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Bacteriophages displaying anticancer peptides in combined antibacterial and anticancer treatment Krystyna Dąbrowska*,1, Zuzanna Kaźmierczak‡,1, Joanna Majewska‡,1, Paulina Miernikiewicz‡,1, Agnieszka Piotrowicz1, Joanna Wietrzyk1, Dorota Lecion1, Katarzyna Hodyra1, Anna Nasulewicz-Goldeman1, Barbara Owczarek1 & Andrzej Górski1 Abstract: Aims: Novel anticancer strategies have employed bacteriophages as drug carriers and display platforms for anticancer agents; however, bacteriophage-based platforms maintain their natural antibacterial activity. This study provides the assessment of combined anticancer (engineered) and antibacterial (natural) phage activity in therapies. Materials & methods: An in vivo BALB/c mouse model of 4T1 tumor growth accompanied by surgical wound infection was applied. The wounds were located in the areas of tumors. Bacteriophages (T4) were modified with anticancer Tyr–Ile–Gly–Ser–Arg (YIGSR) peptides by phage display and injected intraperitoneally. Results & conclusion: Tumor growth was decreased in mice treated with YIGSR-displaying phages. The acuteness of wounds, bacterial load and inflammatory markers in phages-treated mice were markedly decreased. Thus, engineered bacteriophages combine antibacterial and anticancer activity. The potential of peptides in cancer treatment is evident due to many studies evolving therapeutic strategies targeting the progression of tumor growth or metastasis formation. Peptides that can target cancer cells or interfere with their functions without affecting normal cells are being developed as an alternate strategy to conventional chemotherapy. Some of them have even entered Phase II clinical trials, becoming promising tools for medicine [1,2] , while others are constantly designed, developed and studied. One of the investigated classes of anticancer peptides are those interfering with adhesive interactions of cancer and normal cells. They are usually derived from extracellular matrix proteins mediating cell adhesion, such as fibronectin or laminin. Systematic screening of peptide motifs within laminin revealed that Tyr–Ile–Gly–Ser–Arg (YIGSR) peptide can substantially decrease tumor growth and experimental metastasis [3,4] ; thus, YIGSR represents a group of small but highly active peptides that can be used against cancer. Dynamic development of novel anticancer strategies has also applied bacteriophages: bacterial viruses are proposed as drug carriers and/or display platforms for various anticancer agents [5,6] . Phage display has become a powerful technology for selecting and amplifying peptides; it is also proposed as a relatively cheap and easy technology for production of active peptides in bulk amounts. Peptides (or proteins) can be presented on the surface of phage capsids as a result of molecular engineering. This may employ introduction of new sequences into the phage genome or incorporation of recombined proteins during capsid assembly [7–9] . The history of phage display started with filamentous phages [7] ; later large tailed phages were also employed, for example, T4 phage. T4 phage allows for exposure of both large proteins and small peptides as well as for dual exposure of two different active elements on the same phage [10,11] . Ren et al. showed that the T4
Keywords
• antibacterial • anticancer • infection • phage display • T4 phage • wound
1 Institute of Immunology & Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland *Author for correspondence: Tel.: +48 713 371 172 ext. 316; [email protected] ‡ Authors contributed equally. They all occupy second position and are listed in alphabetical order.
10.2217/FMB.14.50 © 2014 Future Medicine Ltd
Future Microbiol. (2014) 9(7), 861–869
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Research Article Dąbrowska, Kaźmierczak, Majewska et al. phage display system can be used for anticancer therapy by employment of the VEGF receptor 2 (VEGFR2) and construction of an anticancer vaccine [12] . At the same time, bacteriophage-based platforms or carriers maintain their natural antibacterial activity that is necessary for phage amplification. Antibacterial activity of phages makes these entities natural enemies of bacteria. Since phages kill bacteria in mechanisms different to those of antibiotics, they can be effective against multidrug-resistant bacterial strains. Nowadays, as we face an emerging problem of antibiotic resistance, bacteriophages are a serious alternative to the insufficient antibacterial arsenal [13,14] . Bacteriophage efficacy has been shown in combating various bacterial infections, including diarrhea, respiratory infections, wounds, urinary tract, bronchitis, otitis media, septicemia, encephalitis and bacterial meningitis, and others [13–15] . However, data on natural phage activity in a wide variety of medical problems are still limited; among those poorly recognized are infections accompanying cancer disease. Most life-endangering multidrug-resistant infections are acquired in hospitals and other healthcare units and cancer patients are often subjected to hospital treatment [16] , including surgery, injection or other invasive treatment. These procedures are related to a serious risk of acquiring drug-resistant bacterial infections. Thus antibacterial activity of bacteriophages may be particularly useful for this group of patients. These facts raise questions about the possibility of combining anticancer (engineered) and antibacterial (natural) phage activity in therapies. If effective, they can offer a novel strategy for anticancer therapy together with antibacterial protection. Here we present the assessment of such combined treatment in an in vivo model of tumor accompanied by surgical wound infection. Material & methods ●●Bacteriophages & bacteria
The T4 phage mutant T4Δhoc served as the platform for phage display (Microorganisms Collection, IIET: Institute of Immunology and Experimental Therapy, Wrocław, Poland) [17] . The host for phage propagation was Escherichia coli expression strain B834 (Novagen; Merck Millipore, Darmstadt, Germany) transformed with expression plasmids carrying the hoc gene in N-terminal fusion with anticancer
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peptides. GATEWAY recombination technology (Invitrogen; Life Technologies Polska, Warszawa, Poland) was used to prepare expression vectors (according to manufacturer’s instructions) in the expression vector pDEST24. As the control, Histag–Hoc fusion expressed from pDEST17 was used, Bacteriophage Laboratory IIET collection. All protein expressions and phage display cycles were conducted, as previously described [9,18] . Lipopolysaccharide (LPS) removal was performed with EndoTrap™ Blue (HyglosGmbH, Bernried am Starnberger See, Germany). ●●Animal model
