- Email: [email protected]
Expressing cytotoxic compounds in Escherichia coli Nissle 1917 for tumor-targeting therapy
Accepted Manuscript Expressing Cytotoxic Compounds in Escherichia coli Nissle 1917 for Tumor-targeting Therapy Ruijuan Li, Linda Helbig, Jun Fu, Xiaoying Bian, Jennifer Herrmann, Michael Baumann, A. Francis Stewart, Rolf Müller, Aiying Li, Daniel Zips, Youming Zhang PII:
S0923-2508(18)30147-5
DOI:
https://doi.org/10.1016/j.resmic.2018.11.001
Reference:
RESMIC 3698
To appear in:
Research in Microbiology
Received Date: 8 May 2018 Revised Date:
16 October 2018
Accepted Date: 3 November 2018
Please cite this article as: R. Li, L. Helbig, J. Fu, X. Bian, J. Herrmann, M. Baumann, A.F. Stewart, R. Müller, A. Li, D. Zips, Y. Zhang, Expressing Cytotoxic Compounds in Escherichia coli Nissle 1917 for Tumor-targeting Therapy, Research in Microbiologoy, https://doi.org/10.1016/j.resmic.2018.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Expressing Cytotoxic Compounds in Escherichia coli Nissle 1917 for Tumor-targeting Therapy
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Ruijuan Lia, Linda Helbigb, Jun Fua,c, Xiaoying Biana,d, Jennifer Herrmannd, Michael Baumannb, A. Francis Stewartc, Rolf Müllerd, Aiying Lia,*, Daniel Zipsb,*, and
Shandong University–Helmholtz Institute of Biotechnology, State Key Laboratory of
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a
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Youming Zhanga,*
Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao 266237, P.R. China b
Experimental Radiotherapy of Tumours, OncoRay National Center for Radiation
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Research in Oncology, Medical Faculty and University Hospital, Dresden University of Technology, Germany c
Department of Genomics, Biotechnology Center, Dresden University of Technology,
Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical
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d
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Dresden, Germany
Research Saarland, Helmholtz Centre for Infection Research and Pharmaceutical Biotechnology, Saarland University, Saarbrücken, Germany
Running title: E. coli Nissle 1917 for tumor-targeting therapy
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ACCEPTED MANUSCRIPT Ruijuan Li and Linda Helbig contribute equally. *
Address for corresponding authors: Youming Zhanga,*, Daniel Zipsb,*, and Aiying
Lia,*
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Email: [email protected], [email protected] and
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[email protected]
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ACCEPTED MANUSCRIPT ABSTRACT Abnormal blood vessels and hypoxic and necrotic regions are common features of solid tumors and related to the malignant phenotype and therapy resistance. Certain
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obligate or facultative anaerobic bacteria exhibit inherent ability to colonize and proliferate within solid tumors in vivo. Escherichia coli Nissle 1917 (EcN), a non-pathogenic probiotic in European markets, has been known to proliferate
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selectively in the interface between the viable and necrotic regions of solid tumors.
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The objective of this study was to establish a tumor-targeting therapy system using the genetically engineered EcN for targeted delivery of cytotoxic compounds, including colibactin, glidobactin and luminmide. Biosynthetic gene clusters of these cytotoxic compounds were introduced into EcN and the corresponding compounds were
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detected in the resultant recombinant EcN strains. The recombinant EcN showed significant cytotoxic activity in vitro and in vivo as well, and significantly suppressed the tumor growth. Together, this study confirmed efficient tumor-targeting
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colonization of EcN and demonstrated its potentiality in the tumor-specific delivery of
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cytotoxic compounds as a new tumor-targeting therapy system. Keywords: Escherichia coli Nissle 1917 (EcN); tumor-targeting therapy; biosynthetic gene cluster; cytotoxic compounds; solid tumors
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ACCEPTED MANUSCRIPT 1. Introduction Tumor-targeting bacterial therapy was firstly reported more than 100 years ago [1, 2]. Obligate anaerobic bacteria such as Bifidobacterium [3] and Clostridium [4] as
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well as facultative anaerobes such as Salmonella [4, 5]and Escherichia [6, 7] can specifically target solid tumors in mice. It was believed that these obligate or facultative anaerobic bacteria evolved adaptability to hypoxia, a common feature of
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solid tumors and associated with malignant phenotype and therapy resistance. Besides
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the capability of tumor-targeted proliferation, these bacteria could regulate tumor micro-environment by promoting macrophage infiltration [8].
E. coli Nissle 1917 (EcN) has been reported to selectively colonize and replicate within solid tumors [7] [9, 10] [11, 12]. EcN has been widely used as a probiotic in
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human medicine in Germany and other countries and sold under the trade name of MutaflorTM to treat intestinal disorders including diarrhea, ulcerative colitis, and Crohn’s disease [13]. It was demonstrated that intestinal recombinant EcN has no
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effect on migration and clonal expansion, or the induction or breakdown of peripheral
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T-cell tolerance in an autoimmune environment [14, 15]. Based on the safety and incredible tumor-colonization, many trials have been
reported using these tumor-colonizing bacteria as drug-carriers to delivery or express cytotoxic proteins, cytokines, tumor-specific antibodies, or small hairpin RNAs and to activate prodrugs in situ in the tumors [16-18]. Recently, by engineering attenuated tumor-colonizing Salmonella enterica subsp to harbor an anti-tumor-Haemolysin 4
ACCEPTED MANUSCRIPT E-encoding gene and a synchronized lysis circuit, a new bacterial drug delivery strategy combining both circuit-engineered bacteria and chemotherapy was set up in situ [19].
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However, no work has been reported using these tumor-targeting strains to express cytotoxic compounds such as polyketides (PKs), non-ribosomal peptides (NRPs) or hybrids which are biosynthesized by multifunctional megasynthases,
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including polyketide synthases (PKS), and non-ribosomal peptide synthases (NRPS),
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or hybrid enzymes (Fischbach and Walsh, 2006), respectively. To reduce the adverse effects of chemotherapy, it is worth establishing a new tumor-targeting drug-delivery system using tumor-colonizing bacteria.
