Bacteriophages in Cancer Biology and Treatment

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Electron micrograph of the Ebola Zaire virus. This was the first photo ever taken of the virus, on 10/13/1976. By Dr. F.A. Murphy, now at U.C. Davis, then at the CDC.

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The use of Bacteriophages for drug delivery

Figure 1: fUSE5-ZZ-chFRP5, fUSE5-ZZ-trastuzumab and control phages fUSE5-ZZ-human IgG were added into wells that contain ErbB2 over-expressing SKBR3 cell line (black bars) or human mammary carcinoma MDA-MB231 cells as control cell line (gray bars) that express a low level of ErbB2.

Figure 2: A. Immunofluorescence Staining: (a) control fUSE5-ZZ-human IgG phages (b), no phage, only antibodies (c-l) serial cuts of A431 cells that were treated with fUSE5-ZZ-chFRP5 phages. Membranes were labelled by using red fluorescent CM-Dil cell tracker (MoBiTec, Göttingen, Germany). Phages were detected by monoclonal mouse anti-M13 followed by incubation with FITC conjugated goat anti-mouse IgG (green fluorescence). B. Evaluation of internalization of FITC labelled, drug conjugated phages into SKBR3 cells using confocal microscopy. a and b fUSE5-ZZ-chFRP5 phages, c and d control fUSE5-ZZ complexed with normal human IgG. As a reference, actin filaments with the cells were labelled by using red fluorescent dye Phalloidin (Sigma, Israel). Phages were labelled directly by FITC. Phages were added to the cells for 2 hr before analysis by confocal microscopy. Panels a and c show the green fluorescence of Fluorescein while in panels b and d the green fluorescence is overlaid on the red fluorescence.

Chemotherapy drugs, while shown to have anti-tumor effects, tend to result in severe toxicity and widespread distribution throughout the body: notably damaging healthy and malignant cells. New research has started to focus on using bacteriophages as an individualized drug-carrying anti-cancer therapy. The therapy would be targeted, based on genetically-modifying and chemically manipulating filamentous bacteriophages. In Bar et al. 2008, the phages were modified to display a host-specificity-conferring ligand, and carry a cytotoxic drug by chemical conjugation

Antibodies anti ErbB2 and anti ERGR were used to direct bacteriophages to cancer cells. ErbB2 is a a well known protein that is found to be over expressed in approximately 20% of invasive breast cancers. ERGR is a receptor protein that is found on the surface of cells that causes some cells to duplicate if an epidermal growth factor binds to it. They both belong to the epidermal growth factor receptor (EGFR) family (19). They are both common proteins that tend to be associated with the growth of cancers. The cell lines used in Bar et al. 2008 were SKBR3 and MDA-MB23 (human breast carcinoma cell lines), A431 (human epidermoid carcinoma), and HEK293 (human kidney). Phages were engineered to be linked to respective antibodies.

Assessing The Ability of the Engineered Antibody-Phages to go to Their Respective Receptors A whole cell ELISA was performed in order to assess the binding of phage-attached antibodies (see figure 1.) MDA-MB231 cells express a low level of ErbB2 proteins, while SKBR3 express a much higher level of ErbB2 proteins. As shown, one can see that the antibody-complexed phages showed cell-specific binding. The engineered fUSE5-ZZ-chFRP5 phage had higher rates of binding to receptors on SKBR3 and even MDA-MB231 than the control phage fUSE5-ZZ-human IgG. This shows that the engineered phages are capable of attaching to their respective receptors through the use of antibodies.

Assessing Engineered Antibody-Phages as Drug Carriers In order to check if phages could effectively deliver drugs, researchers had to verify that they could enter into host cells. One way this was done was by visualizing phage internalization using confocal microscopy and immunofluorescence staining (See Figure 2). Bar et al. 2008 engineered its phages to carry hygromycin inside cancer cells. Hygromycin is an antibiotic produced by the bacterium Streptomyces hygroscopicus. It kills bacteria, fungi, and interestingly inhibits the growth of some eukaryotic cells. Therefore, by engineering phages to carry the this drug into cancer cells, it is possible further growth can be stopped. The assay was done at 2, 12, and 24 hours in order to track the rate at which phages entered malignant cells. Surprisingly, internalization of hygromycin was the greatest at 2 hours, and diminished over time. Figure 2, images "c" and "d" show the attempt of internalization of hygromycin conjugated phages complexed with with human IgG. They were unsuccessful, filling their role as controls. Therefore, data supports that engineered antibody carrying phages are capable of transporting large amounts of shipments of a drug into a compromised cell.

Changing the Tumor Microenvironment

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Phage Display Methods and Tumor-specific Antibody–receptor Pairs

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Activating the Innate Immune System

Fig. 1 Tumor growth and immune response. An overview of the different key factors governing tumor formation, progression, and immune evasion. The numbers in parentheses represent the relevant references in support of the statements made.

Tumors have become increasingly more clever in their ways of evading the immune system.

In both animal and human models, tumors have been discovered to actively suppress and manipulate immune responses. Usually our immune system's CD8+ cytotoxic T cells (CTL) and CD4 + helper T (Th)1 cells are capable of warding off cancer development by producing interferons (IFN)-�and cytotoxins (SCB). Cytotoxic T cells are a type of white blood cell that kill cancer cells, or cells that are dysfunctional. Interferons are signaling proteins released by infected host cells that alert nearby cells to strengthen up their defenses. The continual production of these type of cells leads to chronic inflammation, which has been tied to the spread of cancer. Therefore, evidence shows that when our immune system's initial responses fail to eradicate tumor cells, the flood of immunoregulatory cells leads to the resurgence of a "smarter" cancer.

Tumors have found a way to remain "hidden" from immune cells by utilizing

References

Authored for BIOL 238 Microbiology, taught by Joan Slonczewski, 2018, Kenyon College.