All animal experiments were performed according to European Union Directive 2010/63/EU for animal experiments and were approved by the 1st Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland. The mice were bred in the Animal Breeding Centre of the IIET in SPF (specific pathogen free) conditions. The 4T1 mouse mammary gland carcinoma cell line was obtained from the American Type Culture Collection (MD, USA). The line is maintained in the Cell Culture Collection of our Institute (IIET). The model of tumor and metastases: 6-weekold BALB/c female mice (groups of 8–10 animals) were inoculated subcutaneously with 3–5 × 105 4T1 cells collected from in vitro culture in 0.1 ml of Hanks buffer. One week to 10 days later tumors become easily palpable with approx. diameter 5 mm. All tumors were measured and mice were divided into groups with similar mean tumor volume. Then mice were pre-treated intraperitoneally (ip.) with bacteriophage preparations (YIGSR phage: T4Δhoc phage displaying YIGSR peptide in N-terminal fusion with gpHoc, or control His phage: T4Δhoc phage displaying hexa-histidine peptide in N-terminal fusion with gpHoc) of 5 × 1010 PFU in 0.25 ml per mouse or with phosphate buffered saline (PBS). Next procedures were performed under general anesthesia with a mixture of ketamine hydrochloride (50 mg/kg, Ketamina 10%; Biowet, Puławy, Poland) and xylazine hydrochloride (20 mg/kg; XylaRiem, Riemser Arzneimittel AG, Germany). Fur was removed in the area of the tumor, and the skin was disinfected. Skin and tumor tissue were incised using a scalpel (the length of incision was approx. 4–5 mm). Afterward skin was sutured with 4–0 surgical sutures (Dexon-’S’; Polfa,
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Bacteriophages displaying anticancer peptides in combined antibacterial & anticancer treatment Poznań, Poland). Next, E. coli sensitive to T4 phage was introduced into the wound under the stitch (2 × 109 CFU/mouse in 0.05 ml of PBS). The day of surgery was designated as ‘day zero’ of the experiment. Phages YIGSR phage and control His phage (5 × 1010 PFU in 0.25 ml per mouse) were injected ip. daily for 14 days after the surgery (from day 1 to day 14). Tumors were measured in their maximum lengths and widths and the tumor volume was calculated as a2 x b/2 (a = smaller diameter, b = larger diameter) during the whole experiment [19] . The experiments were ended 21 days after the surgery; mice were sacrificed by cervical dislocation and the metastatic lung colonies were counted. Each experiment was repeated three-times; mean values and exemplary experiments are presented. ●●Cytokine assay
The progress of bacterial infection in wounds was assessed by monitoring inf lammation markers present in the blood. Murine blood was collected from the tail vein into heparinized tubes, under anesthesia. Concentrations of TNF-α were measured by commercially available ELISA kits (PeproTech). Mean values per group of animals are presented. ●●Microbiological & macroscopic assessment
of the infection progress in wounds
Effectiveness of antibacterial treatment with bacteriophages was assessed by the quantitative method for evaluation of bacteria in wounds as described by Lecion et al. [20] . Additionally, wounds were assessed every second day according to a lesion score. The score was calculated by summarizing three aspects of wound status, each rated from 0 to 3 (with mid-values possible). The assessed aspects were as follows: drainage (0: no exudates observed; 3: all surrounding skin covered by suppurative exudate), skin color around the wound (0: pale/normal; 3: intense red), size (0: wound completely closed/resurfaced; 3: wound with no signs of being covered by new skin); this scoring system was based on literature data with modifications [21,22] . Results ●●Phage-based platform displaying
anticancer peptides
T4 phage, an obligatory lytic parasite of E. coli, was used as the platform presenting anticancer peptide YIGSR. Previously, we recognized
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optimal localization and exposure of foreign elements on T4 capsid proteins as N-terminal fusion to Hoc protein [9] . Therefore, YIGSR was incorporated to the T4 head in this configuration. In order to obtain functional E. coli clones expressing YIGSR-Hoc fusion that can be assembled to the phage particles during phage construction inside bacteria (phage display in vivo), a new expression vector was created. Expression of YIGSR-Hoc in E. coli was tested and confirmed before it was used in the procedure of phage capsid modification by phage display (Figure 1) . E. coli strain effectively expressing YIGSRHoc was used for propagation of T4 phage mutant that did not produce wild Hoc protein (T4Δhoc); therefore YIGSR-Hoc fusions expressed from the expression vector could be effectively incorporated to the phage head (phage display in vivo) [8] . To amplify a control phage, an identical system was used but the Hoc protein fusion that was expressed in E. coli contained the His tag (a similar size peptide but without anticancer activity). M
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Figure 1. Expression of recombinant fusion of anticancer YIGSR peptide to bacteriophage Hoc protein. Bacterial cells Escherichia coli B834 were transformed with the expression vector containing hoc gene fused to YIGSR-coding sequence. Bacteria were grown at 37°C until OD600 0.7 was reached, then induced with 0.1 mM IPTG, expression was conducted in 37°C for 4 h. Then bacteria were harvested and analyzed by SDS-PAGE. In the first lane (M) marker of protein mass was presented, the second lane (−) presents the protein profile of bacteria that were not induced with IPTG (control), the third lane (+) presents the protein profile of bacteria that were induced with IPTG, YIGSR-Hoc fusion band was marked with an arrow. IPTG: Isopropyl β- d-1-thiogalactopyranoside; YIGSR: Tyr–Ile–Gly–Ser–Arg.
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Research Article Dąbrowska, Kaźmierczak, Majewska et al. Lysates produced by each type of in vivo phage display (YIGSR phage and His phage) were concentrated and purified by size exclusion chromatography [23] and large-pore membrane dialysis. Lipopolisaccharide (LPS) content in final preparations was determined, since LPS is a potent activator of many physiological processes and it may influence preparation action in vivo. Bacteriophage titers [24] obtained in the purified preparation ranged from 1011 to 1013 PFU/ml. LPS content normalized to 1011 PFU was 10 EU or lower, in order words, 5 EU per mouse or lower. This activity is an approx. equivalent of 0.5 μg of LPS per mouse or less (endotoxin activity in crude lysates was 105 –106 U per 1011 PFU). ●●Anticancer activity of YIGSR-presenting
bacteriophages
Anticancer activity of YIGSR-presenting bacteriophages (YIGSR phage) was investigated in vivo in a model of tumor surgery-related infection accompanied by a tumor recurrence. A
B 1400 Control E. coli YIGSR phage His phage YIGSR phage + E. coli His phage + E. coli
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Mice were bearing subcutaneous 4T1 tumors (murine mammary gland cancer); when the tumor become palpable, they were incised but not removed, skin was sutured with surgical sutures, and E. coli sensitive to T4 phage was introduced into the wound under the stitch. Tumor size was measured from day 1 to day 19 after the surgery. Tumor growth was decreased in mice treated with YIGSR phages (Figure 2), both in the animals whose wounds were infected with E. coli during the surgery and in the noninfected ones. At the end of the experiment (day 17 and day 19) the difference observed in tumor size between YIGSR phage-treated mice and control animals (nontreated or treated with His phage) was significant. This was correlated with higher accumulation of YIGSR phages in tumor in comparison to the control phage (2.8 × 108 PFU/g and 1.7 × 108 PFU/g, respectively). No significant differences were observed in m etastases formation in lungs.