Generally, E. coli strains are easy-to-handle hosts with a clear genetic
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background for heterologous expression of a large amount of proteins. Regardless of codon usage bias, E. coli strains are considered to lack the essential phosphopantetheine transferase (PPTase) for post-translational priming of thiolation
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domain of PKS and NRPS [20]. However, unlike other E. coli strains, EcN is
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naturally capable of producing NRP-derived siderophore, PK-NRP hybrid yesiniabactin [21] and cytotoxin colibactin, suggesting that its metabolic system would probably be compatible to the biosynthesis of PKs or NRPs. In colibactin biosynthetic gene cluster (Figure S1), clbA encodes a putative PPTase which can evade this issue and make EcN be a potential expression host for PK or NRP-derived natural products [22, 23] [24, 25]. These facts showed that EcN could be used as a 5
ACCEPTED MANUSCRIPT safe bacterial carrier for targeted delivery of cytotoxic PK or NRP-derived compounds. Glidobactins, hybrid NRP/PK-derived natural products, discovered from
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Burkholderia K481-B101 (DSM 7029) two decades ago, are cytotoxic to cancer cell lines and prolong the life span of P388 leukaemia-bearing mice [26]. Luminmide A was biosynthesized by a single NRPS cluster plu3263 from Photorabdus luminescens
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TT01 [24, 27] and exhibited cytotoxicity to tumor cell line HCT-116 (IC50 = 27.5
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µg/ml). Those two gene clusters were successfully cloned and transformed into EcN and the corresponding compounds were detected in the resultant recombinant EcN strains (Figure S1-S2).
In this study, we investigated the efficiency of EcN for tumor specific
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colonization and evaluated therapy of EcN as a tumor-targeting drug carrier by expressing cytotoxic compounds-colibactin, glidobactin, and luminmide (Figure S3). The results confirmed the efficient tumor-targeting colonization of EcN and
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demonstrated its potentiality as a new tumor-targeting therapy system.
2. Material and methods 2.1 Bacteria and plasmids Plasmid pGB-tet-cm-luxABECD (IVIS® Spektrum, Xenogen) was transformed
into EcN to express bioluminescence in vivo. pSUMtaA contains the gene mtaA encoding a broad-substrate PPTase from Stigmatella aurantiaca DW4/3-1 [28]. Two 6
ACCEPTED MANUSCRIPT vectors, pACYC184 (New England Biolabs) and pGB-amp-zeo-Ptet, were transformed into EcN as controls, respectively. pGB-amp-zeo-Ptet-glb and pGB-amp-zeo-Ptet-plu3263 were constructed by Bian and Fu, respectively [24, 26].
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E. coli GB05-dir was a general host for DNA recombination experiments [24]. EcN transformed with plasmid pSUMtaA was used for tumor-colonization test. EcN bearing pSUMtaA (∆clb), generated by Red/ET recombineering to completely replace
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the clbA–clbQ gene sequence with a zeocin resistance marker by PCR [25] (Figure
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S4), was used as a control in this study, which lost ability to produce colibactin. Strains EcN/pSUMtaA and EcN/pSUMtaA (∆clb) were constructed by Bian [25]. LB medium for E. coli cultivation was used and supplemented with antibiotics when required.
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2.2 Cell lines and cell cultivation
U-2 OS osteosarcoma cells were purchased from the American Type Culture Collection (ATCC; HTB-96). The base medium for human U-2 OS osteosarcoma cells
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is ATCC-formulated McCoy’s 5a Medium Modified, Catalog No. 30-2007. To prepare
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the complete growth medium, the following components were added to the base medium: fetal bovine serum to a final concentration of 10%. U-2 OS osteosarcoma cells were specially used in transient infection assay to detect the expression of colibactin by EcN.
UT-SCC-5, human head and neck squamous cell carcinoma cell line, was kindly provided by Dr. Grenman, University of Turku, Finnland. This cell line was cultured 7
ACCEPTED MANUSCRIPT in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 400 ng/ml hydrocortisone, 90 % and fetal
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bovine serum, 10 %. 2.3 Animal models
All animal experiments performed as the guideline provided by the National
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Institutes of Health Guide for the Care and Use of Laboratory Animals and the study
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was approved by the Animal Ethics Committee of Shandong University. Specific-pathogen-free (SPF) female NMRI nude mice, 6 to 8 weeks old, were purchased from Shandong University Laboratory Animal Center, Shandong, China. Before experiments, the animals were maintained in SPF conditions for at least 3 days.
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UT-SCC-5 was transplanted subcutaneously into the right hid-leg of female NMRI nude mice. Once the tumor volume reached about 0.2 cm3, mice were randomly assigned to several groups and were observed every day. At a defined time, the mice
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times.
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were sacrificed by cervical dislocation. All animal experiments were replicated three
2.4 Quantification of EcN in mice Quantification of tumor and normal tissue colonization was performed by colony
assays and by in vivo bioluminescence (pGB-tet-cm-luxABECD, IVIS® Spektrum, Xenogen). From the 3rd day after intravenous (i.v.) injection, tumors and livers were isolated, spread on LB plates, and the number of EcN in tissues were counted and 8
ACCEPTED MANUSCRIPT calculated (Figure 1). Luminescence signals at the 3rd day and 11th day after i.v. injection of EcN containing pGB-tet-cm-luxABECD plasmid were detected by using IVIS Spectrum system (Figure 2).
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2.5 Activation of colibactin biosynthetic pathway in EcN Transient infection of human U-2 OS osteosarcoma cells with E. coli strains was performed as reported earlier [22] (Figure 3A). For co-cultivation, human U-2 OS
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osteosarcoma cells were seeded at 2.5 × 104 cells mL−1 in a tissue culture plate
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containing 9 mL of complete medium (McCoys5A, 10% FBS). After 2 days incubation, the approximately 50–60 % of cells were confluent. Then, 1 mL of EcN culture (OD600 = 0.5) was added to the sarcoma cells to achieve a ratio of U-2 OS osteosarcoma and EcN cells of 1:200. The co-cultivation was carried out for 5 hours
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at 37 °C [25].
