Bacteriophages Displaying Anticancer Peptides in Combined Antibacterial and Anticancer Treatment

Krystyna Dąbrowska; Zuzanna Kaźmierczak; Joanna Majewska; Paulina Miernikiewicz; Agnieszka Piotrowicz; Joanna Wietrzyk; Dorota Lecion; Katarzyna Hodyra; Anna Nasulewicz-Goldeman; Barbara Owczarek; Andrzej Górski

Disclosures

Future Microbiol. 2014;9(7):861-869. 

In This Article

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 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).

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.

E. coli strain effectively expressing YIGSR-Hoc 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).

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. 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 metastases formation in lungs.

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.

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 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).

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.

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.

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 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.

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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