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Figure 2. Mammary gland tumor growth in mice treated with YIGSR phages. Subcutaneous growth of 4T1 tumors (murine mammary gland cancer) was assessed in mice treated with YIGSR-presenting (or control) phages. Additionally, in the area of tumors surgery wounds were located, the wounds were infected or noninfected with Escherichia coli during the surgery. Panel (A) presents increase of the mean tumor weight in the course of time (from day 1 to day 19, then the experiment was terminated due to ethical reasons); panel (B) presents mean tumor weight in groups (bars) with SD (whiskers) on day 19 after the surgery. Control: noninfected mice injected daily with phosphate-buffered saline (PBS). E. coli: E. coli-infected mice injected daily with PBS. YIGSR phage: noninfected mice injected daily with YIGSR phage. His phage: noninfected mice injected daily with His phage (control). YIGSR phage + E. coli: E. coli-infected mice injected daily with YIGSR phage. His phage + E. coli: E. coli-infected mice injected daily with His phage (control). *Tumor growth was decreased in mice treated with YIGSR phages (both infected and noninfected mice) in comparison to control groups (ANOVA). YIGSR: Tyr–Ile–Gly–Ser–Arg.
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Bacteriophages displaying anticancer peptides in combined antibacterial & anticancer treatment
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Figure 3. Evaluation of wound status in mice infected with Escherichia coli and treated with phages by macroscopic assessment and scoring. Infected post-surgery wounds located in the areas of tumors were monitored with regard to their acuteness and the progress of healing; the assessed aspects were as follows: drainage (0–3), skin color/redness around the wound (0–3), wound size (0–3); maximum summarized score could be 9. (A) Presents mean score values in groups (bars) with SD (whiskers) 1 day after surgery; (B) Presents a decrease in the mean score values over time (from day 1 to day 13). Control: noninfected mice injected daily with phosphate-buffered saline (PBS). E. coli: E. coli-infected mice injected daily with PBS. YIGSR phage: noninfected mice injected daily with YIGSR phage. His phage: noninfected mice injected daily with His phage (control). YIGSR phage + E. coli: E. coli-infected mice injected daily with YIGSR phage. His phage + E. coli: E. coli-infected mice injected daily with His phage (control). *The wound score in E. coli-infected mice without phage treatment (‘E. coli’) was significantly higher in comparison to all other groups, n = 8 (ANOVA). YIGSR: Tyr–Ile–Gly–Ser–Arg. ●●Infected wound healing by the phage
applied for anticancer treatment
In parallel, infected post-surgery wounds located in the areas of tumors were monitored with regard to their acuteness and the progress of healing. Simple evaluation by macroscopic assessment and scoring revealed high effectiveness of systemically applied YIGSR-presenting bacteriophages in combating bacterial infection in wounds. Wounds in mice infected with E. coli but not treated with phages were in significantly worse condition in comparison to those subjected to phage treatment. Wounds in mice infected with E. coli and treated with phages were in similar condition to those in noninfected animals (Figure 3) . Macroscopic evaluation was in agreement with microbiological monitoring: the bacterial number was decreased in wounds
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of animals subjected to bacteriophage treatment (four- to nine-times lower) in comparison to those not treated (p = 0.041), although in mice without treatment bacterial load in wound was highly differentiated. Antibacterial activity of the phage modified with anticancer peptide (YIGSR phage) was similar to that modified with the control peptide (His phage); in noninfected animals’ wounds no E. coli was found (Figure 4) . Host reactivity to the progress of bacterial infection is correlated with production of inflammation markers, for example, TNF-α. Its systemic level rises even in response to a site-located infection. Thus, TNF-α was controlled in murine blood 1 and 5 days after the surgery. One day after the surgery, no significant differences between particular groups of mice
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Abundance of E. coli cells (cfu/ml)
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Figure 4. Bacterial levels in wounds of mice infected with Escherichia coli and treated with phages. A quantitative method for evaluation of bacteria in wounds by swabbing was applied; standard units equaling the number of bacteria in 1 ml of a test sample were presented (as described by Lecion et al.) [20]. Mean values (bars) and standard deviation (whiskers) were presented. Control: noninfected mice injected daily with phosphate-buffered saline (PBS). E. coli: E. coli-infected mice injected daily with PBS. YIGSR phage: noninfected mice injected daily with YIGSR phage. His phage: noninfected mice injected daily with His phage (control). YIGSR phage + E. coli: E. coli-infected mice injected daily with YIGSR phage. His phage + E. coli: E. coli-infected mice injected daily with His phage (control). *The difference was statistically significant in comparison to nontreated mice (group ‘E. coli’) (ANOVA). YIGSR: Tyr–Ile–Gly–Ser–Arg.
were noted. However, 5 days after the surgery, a marked increase in TNF-α level in the blood was observed in the group of animals with infected wounds without phage treatment. The inflammatory marker in infected animals which received phage treatment was significantly lower (in comparison to nontreated ones) and similar to that in noninfected groups (Figure 5) . Thus, evaluation of the selected inflammatory marker also confirmed good effectiveness of bacteriophages in antibacterial treatment. Discussion In this work, a complex in vivo model of tumors accompanied by surgical wound infection was applied. Bacteriophages modified with anticancer YIGSR peptides acted as the active agents. Evaluation of simultaneous anticancer and antibacterial activity of these bacteriophages revealed that both activities were demonstrated. Tumor growth was decreased in mice treated
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with YIGSR-displaying phages which correlated with phage accumulation in tumors. In the same mice, wounds infected with bacteria but not treated with phages were in significantly worse condition in comparison to those subjected to phage treatment. This worse condition was correlated with the higher bacterial load in the wound and with the higher systemic levels of inflammatory markers. These observations show the possibility of combination of anticancer (engineered) and antibacterial (natural) phage activity in therapies. Bacterial viruses are proposed as drug carriers and/or display platforms for various anticancer agents [5,6] ; their use in novel strategies of anticancer treatment probably offers more than anticancer tools. At the same time they may act as infection preventing or combating agents. In this work, bacteriophages were used before the surgery, so prevention is one of the mechanisms that may contribute to the final effect.
