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

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 ref-834 (Novagen; Merck Millipore, Darmstadt, Germany) transformed with expression plasmids carrying the hoc gene in N-terminal fusion with anticancer 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-week-old 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, 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 inflammation 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]

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....