The relative cell vitality assay (Figure 3B) treated with different bacterial cultures was analyzed using the MTT assay [29].
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2.6 In vivo expression of cytotoxic compounds for cancer therapy
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The recombinant strains, EcN/pSUMtaA, EcN/pGB-amp-zeo-Ptet-glb, and EcN/pGB-amp-zeo-Ptet-plu3263, respectively, were used to test the effect on tumor growth. LB medium and PBS were used as blank control. EcN/pACYC184, EcN/pSUMtaA (∆clb), and EcN/pGB-amp-zeo-Ptet were used as negative controls. When tumor volume reached around 0.2 cm3, female NMRI nude mice bearing UT-SCC-5 human head and neck squamous tumors were i.v. administered with PBS 9
ACCEPTED MANUSCRIPT or 1 × 107 CFU EcN bearing constructed plasmids. The tumor weight and mice body weight were measured after 18 days (Figures 4 and 5). Relative tumor size at the at the day (D0) when 1 × 107 CFU bacterial cells per
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mouse was i.v. injected was set as 1. Then, the relative tumor volume in different stages was calculated by dividing the tumor volume at the indicated day by that at the day (D0). At least 8 mice were used per group. The relative tumor size of each group
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was plotted (Figure 5A).
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2.7 Statistical analysis
Statistical significance for the experiments was determined by using F-test or Student’s t-test. If the p-value was below 0.01, the differences between the
3. Results
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experimental groups were considered significant.
3.1 Tumor-specific colonization of EcN in vivo
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The preferential proliferation of tumor-colonizing bacteria in tumor is pivotal for
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their uses as hosts to express and release cytotoxic compounds for tumor-targeting therapy. At the 3rd day after 1 × 107 CFU EcN was injected into the mouse tail vein, bacterial cell density in tumors and normal tissues was counted. The ratio of EcN in tumors and normal tissues is almost 1000:1 (Figure 1). Tumor-specific colonization in tumors was further confirmed by in vivo imaging after i.v. administration of EcN (Figure 2). The luminescence signal was recorded at 10
ACCEPTED MANUSCRIPT the 3rd day after injection and still can be detectable at the 11th day after injection. The observed tumor-specific colonization of EcN and the tumor-to-normal tissue ratio coheres with the results of Stritzker et al [7]. This result demonstrated that EcN could
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be an ideal tumor-targeting bacterial for the delivery of cytotoxic compounds. 3.2 Activation of colibactin biosynthetic gene cluster by over-expression of MtaA
To investigate the possibilities to use EcN as a heterologous host to express
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foreign PKs or NRPs gene clusters, the first trial was to activate the endogenous
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colibactin gene cluster, which is silent under normal conditions. It was reported that expression of pPant transferase MtaA encoding by myxothiazole gene cluster might be sufficient to activate some key enzymes in the colibactin pathway. To prove this hypothesis, MtaA expression plasmid (called pSUMtaA) was transformed into EcN to
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obtain the strain EcN/pSUMtaA. In the following transient infection experiment, the over-expression of MtaA was determined by MTT assay and cytotoxicity analysis as shown in Figure 3. The results (Figure 3A) showed that EcN/pSUMtaA could
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significantly inhibit the growth of the U-2 OS osteosarcoma cells. The cell relative
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vitality of the EcN/pSUMtaA groups was lower than that of the control group (Figure 3B). This data suggested that in the recombinant strain EcN/pSUMtaA, the presence of MtaA could activate the gene cluster of PKs or NRPs. 3.3 In vivo expression of cytotoxic compounds inhibiting tumor growth UT-SCC-5 human head and neck squamous cell carcinoma model was used to investigate the anti-tumor effects of colibactin/glidobactins/lumimides-expressing 11
ACCEPTED MANUSCRIPT EcN. After the tumor grew to about 0.2 cm3, EcN and its variants were i.v. administered into female NMRI nude mice at a dose of 1 × 107 CFU per mouse. Bacterial level of 1 × 107 CFU colibactin-expressing EcN remarkably inhibited
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tumor growth (Figure 4). At sacrifice after 18 days, the tumor weight in the treatment group ECN-pSUMtaA reached 327 mg. These changes corresponded to 71% reduction compared to that in the PBS-treated group. Furthermore, almost 20%
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reduction of the tumor weight in the EcN treatment group indicated that EcN alone
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can also suppress tumor growth.
Cancer therapeutic effect of glidobactin- or lumimide-expressing EcN strains on tumor growth has been shown in Figure 5. The administration of glidobactin- or lumimide-expressing EcN resulted in a significant reduction of tumor volume, when
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compared to EcN with empty plasmid vector pGB-amp-zeo-Ptet (Figure 5A). Moreover, lumimide-expressing EcN group has showed superior effect on tumor
3.4 Toxicity
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growth than glidobactin-expressing EcN group.
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No animals died during the course of the experiments, and the EcN -treated mice behaved normally, alike the PBS-treated control. The body weight of the treated mice showed that administration of engineered EcN has no side effects on animals (Figure 5B). Hence, the systemic administration of EcN did not show any obvious pathogenic effects on the mice.
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ACCEPTED MANUSCRIPT 4. Discussion In this study, we evaluated the potential of non-pathogenic and probiotic EcN for tumor-targeting bacterial therapy. Our data demonstrated that it could colonize solid
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tumors efficiently and remain in the tumor tissue for long periods of time. Moreover, this is the first attempt to use EcN as a tumor-targeting drug carrier to express and release cytotoxic compounds in situ to significantly restrain the growth of tumors.