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Bacteriophages displaying anticancer peptides in combined antibacterial & anticancer treatment Importantly, phages were applied systemically (ip.), while the infection was located in a wound, but the beneficial effect of bacteriophages was still observed. This is in line with other studies that document good penetration of phages in mammalian tissues and organs [25] , which can be of relevance for the applicability of this combined activity in other locations of tumors and infections. One of limitations of this study that must be noticed is the fact that murine models cannot be directly transferred to human patients. Mice are known to differ in some elements of cancer processes as well as in their reactions to inflammation (e.g., other type of LPS receptors is engaged in the response at the molecular level). Also the
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type of tumor: the murine mammary gland cancer is a selected model while we bear in mind the multiplicity of possible cancer disease types. However, this model offered a good example of effective combination of anticancer and antibacterial activity exerted by engineered bacteriophages. Future studies of this activity should include human cancer cell lines, which will enable studies that are closer to human, as well as studies of other types of tumors. A further limitation for the beneficial ‘additional antibacterial effects’ of phage particles used as carriers is their specificity. Bacteriophages are usually highly specific, which allows the phage to counteract only infections caused by sensitive bacteria. Eventually, prevention of infection in patients will be limited. The
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Figure 5. Inflammatory marker (TNF-α) in mice with post-surgery wounds infected with Escherichia coli and treated with phages. The progress of bacterial infection in wounds was assessed by monitoring inflammation marker: TNF-α in the blood of infected and noninfected mice treated with bacteriophages; the assay was based on ELISA with a standard probe. Mean values (bars) and standard deviation (whiskers) were presented. Control: noninfected mice injected daily with phosphate-buffered saline (PBS). E. coli: E. coli-infected mice injected daily with PBS. YIGSR phage: noninfected mice injected daily with YIGSR phage. His phage: noninfected mice injected daily with His phage (control). YIGSR phage + E. coli: E. coli-infected mice injected daily with YIGSR phage. His phage + E. coli: E. coli-infected mice injected daily with His phage (control). *Serum TNF-α level in E. coli infected mice without phage treatment (‘E. coli’) was significantly higher in comparison to all other groups (ANOVA). YIGSR: Tyr–Ile–Gly–Ser–Arg.
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Research Article Dąbrowska, Kaźmierczak, Majewska et al. range of this limitation will be to some extend related to the range of a particular phage used as a display platform, since bacteriophages are differentiated in the range of their specificity. Here we proposed T4; in the case of T4-like phages this is E. coli and related bacteria. E. coli is also a pathogen responsible for post-surgery infections, including drug-resistant infections. However, in the future other types of bacteriophages used as platforms may widen or change the range of antibacterial protection in cancer patients. In this case, a wide range of antibacterial activity (in comparison to other phage strains) would be a positive designation for a phage as a platform or a carrier. In addition to the possible antibacterial and anticancer co-activity, this work also contributes to studies of safety aspects in bacteriophage therapy. No negative effects of the use of phages were observed in terms of tumor growth and metastases formation after treatment with neutral (His-modified) bacteriophages. This brings new data on natural phage activity in cancer disease, which further contributes to the prospect of antibacterial use of natural phages in cancer patients. Most life-endangering multidrugresistant infections are acquired in hospitals and other healthcare units, which often affect cancer patients. Therefore, alternative antibacterial strategies can be useful for this group of patients, both taking into account engineered phages and that of natural ones.
treatment due to combination of their natural and engineered activity. Both aspects should be considered in potential applications of phages as phage display platforms or drug carriers. Combining anticancer (engineered) and antibacterial (natural) phage activity in therapies offers a potential solution for the medicine. This study also suggests safety of antibacterial use of natural bacteriophages in coexisting cancer problems. Future perspective Bacteriophages have already offered innovative solutions in anticancer therapies, in vaccine design and other medical problems, so they will be proposed for increasing number of medical applications. Since no decrease in cancer disease frequency can be expected in realistic prognoses for upcoming years, all novel strategies will be urgently needed. According to recent prognostics, multidrug resistance will also be increasing challenge for the medicine and bacteriophages are recently proposed as a serious alternative to ineffective antibacterials. Most life-endangering multidrug-resistant infections are acquired in hospitals and other healthcare units, which often affect cancer patients. Therefore, medicine will take advantage of combination of anticancer (engineered) and antibacterial (natural) phage activity in therapies. Financial & competing interests disclosure
Conclusion Engineered bacteriophages provide a solution for combined antibacterial and anticancer
This work was supported by the Polish Ministry of Science (grant no. N N401 147539). P Miernikiewicz is a recipient of the ‘Start’ grant Foundation for Polish Science. The
Executive summary Anticancer activity of YIGSR-presenting bacteriophages ●●
Tumor growth was decreased in mice treated with Tyr–Ile–Gly–Ser–Arg (YIGSR)-displaying T4-like phages.
●●
No increase of tumor growth or metastases formation after the treatment with natural bacteriophages was observed.
Infected wound healing by the phage applied for anticancer treatment ●●
Both YIGSR-displaying and control phages helped in wound healing in mice infected with Escherichia coli.
●●
Improvement in wound healing was correlated with decreased systemic level of inflammatory markers.
Conclusion ●●
Engineered bacteriophages can be used for combined antibacterial and anticancer treatment due to combination of their natural and engineered activity.
●●
Dual activity of engineered bacteriophages must be considered in potential applications of phages as phage display platforms or drug carriers.
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This study suggests safety of antibacterial use of natural bacteriophages in coexisting cancer problems.
Future Microbiol. (2014) 9(7)
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Bacteriophages displaying anticancer peptides in combined antibacterial & anticancer treatment authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
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Miedzybrodzki R, Weber-Dabrowska B, Górski A. Application of microbiological quantitative methods for evaluation of changes in the amount of bacteria in patients with wounds and purulent fistulas subjected to phage therapy and for assessment of phage preparation effectiveness (in vitro studies). Adv. Med. Sci. 58, 257–264 (2013). 21 Bates-Jensen B, Sussman C. Tools to Measure
Wound Healing, Wound Care, a Collaborative Practice Manual for Health Professionals (4th Edition). Lippincott Williams and Wilkins, Baltimore, MD, USA (2012). 22 Sussman C, Swanson G. Utility of the
Sussman wound healing tool in predicting wound healing outcomes in physical therapy. Adv. Wound Care 10, 74–77 (1997). 23 Boratyński J, Syper D, Weber-Dąbrowska B,
Łusiak-Szelachowska M, Późniak G, Górski A. Preparation of endotoxin-free bacteriophages. Cell. Mol. Biol. Lett. 9, 253–259 (2004). 24 Collier LH. Topley and Wilson´s Principles of
Bacteriology, Virology and Immunity (8th Edition). Edward Arnold, London, UK (1990). 25 Dabrowska K, Switala-Jelen K, Opolski A,
Weber-Dabrowska B, Gorski A. Bacteriophage penetration in vertebrates. J. Appl. Microbiol. 98, 7–13 (2005).
