraacetic acid\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eSDS-PAGE\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eSDS-poly acrylamidegel electrophoresis\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eTris\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eTris (hydroxymethyl) aminomethane\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003ekDa\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003ekiloDalton\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eH\u0026amp;E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eHematoxylin-eosin stainin\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eIHC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eImmunohistochemistry\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003e\u003cem\u003eE.coli\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eEcN\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003e\u003cem\u003eE.coli \u003c/em\u003eNissle 1917\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eIL-2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eInterleukin-2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"64\"\u003e\n\u003cp\u003eELISA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"489\"\u003e\n\u003cp\u003eenzyme linked immunosorbent assay\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31770106),the National Basic Research Program (973) of China (2012CB722301), the International Cooperation Project (0102011DFA32610) and the Cooperative Innovation Center of Engineering and New Products for Developmental Biology of Hunan Province (20134486).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceived the research idea: BHL, LQX, HJY. Designed the experiments: BHL, HJY, LQX, YJS. Methodology: BHL, FL, XZD, HCH. Performed the experiments: BHL, HJY, HCH. Discussed the results: BHL, HJY, LQX. Analyzed the data: BHL, HJY, SBH. Wrote the manuscript: BHL,HJY.\u003c/p\u003e\n\u003cp\u003eDiscussed and revised the manuscript: BHL, HJY, HCH, YJS, XZD, LQX. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in thispublished article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e Hunan Provincial Key Laboratory of Microbial Molecular Biology, State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha 410081, People\u0026rsquo;s Republic of China..\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMlecnik B, Bindea G, Kirilovsky A, Angell HK, Obenauf AC, Tosolini M\u003cem\u003e et al\u003c/em\u003e: The tumor microenvironment and Immunoscore are critical determinants of dissemination to distant metastasis. 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Clinical orthopaedics and related research. 2008, 466(6):1285-1291.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"E.coli Nissle 1917, IL-2, Anti-tumor engineering bacteria, Targeted cancer therapy","lastPublishedDoi":"10.21203/rs.3.rs-27294/v4","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-27294/v4","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Escherichia coli Nissle 1917 (EcN) is non-pathogenic probiotic bacteria. Previous studies have indicated that EcN can accumulate and proliferate selectively in solid tumours in BALB/c mouse models. In this study, EcN was engineered to express human interleukin 2 (hIL-2), which is known to enhance immune responses to tumours by activating a variety of immune cells. IL-2 expressed by EcN was proven to activate PBMCs in vitro. Compared to control EcN, intraperitoneally injected EcN expressing hIL-2 (EcN(hIL-2)) was selectively distributed in the tumour microenvironment and inhibited the growth of CT26 tumours in a tumour-bearing mouse model. Antitumour activity was achieved without toxicity to key normal organs and tissues, such as liver, spleen and kidneys. The antitumour mechanism was associated with the infiltration of inflammatory cells, such as T cells, neutrophils and macrophages. These findings provide evidence that the combination of tumour-targeting EcN bacteria and delivery of the immunostimulatory factor IL-2 can be exploited as a promising tumour immunotherapy.","manuscriptTitle":"Escherichia coli Nissle 1917 secreting functional interleukin 2 targets tumours and enhances the immune response to suppress tumours","msid":"","msnumber":"","nonDraftVersions":[{"code":4,"date":"2020-12-01 18:06:46","doi":"10.21203/rs.3.rs-27294/v4","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":3,"date":"2020-09-03 16:24:22","doi":"10.21203/rs.3.rs-27294/v3","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":2,"date":"2020-08-03 15:26:30","doi":"10.21203/rs.3.rs-27294/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2020-05-11 19:41:34","doi":"10.21203/rs.3.rs-27294/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56fae1b2-241c-4e10-b33f-5c023488d679","owner":[],"postedDate":"December 1st, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":1286834,"name":"Biotechnology and Bioengineering"},{"id":1286835,"name":"Environmental Engineering"}],"tags":[],"updatedAt":"2020-11-25T23:57:18+00:00","versionOfRecord":[],"versionCreatedAt":"2020-12-01 18:06:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v4","identity":"rs-27294","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-27294","identity":"rs-27294","version":["v4"]},"buildId":"k-LANOkFix9YAoV-Y-q_i","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}