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Here, we confirmed that PPTase MtaA expression in EcN is quite critical for the
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accumulation of several cytotoxic compounds. The endogenous gene cluster colibactin was activated in EcN by over-expression of MtaA [30]. Though colibactin has been reported to be a human gut bacterial genotoxin linked to colon cancer [31-32], transient infection of human U-2 OS osteosarcoma cells with E. coli strains
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showed that EcN expressing colibactin could significantly inhibit the growth of the U-2 OS osteosarcoma cells. Thus, in this study, we combined the tumor inhibition activity of colibactin and tumor-targeting activity of EcN to build a tumor-targeting
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bacterial therapy system.
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EcN was proved to be as heterologous host which can express cytotoxic compounds of polyketides or non-ribosomal peptides. Our experiments evidence that, it is feasible to express glidobactins and lumimides in EcN. Lumimides, produced by plu3263 showed less cytotoxic activity than glidobactins, but they have better solubility in water than glidobactins. This might be one of the reasons for their better tumor therapy than that glidobactins in vivo. 13
ACCEPTED MANUSCRIPT Based on our data, EcN has several advantages used as potential bacteria therapy: (1) possessing specific and efficient tumor-targeting and tumor-colonizing features (about 109 CFU/g of tumor tissue); (2) remaining in the tumor tissue for longer time
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and most importantly, as a non-pathogenic and probiotic; (3) repressing tumor growth by itself; (4) being a suitable host of cytotoxic compound gene cluster and drug-delivery carrier in situ. To explain why tumor-targeting bacteria could inhibit
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tumor growth, researchers proposed that these bacteria might stimulate immune
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responses by regulating tumor microenvironment via macrophage infiltration [33]. Further we would like to investigate the mechanism that EcN against tumor growth. To conclude, we successfully established a pioneer strategy to restrain the growth of solid tumors, by combining the safe tumor-targeting strain EcN with the production
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of several PK- or NRP- derived cytotoxic compounds in situ in tumors for the first time. This approach will certainly promote the development of tumor-targeting bacterial therapy for solid tumors.
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Abbreviations
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ATCC: American Type Culture Collection; CFU: Colony forming units; cm: chloromycetin; i.v.: intravenously; EcN: E. coli Nissle 1917; LB: lysogeny broth; NMRI: Naval Medical Research Institute; NRPS: non-ribosomal peptide synthases; NRPs: non-ribosomal peptides; PBS: phosphate-buffered saline; PKS: polyketide synthases; PKs: polyketides; PPTase: phosphopantetheine transferase; SPF: specific-pathogen-free; tet: tetracycline. 14
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Authors contributions L.H. performed experiments and R.L. wrote the manuscript; R.L., J. F., X.B., J.H.,
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M.B., A.F.S., R.M., A.L., D.Z., and Y.Z. performed research and analyzed data. R.L. and L.H. contribute equally. *
Address for corresponding authors: Youming Zhanga,*, Daniel Zipsb,*, and Aiying
[email protected],
[email protected]
and
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Email:
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Lia,*
[email protected]
Conflict of interest
Acknowledgments
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The authors declare no conflicts of interest.
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This study was supported by National Natural Science Foundation of China (no.
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81502962; 31670097), China Postdoctoral Science Foundation Grant (no. 2015M572017), Major Project of Science and Technology of Shandong Province (2015ZDJS04001), Key Research and Development Program of Shandong Province (2015GSF12101), and International Science & Technology Cooperation Program of China (2015DFE32850).
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Fig. 1. Bacteria colonization at the 3rd day after the i.v. tail vein injection. Colony forming units (CFU) per gram (g) tissue were determined ex vivo. Statistical significance between the CFU of EcN in tumors and normal tissue was determined by F-test. P < 0.005.
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Figure 2. Luminescence signal at the 3rd day (left) and at the 11th day (right) after the i.v. injection of EcN containing pGB-tet-cm-luxABECD plasmid to express bioluminescence detected by using IVIS Spectrum system. The control animal (right position) received no bacteria.
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Figure 3. (A) Transient infection of human U-2 OS osteosarcoma cells with E. coli strains (Giemsa staining, 200 × magnification; phase contrast) [22]. (B) Cell relative vitality was measured by MTT assay. Error bars represented the standard deviation from three replicated experiments. Asterisks indicate significant differences between treatment and control as determined by Student’s t-test: **P < 0.01; ***P < 0.001.
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Figure 4. Therapeutic effect of colibactin-expressing EcN strains on tumor growth. Female NMRI nude mice (n = 8) bearing UT-SCC-5 human head and neck squamous tumors were treated by i.v. administration of PBS or 1 × 107 CFU EcN, EcN bearing pSUMtaA (EcN/pSUMtaA). Asterisks indicate significant differences between treatment and control as determined by Student’s t-test: **P < 0.01; ***P < 0.001.
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Figure 5. (A) Therapeutic effect of glidobactin- or lumimide-expressing EcN strains on tumor growth. Female NMRI nude mice (n = 8) bearing UT-SCC-5 human head and neck squamous tumors were treated by i.v. administration of PBS or 1 × 107 CFU EcN bearing pGB-amp-zeo-Ptet vector (EcN/pGB-amp-zeo-Ptet), EcN bearing glidobactin-expressing plasmid (EcN/pGB-amp-zeo-Ptet-glb), or EcN bearing lumimide-expressing plasmid (EcN/pGB-amp-zeo-Ptet-plu3263). (B) The body weight of the treated mice was measured after 18 days. Error bars represent the standard error of the mean.