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Bacteriophages displaying anticancer peptides in combined antibacterial and anticancer treatment Krystyna Dąbrowska*,1, Zuzanna Kaźmierczak‡,1, Joanna Majewska‡,1, Paulina Miernikiewicz‡,1, Agnieszka Piotrowicz1, Joanna Wietrzyk1, Dorota Lecion1, Katarzyna Hodyra1, Anna Nasulewicz-Goldeman1, Barbara Owczarek1 & Andrzej Górski1 Abstract: Aims: Novel anticancer strategies have employed bacteriophages as drug carriers and display platforms for anticancer agents; however, bacteriophage-based platforms maintain their natural antibacterial activity. This study provides the assessment of combined anticancer (engineered) and antibacterial (natural) phage activity in therapies. Materials & methods: An in vivo BALB/c mouse model of 4T1 tumor growth accompanied by surgical wound infection was applied. The wounds were located in the areas of tumors. Bacteriophages (T4) were modified with anticancer Tyr–Ile–Gly–Ser–Arg (YIGSR) peptides by phage display and injected intraperitoneally. Results & conclusion: Tumor growth was decreased in mice treated with YIGSR-displaying phages. The acuteness of wounds, bacterial load and inflammatory markers in phages-treated mice were markedly decreased. Thus, engineered bacteriophages combine antibacterial and anticancer activity. The potential of peptides in cancer treatment is evident due to many studies evolving therapeutic strategies targeting the progression of tumor growth or metastasis formation. Peptides that can target cancer cells or interfere with their functions without affecting normal cells are being developed as an alternate strategy to conventional chemotherapy. Some of them have even entered Phase II clinical trials, becoming promising tools for medicine [1,2] , while others are constantly designed, developed and studied. One of the investigated classes of anticancer peptides are those interfering with adhesive interactions of cancer and normal cells. They are usually derived from extracellular matrix proteins mediating cell adhesion, such as fibronectin or laminin. Systematic screening of peptide motifs within laminin revealed that Tyr–Ile–Gly–Ser–Arg (YIGSR) peptide can substantially decrease tumor growth and experimental metastasis [3,4] ; thus, YIGSR represents a group of small but highly active peptides that can be used against cancer. Dynamic development of novel anticancer strategies has also applied bacteriophages: bacterial viruses are proposed as drug carriers and/or display platforms for various anticancer agents [5,6] . Phage display has become a powerful technology for selecting and amplifying peptides; it is also proposed as a relatively cheap and easy technology for production of active peptides in bulk amounts. Peptides (or proteins) can be presented on the surface of phage capsids as a result of molecular engineering. This may employ introduction of new sequences into the phage genome or incorporation of recombined proteins during capsid assembly [7–9] . The history of phage display started with filamentous phages [7] ; later large tailed phages were also employed, for example, T4 phage. T4 phage allows for exposure of both large proteins and small peptides as well as for dual exposure of two different active elements on the same phage [10,11] . Ren et al. showed that the T4
Keywords
• antibacterial • anticancer • infection • phage display • T4 phage • wound
1 Institute of Immunology & Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław, Poland *Author for correspondence: Tel.: +48 713 371 172 ext. 316; [email protected] ‡ Authors contributed equally. They all occupy second position and are listed in alphabetical order.
10.2217/FMB.14.50 © 2014 Future Medicine Ltd
Future Microbiol. (2014) 9(7), 861–869
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Research Article Dąbrowska, Kaźmierczak, Majewska et al. phage display system can be used for anticancer therapy by employment of the VEGF receptor 2 (VEGFR2) and construction of an anticancer vaccine [12] . At the same time, bacteriophage-based platforms or carriers maintain their natural antibacterial activity that is necessary for phage amplification. Antibacterial activity of phages makes these entities natural enemies of bacteria. Since phages kill bacteria in mechanisms different to those of antibiotics, they can be effective against multidrug-resistant bacterial strains. Nowadays, as we face an emerging problem of antibiotic resistance, bacteriophages are a serious alternative to the insufficient antibacterial arsenal [13,14] . Bacteriophage efficacy has been shown in combating various bacterial infections, including diarrhea, respiratory infections, wounds, urinary tract, bronchitis, otitis media, septicemia, encephalitis and bacterial meningitis, and others [13–15] . However, data on natural phage activity in a wide variety of medical problems are still limited; among those poorly recognized are infections accompanying cancer disease. Most life-endangering multidrug-resistant infections are acquired in hospitals and other healthcare units and cancer patients are often subjected to hospital treatment [16] , including surgery, injection or other invasive treatment. These procedures are related to a serious risk of acquiring drug-resistant bacterial infections. Thus antibacterial activity of bacteriophages may be particularly useful for this group of patients. These facts raise questions about the possibility of combining anticancer (engineered) and antibacterial (natural) phage activity in therapies. If effective, they can offer a novel strategy for anticancer therapy together with antibacterial protection. Here we present the assessment of such combined treatment in an in vivo model of tumor accompanied by surgical wound infection. Material & methods ●●Bacteriophages & bacteria
The T4 phage mutant T4Δhoc served as the platform for phage display (Microorganisms Collection, IIET: Institute of Immunology and Experimental Therapy, Wrocław, Poland) [17] . The host for phage propagation was Escherichia coli expression strain B834 (Novagen; Merck Millipore, Darmstadt, Germany) transformed with expression plasmids carrying the hoc gene in N-terminal fusion with anticancer
