Purpose

To determine whether a double mutant Herpes Simplex Virus type I, KM110red (KM110r), is an effective and safe oncolytic virus therapy by exploring its effects on cancerous bone (osteosarcoma or U2OS) cells and non-cancerous precursor bone (human fetal osteoblast or hFOB) cells, and to determine whether induced genetic differentiation alters the permissiveness of hFOB cells to KM110r.

Hypotheses

Genetically engineered KM110r will target and selectively destroy all U2OS cells and will not be toxic to hFOB cells.  KM110r will be an effective and safe potential cancer treatment when administered appropriately. U2OS living cell counts will decrease post KM110r infection, while undifferentiated hFOB cells will continue to grow at constant growth rates with no identified infection.

The point during in vitro cell differentiation at which the hFOB cells respond to infection by KM110r will be identified and the potential underlying genetic changes investigated. This will enhance understanding of which genetic changes permit oncolytic virus infection with the intention of identifying the point along the differentiation pathway to maximize treatment efficacy.

Procedure

U2OS cells are moderately differentiated bone cancer cells that are genetically recombined with bioluminescent green fluorescent protein (GFP) to make proteins in the cells glow fluorescent green in order to identify them under ultraviolet light microscopy. hFOB cells are normal undifferentiated pre-cursor bone cells containing a temperature sensitive mutation (tsA58) that drives differentiation in response to temperature change. KM110r is also genetically recombined with a bioluminescent fluorescent protein called red fluorescent protein (RFP) which causes any infected cells to appear red under fluorescent microscopy. 

The cells were cultured in 6-well dishes which contained two wells each of hFOB, U2OS, and MIX (50% U2OS, 50% hFOB) seeded at concentrations of 1.3x10⁵ cells/mL. One of each group was infected using KM110r while their counterparts were mock-infected as the negative-control. Three experimental trials were conducted at incubation temperatures of 34°C, 37°C, and 39°C to induce genetic differentiation in the hFOB cells. Each temperature trial was completely repeated to ensure reliability of the data.

Each experimental trial employed fluorescent microscopy to conduct a proliferation assay over the course of four (4) time periods spanning a five (5) day infection (since full differentiation is expected in this time period). During each time period three (3) areas in each of the wells were imaged in bright field light, GFP and RFP channels. Approximately forty (40) images were taken each day, totaling almost one-thousand (1000) images over the course of experimentation. The cells in each image were counted and morphologically analyzed.

          hFOB cells grew at almost identical rates regardless of incubation temperature or status of infection (Figure 3), as verified by a student’s t-test. Throughout the entire experimentation hFOB+ cells were not permissive to KM110r.                                                                                             


Figure 3

Uninfected co-cultures (MIX-) cells grew at general rates of increase regardless of incubation temperature and were consistently lower than U2OS- and higher than hFOB uninfected (hFOB-).  Infected co-cultures (MIX+) cells, at 37°C and 39°C incubation remain almost constant, but MIX+ cells at 34°C incubation steadily decrease until time period 3 when it plateaus.

            Visual analysis of hFOB cells under bright field microscopy showed evidence of morphological changes among the 3 different incubation temperatures, indicating that genetic differentiation was occurring at 39°C incubation as reported[2].

Conclusions

            The first major finding of this experiment confirms that KM110r is able to target and selectively destroy U2OS cells and is not toxic to hFOB cells over the range of temperatures 34°C, 37°C and 39°C where hFOB cells are differentiating.  At 34°C and 37°C, it was clear that KM110r could successfully eradicate all of the cancerous U2OS cells within the experimentation period.  However, KM110r did not effectively destroy all cancerous cells at 39°C.  This strongly suggests that at 39°C KM110r infection of U2OS has been altered.  Furthermore, at 37°C the cancerous cells were completely eradicated at

has been altered.  Furthermore, at 37°C the cancerous cells were completely eradicated at time period 3, one entire time period earlier than at 34°C. The significant practical application of this discovery is that as a cancer therapy, use of KM110r is optimal when administered at 37°C—or physiological body temperature—since 34°C conditions delay cell destruction and 39°C conditions do not permit successful termination of the cancerous cells. Therefore, the effectiveness of KM110r as an oncolytic virus therapy could be severely impaired if the body temperature of the cancer patient becomes hyperthermic or hypothermic, i.e. the patient spikes a fever or experiences dangerously low body temperatures, respectively.      

            The second major finding is that the results did not support identification of a specific point during in vitro cell differentiation that the hFOB cells were infected by KM110r. hFOB cells were not permissive to KM110r at any temperature condition. This reveals an advantageous quality of the virus from a gene therapeutic perspective. The differentiation that occurred in the hFOB cells at 39°C are changes that would generally occur in a normal precursor bone cells, meaning that the virus is able to continue to ignore these non-cancerous cells despite differentiation. The fact that KM110r was able to ignore hFOB cells over the course of full differentiation strongly affirms the safety of this genetically engineered virus. This is an extremely valuable finding since non-cancerous cell death must be inhibited in order to avoid the negative side effects of conventional cancer therapies such as chemotherapy. This suggests that KM110r satisfies the highly sought-after criteria in the discovery of effective cancer therapies: the ability to completely terminate cancerous cells and the ability to ignore and avoid harm to non-cancerous cells.

Acknowledgments

            The following are acknowledged for their support and contributions to this project.

Dr. J. Hummel – Post-doctoral fellow, Pathology and Molecular Medicine, McMaster University; Dr. K. Mossman – Assistant Professor, Pathology and Molecular Medicine, McMaster University; Dr. J Gauldie – Professor and Chair, Pathology and Molecular Medicine, McMaster University;

K. Moyle – Mentor; C. Raso – Mother.