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S0923-2508(18)30147-5
DOI:
https://doi.org/10.1016/j.resmic.2018.11.001
Reference:
RESMIC 3698
To appear in:
Research in Microbiology
Received Date: 8 May 2018 Revised Date:
16 October 2018
Accepted Date: 3 November 2018
Please cite this article as: R. Li, L. Helbig, J. Fu, X. Bian, J. Herrmann, M. Baumann, A.F. Stewart, R. Müller, A. Li, D. Zips, Y. Zhang, Expressing Cytotoxic Compounds in Escherichia coli Nissle 1917 for Tumor-targeting Therapy, Research in Microbiologoy, https://doi.org/10.1016/j.resmic.2018.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Expressing Cytotoxic Compounds in Escherichia coli Nissle 1917 for Tumor-targeting Therapy
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Ruijuan Lia, Linda Helbigb, Jun Fua,c, Xiaoying Biana,d, Jennifer Herrmannd, Michael Baumannb, A. Francis Stewartc, Rolf Müllerd, Aiying Lia,*, Daniel Zipsb,*, and
Shandong University–Helmholtz Institute of Biotechnology, State Key Laboratory of
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a
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Youming Zhanga,*
Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao 266237, P.R. China b
Experimental Radiotherapy of Tumours, OncoRay National Center for Radiation
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Research in Oncology, Medical Faculty and University Hospital, Dresden University of Technology, Germany c
Department of Genomics, Biotechnology Center, Dresden University of Technology,
Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical
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d
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Dresden, Germany
Research Saarland, Helmholtz Centre for Infection Research and Pharmaceutical Biotechnology, Saarland University, Saarbrücken, Germany
Running title: E. coli Nissle 1917 for tumor-targeting therapy
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ACCEPTED MANUSCRIPT Ruijuan Li and Linda Helbig contribute equally. *
Address for corresponding authors: Youming Zhanga,*, Daniel Zipsb,*, and Aiying
Lia,*
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Email: [email protected], [email protected] and
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[email protected]
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ACCEPTED MANUSCRIPT ABSTRACT Abnormal blood vessels and hypoxic and necrotic regions are common features of solid tumors and related to the malignant phenotype and therapy resistance. Certain
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obligate or facultative anaerobic bacteria exhibit inherent ability to colonize and proliferate within solid tumors in vivo. Escherichia coli Nissle 1917 (EcN), a non-pathogenic probiotic in European markets, has been known to proliferate
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selectively in the interface between the viable and necrotic regions of solid tumors.
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The objective of this study was to establish a tumor-targeting therapy system using the genetically engineered EcN for targeted delivery of cytotoxic compounds, including colibactin, glidobactin and luminmide. Biosynthetic gene clusters of these cytotoxic compounds were introduced into EcN and the corresponding compounds were
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detected in the resultant recombinant EcN strains. The recombinant EcN showed significant cytotoxic activity in vitro and in vivo as well, and significantly suppressed the tumor growth. Together, this study confirmed efficient tumor-targeting
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colonization of EcN and demonstrated its potentiality in the tumor-specific delivery of
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cytotoxic compounds as a new tumor-targeting therapy system. Keywords: Escherichia coli Nissle 1917 (EcN); tumor-targeting therapy; biosynthetic gene cluster; cytotoxic compounds; solid tumors
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ACCEPTED MANUSCRIPT 1. Introduction Tumor-targeting bacterial therapy was firstly reported more than 100 years ago [1, 2]. Obligate anaerobic bacteria such as Bifidobacterium [3] and Clostridium [4] as
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well as facultative anaerobes such as Salmonella [4, 5]and Escherichia [6, 7] can specifically target solid tumors in mice. It was believed that these obligate or facultative anaerobic bacteria evolved adaptability to hypoxia, a common feature of
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solid tumors and associated with malignant phenotype and therapy resistance. Besides
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the capability of tumor-targeted proliferation, these bacteria could regulate tumor micro-environment by promoting macrophage infiltration [8].
E. coli Nissle 1917 (EcN) has been reported to selectively colonize and replicate within solid tumors [7] [9, 10] [11, 12]. EcN has been widely used as a probiotic in
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human medicine in Germany and other countries and sold under the trade name of MutaflorTM to treat intestinal disorders including diarrhea, ulcerative colitis, and Crohn’s disease [13]. It was demonstrated that intestinal recombinant EcN has no
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effect on migration and clonal expansion, or the induction or breakdown of peripheral
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T-cell tolerance in an autoimmune environment [14, 15]. Based on the safety and incredible tumor-colonization, many trials have been
reported using these tumor-colonizing bacteria as drug-carriers to delivery or express cytotoxic proteins, cytokines, tumor-specific antibodies, or small hairpin RNAs and to activate prodrugs in situ in the tumors [16-18]. Recently, by engineering attenuated tumor-colonizing Salmonella enterica subsp to harbor an anti-tumor-Haemolysin 4
ACCEPTED MANUSCRIPT E-encoding gene and a synchronized lysis circuit, a new bacterial drug delivery strategy combining both circuit-engineered bacteria and chemotherapy was set up in situ [19].
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However, no work has been reported using these tumor-targeting strains to express cytotoxic compounds such as polyketides (PKs), non-ribosomal peptides (NRPs) or hybrids which are biosynthesized by multifunctional megasynthases,
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including polyketide synthases (PKS), and non-ribosomal peptide synthases (NRPS),
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or hybrid enzymes (Fischbach and Walsh, 2006), respectively. To reduce the adverse effects of chemotherapy, it is worth establishing a new tumor-targeting drug-delivery system using tumor-colonizing bacteria.
Generally, E. coli strains are easy-to-handle hosts with a clear genetic
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background for heterologous expression of a large amount of proteins. Regardless of codon usage bias, E. coli strains are considered to lack the essential phosphopantetheine transferase (PPTase) for post-translational priming of thiolation
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domain of PKS and NRPS [20]. However, unlike other E. coli strains, EcN is
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naturally capable of producing NRP-derived siderophore, PK-NRP hybrid yesiniabactin [21] and cytotoxin colibactin, suggesting that its metabolic system would probably be compatible to the biosynthesis of PKs or NRPs. In colibactin biosynthetic gene cluster (Figure S1), clbA encodes a putative PPTase which can evade this issue and make EcN be a potential expression host for PK or NRP-derived natural products [22, 23] [24, 25]. These facts showed that EcN could be used as a 5
ACCEPTED MANUSCRIPT safe bacterial carrier for targeted delivery of cytotoxic PK or NRP-derived compounds. Glidobactins, hybrid NRP/PK-derived natural products, discovered from
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Burkholderia K481-B101 (DSM 7029) two decades ago, are cytotoxic to cancer cell lines and prolong the life span of P388 leukaemia-bearing mice [26]. Luminmide A was biosynthesized by a single NRPS cluster plu3263 from Photorabdus luminescens
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TT01 [24, 27] and exhibited cytotoxicity to tumor cell line HCT-116 (IC50 = 27.5
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µg/ml). Those two gene clusters were successfully cloned and transformed into EcN and the corresponding compounds were detected in the resultant recombinant EcN strains (Figure S1-S2).