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peptides. GATEWAY recombination technology (Invitrogen; Life Technologies Polska, Warszawa, Poland) was used to prepare expression vectors (according to manufacturer’s instructions) in the expression vector pDEST24. As the control, Histag–Hoc fusion expressed from pDEST17 was used, Bacteriophage Laboratory IIET collection. All protein expressions and phage display cycles were conducted, as previously described [9,18] . Lipopolysaccharide (LPS) removal was performed with EndoTrap™ Blue (HyglosGmbH, Bernried am Starnberger See, Germany). ●●Animal model
All animal experiments were performed according to European Union Directive 2010/63/EU for animal experiments and were approved by the 1st Local Committee for Experiments with the Use of Laboratory Animals, Wroclaw, Poland. The mice were bred in the Animal Breeding Centre of the IIET in SPF (specific pathogen free) conditions. The 4T1 mouse mammary gland carcinoma cell line was obtained from the American Type Culture Collection (MD, USA). The line is maintained in the Cell Culture Collection of our Institute (IIET). The model of tumor and metastases: 6-weekold BALB/c female mice (groups of 8–10 animals) were inoculated subcutaneously with 3–5 × 105 4T1 cells collected from in vitro culture in 0.1 ml of Hanks buffer. One week to 10 days later tumors become easily palpable with approx. diameter 5 mm. All tumors were measured and mice were divided into groups with similar mean tumor volume. Then mice were pre-treated intraperitoneally (ip.) with bacteriophage preparations (YIGSR phage: T4Δhoc phage displaying YIGSR peptide in N-terminal fusion with gpHoc, or control His phage: T4Δhoc phage displaying hexa-histidine peptide in N-terminal fusion with gpHoc) of 5 × 1010 PFU in 0.25 ml per mouse or with phosphate buffered saline (PBS). Next procedures were performed under general anesthesia with a mixture of ketamine hydrochloride (50 mg/kg, Ketamina 10%; Biowet, Puławy, Poland) and xylazine hydrochloride (20 mg/kg; XylaRiem, Riemser Arzneimittel AG, Germany). Fur was removed in the area of the tumor, and the skin was disinfected. Skin and tumor tissue were incised using a scalpel (the length of incision was approx. 4–5 mm). Afterward skin was sutured with 4–0 surgical sutures (Dexon-’S’; Polfa,
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Bacteriophages displaying anticancer peptides in combined antibacterial & anticancer treatment Poznań, Poland). Next, E. coli sensitive to T4 phage was introduced into the wound under the stitch (2 × 109 CFU/mouse in 0.05 ml of PBS). The day of surgery was designated as ‘day zero’ of the experiment. Phages YIGSR phage and control His phage (5 × 1010 PFU in 0.25 ml per mouse) were injected ip. daily for 14 days after the surgery (from day 1 to day 14). Tumors were measured in their maximum lengths and widths and the tumor volume was calculated as a2 x b/2 (a = smaller diameter, b = larger diameter) during the whole experiment [19] . The experiments were ended 21 days after the surgery; mice were sacrificed by cervical dislocation and the metastatic lung colonies were counted. Each experiment was repeated three-times; mean values and exemplary experiments are presented. ●●Cytokine assay
The progress of bacterial infection in wounds was assessed by monitoring inf lammation markers present in the blood. Murine blood was collected from the tail vein into heparinized tubes, under anesthesia. Concentrations of TNF-α were measured by commercially available ELISA kits (PeproTech). Mean values per group of animals are presented. ●●Microbiological & macroscopic assessment
of the infection progress in wounds
Effectiveness of antibacterial treatment with bacteriophages was assessed by the quantitative method for evaluation of bacteria in wounds as described by Lecion et al. [20] . Additionally, wounds were assessed every second day according to a lesion score. The score was calculated by summarizing three aspects of wound status, each rated from 0 to 3 (with mid-values possible). The assessed aspects were as follows: drainage (0: no exudates observed; 3: all surrounding skin covered by suppurative exudate), skin color around the wound (0: pale/normal; 3: intense red), size (0: wound completely closed/resurfaced; 3: wound with no signs of being covered by new skin); this scoring system was based on literature data with modifications [21,22] . Results ●●Phage-based platform displaying
anticancer peptides
T4 phage, an obligatory lytic parasite of E. coli, was used as the platform presenting anticancer peptide YIGSR. Previously, we recognized
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optimal localization and exposure of foreign elements on T4 capsid proteins as N-terminal fusion to Hoc protein [9] . Therefore, YIGSR was incorporated to the T4 head in this configuration. In order to obtain functional E. coli clones expressing YIGSR-Hoc fusion that can be assembled to the phage particles during phage construction inside bacteria (phage display in vivo), a new expression vector was created. Expression of YIGSR-Hoc in E. coli was tested and confirmed before it was used in the procedure of phage capsid modification by phage display (Figure 1) . E. coli strain effectively expressing YIGSRHoc was used for propagation of T4 phage mutant that did not produce wild Hoc protein (T4Δhoc); therefore YIGSR-Hoc fusions expressed from the expression vector could be effectively incorporated to the phage head (phage display in vivo) [8] . To amplify a control phage, an identical system was used but the Hoc protein fusion that was expressed in E. coli contained the His tag (a similar size peptide but without anticancer activity). M
–
+
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50 kDa YIGSR-Hoc
Figure 1. Expression of recombinant fusion of anticancer YIGSR peptide to bacteriophage Hoc protein. Bacterial cells Escherichia coli B834 were transformed with the expression vector containing hoc gene fused to YIGSR-coding sequence. Bacteria were grown at 37°C until OD600 0.7 was reached, then induced with 0.1 mM IPTG, expression was conducted in 37°C for 4 h. Then bacteria were harvested and analyzed by SDS-PAGE. In the first lane (M) marker of protein mass was presented, the second lane (−) presents the protein profile of bacteria that were not induced with IPTG (control), the third lane (+) presents the protein profile of bacteria that were induced with IPTG, YIGSR-Hoc fusion band was marked with an arrow. IPTG: Isopropyl β- d-1-thiogalactopyranoside; YIGSR: Tyr–Ile–Gly–Ser–Arg.