In this study, we investigated the efficiency of EcN for tumor specific
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colonization and evaluated therapy of EcN as a tumor-targeting drug carrier by expressing cytotoxic compounds-colibactin, glidobactin, and luminmide (Figure S3). The results confirmed the efficient tumor-targeting colonization of EcN and
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demonstrated its potentiality as a new tumor-targeting therapy system.
2. Material and methods 2.1 Bacteria and plasmids Plasmid pGB-tet-cm-luxABECD (IVIS® Spektrum, Xenogen) was transformed
into EcN to express bioluminescence in vivo. pSUMtaA contains the gene mtaA encoding a broad-substrate PPTase from Stigmatella aurantiaca DW4/3-1 [28]. Two 6
ACCEPTED MANUSCRIPT vectors, pACYC184 (New England Biolabs) and pGB-amp-zeo-Ptet, were transformed into EcN as controls, respectively. pGB-amp-zeo-Ptet-glb and pGB-amp-zeo-Ptet-plu3263 were constructed by Bian and Fu, respectively [24, 26].
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E. coli GB05-dir was a general host for DNA recombination experiments [24]. EcN transformed with plasmid pSUMtaA was used for tumor-colonization test. EcN bearing pSUMtaA (∆clb), generated by Red/ET recombineering to completely replace
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the clbA–clbQ gene sequence with a zeocin resistance marker by PCR [25] (Figure
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S4), was used as a control in this study, which lost ability to produce colibactin. Strains EcN/pSUMtaA and EcN/pSUMtaA (∆clb) were constructed by Bian [25]. LB medium for E. coli cultivation was used and supplemented with antibiotics when required.
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2.2 Cell lines and cell cultivation
U-2 OS osteosarcoma cells were purchased from the American Type Culture Collection (ATCC; HTB-96). The base medium for human U-2 OS osteosarcoma cells
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is ATCC-formulated McCoy’s 5a Medium Modified, Catalog No. 30-2007. To prepare
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the complete growth medium, the following components were added to the base medium: fetal bovine serum to a final concentration of 10%. U-2 OS osteosarcoma cells were specially used in transient infection assay to detect the expression of colibactin by EcN.
UT-SCC-5, human head and neck squamous cell carcinoma cell line, was kindly provided by Dr. Grenman, University of Turku, Finnland. This cell line was cultured 7
ACCEPTED MANUSCRIPT in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES and 0.5 mM sodium pyruvate supplemented with 400 ng/ml hydrocortisone, 90 % and fetal
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bovine serum, 10 %. 2.3 Animal models
All animal experiments performed as the guideline provided by the National
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Institutes of Health Guide for the Care and Use of Laboratory Animals and the study
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was approved by the Animal Ethics Committee of Shandong University. Specific-pathogen-free (SPF) female NMRI nude mice, 6 to 8 weeks old, were purchased from Shandong University Laboratory Animal Center, Shandong, China. Before experiments, the animals were maintained in SPF conditions for at least 3 days.
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UT-SCC-5 was transplanted subcutaneously into the right hid-leg of female NMRI nude mice. Once the tumor volume reached about 0.2 cm3, mice were randomly assigned to several groups and were observed every day. At a defined time, the mice
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times.
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were sacrificed by cervical dislocation. All animal experiments were replicated three
2.4 Quantification of EcN in mice Quantification of tumor and normal tissue colonization was performed by colony
assays and by in vivo bioluminescence (pGB-tet-cm-luxABECD, IVIS® Spektrum, Xenogen). From the 3rd day after intravenous (i.v.) injection, tumors and livers were isolated, spread on LB plates, and the number of EcN in tissues were counted and 8
ACCEPTED MANUSCRIPT calculated (Figure 1). Luminescence signals at the 3rd day and 11th day after i.v. injection of EcN containing pGB-tet-cm-luxABECD plasmid were detected by using IVIS Spectrum system (Figure 2).
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2.5 Activation of colibactin biosynthetic pathway in EcN Transient infection of human U-2 OS osteosarcoma cells with E. coli strains was performed as reported earlier [22] (Figure 3A). For co-cultivation, human U-2 OS
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osteosarcoma cells were seeded at 2.5 × 104 cells mL−1 in a tissue culture plate
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containing 9 mL of complete medium (McCoys5A, 10% FBS). After 2 days incubation, the approximately 50–60 % of cells were confluent. Then, 1 mL of EcN culture (OD600 = 0.5) was added to the sarcoma cells to achieve a ratio of U-2 OS osteosarcoma and EcN cells of 1:200. The co-cultivation was carried out for 5 hours
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at 37 °C [25].
The relative cell vitality assay (Figure 3B) treated with different bacterial cultures was analyzed using the MTT assay [29].
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2.6 In vivo expression of cytotoxic compounds for cancer therapy
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The recombinant strains, EcN/pSUMtaA, EcN/pGB-amp-zeo-Ptet-glb, and EcN/pGB-amp-zeo-Ptet-plu3263, respectively, were used to test the effect on tumor growth. LB medium and PBS were used as blank control. EcN/pACYC184, EcN/pSUMtaA (∆clb), and EcN/pGB-amp-zeo-Ptet were used as negative controls. When tumor volume reached around 0.2 cm3, female NMRI nude mice bearing UT-SCC-5 human head and neck squamous tumors were i.v. administered with PBS 9
ACCEPTED MANUSCRIPT or 1 × 107 CFU EcN bearing constructed plasmids. The tumor weight and mice body weight were measured after 18 days (Figures 4 and 5). Relative tumor size at the at the day (D0) when 1 × 107 CFU bacterial cells per
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mouse was i.v. injected was set as 1. Then, the relative tumor volume in different stages was calculated by dividing the tumor volume at the indicated day by that at the day (D0). At least 8 mice were used per group. The relative tumor size of each group
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was plotted (Figure 5A).
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2.7 Statistical analysis
Statistical significance for the experiments was determined by using F-test or Student’s t-test. If the p-value was below 0.01, the differences between the
3. Results
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experimental groups were considered significant.
3.1 Tumor-specific colonization of EcN in vivo
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The preferential proliferation of tumor-colonizing bacteria in tumor is pivotal for
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their uses as hosts to express and release cytotoxic compounds for tumor-targeting therapy. At the 3rd day after 1 × 107 CFU EcN was injected into the mouse tail vein, bacterial cell density in tumors and normal tissues was counted. The ratio of EcN in tumors and normal tissues is almost 1000:1 (Figure 1). Tumor-specific colonization in tumors was further confirmed by in vivo imaging after i.v. administration of EcN (Figure 2). The luminescence signal was recorded at 10
ACCEPTED MANUSCRIPT the 3rd day after injection and still can be detectable at the 11th day after injection. The observed tumor-specific colonization of EcN and the tumor-to-normal tissue ratio coheres with the results of Stritzker et al [7]. This result demonstrated that EcN could
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be an ideal tumor-targeting bacterial for the delivery of cytotoxic compounds. 3.2 Activation of colibactin biosynthetic gene cluster by over-expression of MtaA
To investigate the possibilities to use EcN as a heterologous host to express
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foreign PKs or NRPs gene clusters, the first trial was to activate the endogenous
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colibactin gene cluster, which is silent under normal conditions. It was reported that expression of pPant transferase MtaA encoding by myxothiazole gene cluster might be sufficient to activate some key enzymes in the colibactin pathway. To prove this hypothesis, MtaA expression plasmid (called pSUMtaA) was transformed into EcN to
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obtain the strain EcN/pSUMtaA. In the following transient infection experiment, the over-expression of MtaA was determined by MTT assay and cytotoxicity analysis as shown in Figure 3. The results (Figure 3A) showed that EcN/pSUMtaA could
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significantly inhibit the growth of the U-2 OS osteosarcoma cells. The cell relative
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vitality of the EcN/pSUMtaA groups was lower than that of the control group (Figure 3B). This data suggested that in the recombinant strain EcN/pSUMtaA, the presence of MtaA could activate the gene cluster of PKs or NRPs. 3.3 In vivo expression of cytotoxic compounds inhibiting tumor growth UT-SCC-5 human head and neck squamous cell carcinoma model was used to investigate the anti-tumor effects of colibactin/glidobactins/lumimides-expressing 11
ACCEPTED MANUSCRIPT EcN. After the tumor grew to about 0.2 cm3, EcN and its variants were i.v. administered into female NMRI nude mice at a dose of 1 × 107 CFU per mouse. Bacterial level of 1 × 107 CFU colibactin-expressing EcN remarkably inhibited
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tumor growth (Figure 4). At sacrifice after 18 days, the tumor weight in the treatment group ECN-pSUMtaA reached 327 mg. These changes corresponded to 71% reduction compared to that in the PBS-treated group. Furthermore, almost 20%
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reduction of the tumor weight in the EcN treatment group indicated that EcN alone
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can also suppress tumor growth.
Cancer therapeutic effect of glidobactin- or lumimide-expressing EcN strains on tumor growth has been shown in Figure 5. The administration of glidobactin- or lumimide-expressing EcN resulted in a significant reduction of tumor volume, when
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compared to EcN with empty plasmid vector pGB-amp-zeo-Ptet (Figure 5A). Moreover, lumimide-expressing EcN group has showed superior effect on tumor
3.4 Toxicity
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growth than glidobactin-expressing EcN group.
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No animals died during the course of the experiments, and the EcN -treated mice behaved normally, alike the PBS-treated control. The body weight of the treated mice showed that administration of engineered EcN has no side effects on animals (Figure 5B). Hence, the systemic administration of EcN did not show any obvious pathogenic effects on the mice.
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ACCEPTED MANUSCRIPT 4. Discussion In this study, we evaluated the potential of non-pathogenic and probiotic EcN for tumor-targeting bacterial therapy. Our data demonstrated that it could colonize solid
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tumors efficiently and remain in the tumor tissue for long periods of time. Moreover, this is the first attempt to use EcN as a tumor-targeting drug carrier to express and release cytotoxic compounds in situ to significantly restrain the growth of tumors.
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Here, we confirmed that PPTase MtaA expression in EcN is quite critical for the
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accumulation of several cytotoxic compounds. The endogenous gene cluster colibactin was activated in EcN by over-expression of MtaA [30]. Though colibactin has been reported to be a human gut bacterial genotoxin linked to colon cancer [31-32], transient infection of human U-2 OS osteosarcoma cells with E. coli strains
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showed that EcN expressing colibactin could significantly inhibit the growth of the U-2 OS osteosarcoma cells. Thus, in this study, we combined the tumor inhibition activity of colibactin and tumor-targeting activity of EcN to build a tumor-targeting
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bacterial therapy system.