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Research Article Dąbrowska, Kaźmierczak, Majewska et al. Lysates produced by each type of in vivo phage display (YIGSR phage and His phage) were concentrated and purified by size exclusion chromatography [23] and large-pore membrane dialysis. Lipopolisaccharide (LPS) content in final preparations was determined, since LPS is a potent activator of many physiological processes and it may influence preparation action in vivo. Bacteriophage titers [24] obtained in the purified preparation ranged from 1011 to 1013 PFU/ml. LPS content normalized to 1011 PFU was 10 EU or lower, in order words, 5 EU per mouse or lower. This activity is an approx. equivalent of 0.5 μg of LPS per mouse or less (endotoxin activity in crude lysates was 105 –106 U per 1011 PFU). ●●Anticancer activity of YIGSR-presenting
bacteriophages
Anticancer activity of YIGSR-presenting bacteriophages (YIGSR phage) was investigated in vivo in a model of tumor surgery-related infection accompanied by a tumor recurrence. A
B 1400 Control E. coli YIGSR phage His phage YIGSR phage + E. coli His phage + E. coli
1000 800 600 400 200
1200 Tumor weight (mg)
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Mice were bearing subcutaneous 4T1 tumors (murine mammary gland cancer); when the tumor become palpable, they were incised but not removed, skin was sutured with surgical sutures, and E. coli sensitive to T4 phage was introduced into the wound under the stitch. Tumor size was measured from day 1 to day 19 after the surgery. Tumor growth was decreased in mice treated with YIGSR phages (Figure 2), both in the animals whose wounds were infected with E. coli during the surgery and in the noninfected ones. At the end of the experiment (day 17 and day 19) the difference observed in tumor size between YIGSR phage-treated mice and control animals (nontreated or treated with His phage) was significant. This was correlated with higher accumulation of YIGSR phages in tumor in comparison to the control phage (2.8 × 108 PFU/g and 1.7 × 108 PFU/g, respectively). No significant differences were observed in m etastases formation in lungs.
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Figure 2. Mammary gland tumor growth in mice treated with YIGSR phages. Subcutaneous growth of 4T1 tumors (murine mammary gland cancer) was assessed in mice treated with YIGSR-presenting (or control) phages. Additionally, in the area of tumors surgery wounds were located, the wounds were infected or noninfected with Escherichia coli during the surgery. Panel (A) presents increase of the mean tumor weight in the course of time (from day 1 to day 19, then the experiment was terminated due to ethical reasons); panel (B) presents mean tumor weight in groups (bars) with SD (whiskers) on day 19 after the surgery. Control: noninfected mice injected daily with phosphate-buffered saline (PBS). E. coli: E. coli-infected mice injected daily with PBS. YIGSR phage: noninfected mice injected daily with YIGSR phage. His phage: noninfected mice injected daily with His phage (control). YIGSR phage + E. coli: E. coli-infected mice injected daily with YIGSR phage. His phage + E. coli: E. coli-infected mice injected daily with His phage (control). *Tumor growth was decreased in mice treated with YIGSR phages (both infected and noninfected mice) in comparison to control groups (ANOVA). YIGSR: Tyr–Ile–Gly–Ser–Arg.
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Bacteriophages displaying anticancer peptides in combined antibacterial & anticancer treatment
A
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Figure 3. Evaluation of wound status in mice infected with Escherichia coli and treated with phages by macroscopic assessment and scoring. Infected post-surgery wounds located in the areas of tumors were monitored with regard to their acuteness and the progress of healing; the assessed aspects were as follows: drainage (0–3), skin color/redness around the wound (0–3), wound size (0–3); maximum summarized score could be 9. (A) Presents mean score values in groups (bars) with SD (whiskers) 1 day after surgery; (B) Presents a decrease in the mean score values over time (from day 1 to day 13). Control: noninfected mice injected daily with phosphate-buffered saline (PBS). E. coli: E. coli-infected mice injected daily with PBS. YIGSR phage: noninfected mice injected daily with YIGSR phage. His phage: noninfected mice injected daily with His phage (control). YIGSR phage + E. coli: E. coli-infected mice injected daily with YIGSR phage. His phage + E. coli: E. coli-infected mice injected daily with His phage (control). *The wound score in E. coli-infected mice without phage treatment (‘E. coli’) was significantly higher in comparison to all other groups, n = 8 (ANOVA). YIGSR: Tyr–Ile–Gly–Ser–Arg. ●●Infected wound healing by the phage
applied for anticancer treatment
In parallel, infected post-surgery wounds located in the areas of tumors were monitored with regard to their acuteness and the progress of healing. Simple evaluation by macroscopic assessment and scoring revealed high effectiveness of systemically applied YIGSR-presenting bacteriophages in combating bacterial infection in wounds. Wounds in mice infected with E. coli but not treated with phages were in significantly worse condition in comparison to those subjected to phage treatment. Wounds in mice infected with E. coli and treated with phages were in similar condition to those in noninfected animals (Figure 3) . Macroscopic evaluation was in agreement with microbiological monitoring: the bacterial number was decreased in wounds
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of animals subjected to bacteriophage treatment (four- to nine-times lower) in comparison to those not treated (p = 0.041), although in mice without treatment bacterial load in wound was highly differentiated. Antibacterial activity of the phage modified with anticancer peptide (YIGSR phage) was similar to that modified with the control peptide (His phage); in noninfected animals’ wounds no E. coli was found (Figure 4) . Host reactivity to the progress of bacterial infection is correlated with production of inflammation markers, for example, TNF-α. Its systemic level rises even in response to a site-located infection. Thus, TNF-α was controlled in murine blood 1 and 5 days after the surgery. One day after the surgery, no significant differences between particular groups of mice
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Research Article Dąbrowska, Kaźmierczak, Majewska et al.
Abundance of E. coli cells (cfu/ml)
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Figure 4. Bacterial levels in wounds of mice infected with Escherichia coli and treated with phages. A quantitative method for evaluation of bacteria in wounds by swabbing was applied; standard units equaling the number of bacteria in 1 ml of a test sample were presented (as described by Lecion et al.) [20]. Mean values (bars) and standard deviation (whiskers) were presented. Control: noninfected mice injected daily with phosphate-buffered saline (PBS). E. coli: E. coli-infected mice injected daily with PBS. YIGSR phage: noninfected mice injected daily with YIGSR phage. His phage: noninfected mice injected daily with His phage (control). YIGSR phage + E. coli: E. coli-infected mice injected daily with YIGSR phage. His phage + E. coli: E. coli-infected mice injected daily with His phage (control). *The difference was statistically significant in comparison to nontreated mice (group ‘E. coli’) (ANOVA). YIGSR: Tyr–Ile–Gly–Ser–Arg.
were noted. However, 5 days after the surgery, a marked increase in TNF-α level in the blood was observed in the group of animals with infected wounds without phage treatment. The inflammatory marker in infected animals which received phage treatment was significantly lower (in comparison to nontreated ones) and similar to that in noninfected groups (Figure 5) . Thus, evaluation of the selected inflammatory marker also confirmed good effectiveness of bacteriophages in antibacterial treatment. Discussion In this work, a complex in vivo model of tumors accompanied by surgical wound infection was applied. Bacteriophages modified with anticancer YIGSR peptides acted as the active agents. Evaluation of simultaneous anticancer and antibacterial activity of these bacteriophages revealed that both activities were demonstrated. Tumor growth was decreased in mice treated
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with YIGSR-displaying phages which correlated with phage accumulation in tumors. In the same mice, wounds infected with bacteria but not treated with phages were in significantly worse condition in comparison to those subjected to phage treatment. This worse condition was correlated with the higher bacterial load in the wound and with the higher systemic levels of inflammatory markers. These observations show the possibility of combination of anticancer (engineered) and antibacterial (natural) phage activity in therapies. Bacterial viruses are proposed as drug carriers and/or display platforms for various anticancer agents [5,6] ; their use in novel strategies of anticancer treatment probably offers more than anticancer tools. At the same time they may act as infection preventing or combating agents. In this work, bacteriophages were used before the surgery, so prevention is one of the mechanisms that may contribute to the final effect.
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Bacteriophages displaying anticancer peptides in combined antibacterial & anticancer treatment Importantly, phages were applied systemically (ip.), while the infection was located in a wound, but the beneficial effect of bacteriophages was still observed. This is in line with other studies that document good penetration of phages in mammalian tissues and organs [25] , which can be of relevance for the applicability of this combined activity in other locations of tumors and infections. One of limitations of this study that must be noticed is the fact that murine models cannot be directly transferred to human patients. Mice are known to differ in some elements of cancer processes as well as in their reactions to inflammation (e.g., other type of LPS receptors is engaged in the response at the molecular level). Also the
Research Article
type of tumor: the murine mammary gland cancer is a selected model while we bear in mind the multiplicity of possible cancer disease types. However, this model offered a good example of effective combination of anticancer and antibacterial activity exerted by engineered bacteriophages. Future studies of this activity should include human cancer cell lines, which will enable studies that are closer to human, as well as studies of other types of tumors. A further limitation for the beneficial ‘additional antibacterial effects’ of phage particles used as carriers is their specificity. Bacteriophages are usually highly specific, which allows the phage to counteract only infections caused by sensitive bacteria. Eventually, prevention of infection in patients will be limited. The
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Figure 5. Inflammatory marker (TNF-α) in mice with post-surgery wounds infected with Escherichia coli and treated with phages. The progress of bacterial infection in wounds was assessed by monitoring inflammation marker: TNF-α in the blood of infected and noninfected mice treated with bacteriophages; the assay was based on ELISA with a standard probe. Mean values (bars) and standard deviation (whiskers) were presented. Control: noninfected mice injected daily with phosphate-buffered saline (PBS). E. coli: E. coli-infected mice injected daily with PBS. YIGSR phage: noninfected mice injected daily with YIGSR phage. His phage: noninfected mice injected daily with His phage (control). YIGSR phage + E. coli: E. coli-infected mice injected daily with YIGSR phage. His phage + E. coli: E. coli-infected mice injected daily with His phage (control). *Serum TNF-α level in E. coli infected mice without phage treatment (‘E. coli’) was significantly higher in comparison to all other groups (ANOVA). YIGSR: Tyr–Ile–Gly–Ser–Arg.
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Research Article Dąbrowska, Kaźmierczak, Majewska et al. range of this limitation will be to some extend related to the range of a particular phage used as a display platform, since bacteriophages are differentiated in the range of their specificity. Here we proposed T4; in the case of T4-like phages this is E. coli and related bacteria. E. coli is also a pathogen responsible for post-surgery infections, including drug-resistant infections. However, in the future other types of bacteriophages used as platforms may widen or change the range of antibacterial protection in cancer patients. In this case, a wide range of antibacterial activity (in comparison to other phage strains) would be a positive designation for a phage as a platform or a carrier. In addition to the possible antibacterial and anticancer co-activity, this work also contributes to studies of safety aspects in bacteriophage therapy. No negative effects of the use of phages were observed in terms of tumor growth and metastases formation after treatment with neutral (His-modified) bacteriophages. This brings new data on natural phage activity in cancer disease, which further contributes to the prospect of antibacterial use of natural phages in cancer patients. Most life-endangering multidrugresistant infections are acquired in hospitals and other healthcare units, which often affect cancer patients. Therefore, alternative antibacterial strategies can be useful for this group of patients, both taking into account engineered phages and that of natural ones.
treatment due to combination of their natural and engineered activity. Both aspects should be considered in potential applications of phages as phage display platforms or drug carriers. Combining anticancer (engineered) and antibacterial (natural) phage activity in therapies offers a potential solution for the medicine. This study also suggests safety of antibacterial use of natural bacteriophages in coexisting cancer problems. Future perspective Bacteriophages have already offered innovative solutions in anticancer therapies, in vaccine design and other medical problems, so they will be proposed for increasing number of medical applications. Since no decrease in cancer disease frequency can be expected in realistic prognoses for upcoming years, all novel strategies will be urgently needed. According to recent prognostics, multidrug resistance will also be increasing challenge for the medicine and bacteriophages are recently proposed as a serious alternative to ineffective antibacterials. Most life-endangering multidrug-resistant infections are acquired in hospitals and other healthcare units, which often affect cancer patients. Therefore, medicine will take advantage of combination of anticancer (engineered) and antibacterial (natural) phage activity in therapies. Financial & competing interests disclosure
Conclusion Engineered bacteriophages provide a solution for combined antibacterial and anticancer
This work was supported by the Polish Ministry of Science (grant no. N N401 147539). P Miernikiewicz is a recipient of the ‘Start’ grant Foundation for Polish Science. The
Executive summary Anticancer activity of YIGSR-presenting bacteriophages ●●
Tumor growth was decreased in mice treated with Tyr–Ile–Gly–Ser–Arg (YIGSR)-displaying T4-like phages.
●●
No increase of tumor growth or metastases formation after the treatment with natural bacteriophages was observed.
Infected wound healing by the phage applied for anticancer treatment ●●
Both YIGSR-displaying and control phages helped in wound healing in mice infected with Escherichia coli.
●●
Improvement in wound healing was correlated with decreased systemic level of inflammatory markers.
Conclusion ●●
Engineered bacteriophages can be used for combined antibacterial and anticancer treatment due to combination of their natural and engineered activity.
●●
Dual activity of engineered bacteriophages must be considered in potential applications of phages as phage display platforms or drug carriers.
●●
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This study suggests safety of antibacterial use of natural bacteriophages in coexisting cancer problems.
Future Microbiol. (2014) 9(7)
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Bacteriophages displaying anticancer peptides in combined antibacterial & anticancer treatment authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research The authors state that they have obtained appropriate insti tutional review board approval or have followed the princi ples outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
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