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EcN was proved to be as heterologous host which can express cytotoxic compounds of polyketides or non-ribosomal peptides. Our experiments evidence that, it is feasible to express glidobactins and lumimides in EcN. Lumimides, produced by plu3263 showed less cytotoxic activity than glidobactins, but they have better solubility in water than glidobactins. This might be one of the reasons for their better tumor therapy than that glidobactins in vivo. 13
ACCEPTED MANUSCRIPT Based on our data, EcN has several advantages used as potential bacteria therapy: (1) possessing specific and efficient tumor-targeting and tumor-colonizing features (about 109 CFU/g of tumor tissue); (2) remaining in the tumor tissue for longer time
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and most importantly, as a non-pathogenic and probiotic; (3) repressing tumor growth by itself; (4) being a suitable host of cytotoxic compound gene cluster and drug-delivery carrier in situ. To explain why tumor-targeting bacteria could inhibit
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tumor growth, researchers proposed that these bacteria might stimulate immune
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responses by regulating tumor microenvironment via macrophage infiltration [33]. Further we would like to investigate the mechanism that EcN against tumor growth. To conclude, we successfully established a pioneer strategy to restrain the growth of solid tumors, by combining the safe tumor-targeting strain EcN with the production
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of several PK- or NRP- derived cytotoxic compounds in situ in tumors for the first time. This approach will certainly promote the development of tumor-targeting bacterial therapy for solid tumors.
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Abbreviations
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ATCC: American Type Culture Collection; CFU: Colony forming units; cm: chloromycetin; i.v.: intravenously; EcN: E. coli Nissle 1917; LB: lysogeny broth; NMRI: Naval Medical Research Institute; NRPS: non-ribosomal peptide synthases; NRPs: non-ribosomal peptides; PBS: phosphate-buffered saline; PKS: polyketide synthases; PKs: polyketides; PPTase: phosphopantetheine transferase; SPF: specific-pathogen-free; tet: tetracycline. 14
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Authors contributions L.H. performed experiments and R.L. wrote the manuscript; R.L., J. F., X.B., J.H.,
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M.B., A.F.S., R.M., A.L., D.Z., and Y.Z. performed research and analyzed data. R.L. and L.H. contribute equally. *
Address for corresponding authors: Youming Zhanga,*, Daniel Zipsb,*, and Aiying
[email protected],
[email protected]
and
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Email:
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Lia,*
[email protected]
Conflict of interest
Acknowledgments
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The authors declare no conflicts of interest.
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This study was supported by National Natural Science Foundation of China (no.
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81502962; 31670097), China Postdoctoral Science Foundation Grant (no. 2015M572017), Major Project of Science and Technology of Shandong Province (2015ZDJS04001), Key Research and Development Program of Shandong Province (2015GSF12101), and International Science & Technology Cooperation Program of China (2015DFE32850).
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maturation. Chembiochem : a European journal of chemical biology 2013, 14(10):1194-1197. Bian X, Huang F, Wang H, Klefisch T, Muller R, Zhang Y. Heterologous production of glidobactins/luminmycins in Escherichia coli Nissle containing the glidobactin biosynthetic gene cluster from Burkholderia DSM7029. Chembiochem : a European journal of chemical biology 2014, 15(15):2221-2224. Bian X, Plaza A, Yan F, Zhang Y, Muller R. Rational and efficient site-directed mutagenesis of adenylation domain alters relative yields of luminmide derivatives in vivo. Biotechnology and bioengineering 2015. Gaitatzis N, Hans A, Muller R, Beyer S. The mtaA gene of the myxothiazol biosynthetic gene cluster from Stigmatella aurantiaca DW4/3-1 encodes a phosphopantetheinyl transferase that activates polyketide synthases and polypeptide synthetases. Journal of biochemistry 2001, 129(1):119-124. Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res 1988, 48(3):589-601. Bian X, Huang F, Stewart FA, Xia L, Zhang Y, Muller R. Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering. Chembiochem : a European journal of chemical biology 2012, 13(13):1946-1952. Li Zha, Matthew R. Wilson, Carolyn A. Brotherton, et al.. Characterization of Polyketide Synthase Machinery from the pks Island Facilitates Isolation of a Candidate Precolibactin. ACS Chem. Biol. 2016, 11(5):1287-95. Francesca Rosadi, Carla Fiorentini and Alessia Fabbri. Bacterial protein toxins in human cancers. FEMS Pathogens and Disease, 74, 2016, ftv105. Lee CH. Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives. Appl Microbiol Biotechnol 2012, 93:517-523.
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Fig. 1. Bacteria colonization at the 3rd day after the i.v. tail vein injection. Colony forming units (CFU) per gram (g) tissue were determined ex vivo. Statistical significance between the CFU of EcN in tumors and normal tissue was determined by F-test. P < 0.005.
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Figure 2. Luminescence signal at the 3rd day (left) and at the 11th day (right) after the i.v. injection of EcN containing pGB-tet-cm-luxABECD plasmid to express bioluminescence detected by using IVIS Spectrum system. The control animal (right position) received no bacteria.
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Figure 3. (A) Transient infection of human U-2 OS osteosarcoma cells with E. coli strains (Giemsa staining, 200 × magnification; phase contrast) [22]. (B) Cell relative vitality was measured by MTT assay. Error bars represented the standard deviation from three replicated experiments. Asterisks indicate significant differences between treatment and control as determined by Student’s t-test: **P < 0.01; ***P < 0.001.
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Figure 4. Therapeutic effect of colibactin-expressing EcN strains on tumor growth. Female NMRI nude mice (n = 8) bearing UT-SCC-5 human head and neck squamous tumors were treated by i.v. administration of PBS or 1 × 107 CFU EcN, EcN bearing pSUMtaA (EcN/pSUMtaA). Asterisks indicate significant differences between treatment and control as determined by Student’s t-test: **P < 0.01; ***P < 0.001.
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Figure 5. (A) Therapeutic effect of glidobactin- or lumimide-expressing EcN strains on tumor growth. Female NMRI nude mice (n = 8) bearing UT-SCC-5 human head and neck squamous tumors were treated by i.v. administration of PBS or 1 × 107 CFU EcN bearing pGB-amp-zeo-Ptet vector (EcN/pGB-amp-zeo-Ptet), EcN bearing glidobactin-expressing plasmid (EcN/pGB-amp-zeo-Ptet-glb), or EcN bearing lumimide-expressing plasmid (EcN/pGB-amp-zeo-Ptet-plu3263). (B) The body weight of the treated mice was measured after 18 days. Error bars represent the standard error of the mean.
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