The three classic arms of cancer therapy are surgical resection, chemotherapeutics, and radiation. As these fields have progressed we have greatly improved cancer survival rates in both companion animals and humans. However, with the complexities of neoplastic disease and their ability to further mutate due to genomic instability, these classic approaches are not always successful and often have undesirable adverse effects. Oncolytic virotherapy has the potential to be highly effective with minimal adverse effects due to targeted lysis of neoplastic cells. Studies were performed to evaluate the oncolytic effects of recombinant myxoma virus (MYXV) on canine osteosarcoma and soft tissue sarcoma primary cell cultures. The tumor samples were obtained following surgical excision. Once cultured, cells were evaluated for vimentin using immunocytochemistry and for alkaline phosphatase activity using nitroblue tetrazolium chloride/5-bromo-4-chloro-3- indolyl phosphate toluidine solution. The cancer cell-lines were then inoculated with recombinant myxoma virus expressing a red fluorescent protein and collected after 48-hours. Cytopathic effects were assessed at 24-hour and 48-hour time points via fluorescent microscopy. Ability of the virus to replicate within the cancer cells was confirmed via Western immunoblot detection of late MYXV protein production. The findings of this study suggest that canine osteosarcoma and soft tissue sarcoma primary cell cultures are lacking antiviral mechanisms and susceptible to MYXV infection leading to oncolysis.

This project was partially funded by the Young Investigator Grant Program in the Center for Companion Animal Studies. Special thanks to: •  Veronica Kinn for all her guidance in the lab. •  Karyn Wesley for the microscopic images of OSA. •  Katelyn Polemi for Cell Viability Assay data. •  Sarah Leavell for Immunocytochemistry instruction.

1.  Canine cancer cell lines can be isolated from resected tumors and cultured successfully.

2.  All the cultured cell lines were vimentin positive, cytokeratin negative indicating they are of mesenchymal origin as suspected.

3.  Alkaline Phosphatase staining of the two OSA cell lines were both weakly positive further supporting the suspicion that they are OSA cells (data not shown).

4.  All the cell lines were susceptible to infection with myxoma virus shown with cytopathic effect assay and Western Immunoblot detection of a late MYXV protein m130R.

5.  Cell viability is significantly decreased when samples are infected with MYXV.

Canine tumor cell isolation •  Canine cancer tissues were surgically removed. •  Tissues were processed and incubated in cell culture growth media until confluent.

Cell characterization •  Cells were evaluated for alkaline phosphatase activity. •  Immunocytochemistry analysis for vimentin and cytokeratin was performed to determine if

cultured cells were of epithelial or mesenchymal origin.

Viral Inoculation Procedures •  Cytopathic Effect Assay – Cultured cells were incubated with MYXV at a multiplicity of

infection (MOI) = 1 for 24 and 48 hours to determine if cells were susceptible to infection and lysis.

•  Bradford Assay – Protein extracted from cells at 48 h post-MYXV inoculation was quantified. This assay detects an absorbance shift of Coomassie Brilliant Blue dye, the protein binds to the dye causing a color shift proportional to the concentration of protein present in each sample.

•  Western Immunoblot – To determine if a MYXV protein expressed at a late time point during MYXV replication (m130R) was present in MYXV infected samples and absent in Mock-infected samples, an equivalent amount of protein for each sample was loaded onto an acrylamide gel. The gel was probed using a primary polyclonal rabbit anti-m130R IgG and a secondary horseradish peroxidase-conjugated goat anti-rabbit IgG.

•  Cell Viability Assay – A Cell Titer Blue Assay (Promega) was used to quantify cell viability in mock-infected cells and cells incubated for 72 hours with MYXV (MOI = 1). This assay uses conversion of resazurin to resorufin which emits fluorescence to measure the metabolic capacity of cells.

Objectives 1.  Culture cell explants from surgically excised canine tumors. 2.  Characterize and compare cultured cells and tumor sections using established

molecular techniques. 3.  Inoculate cells with recombinant myxoma virus (MYXV) and evaluate cultures for

viral replication and cytopathic effects. Hypotheses 1.  Primary canine cancer cells that have the same molecular characteristics as the

tumor they were isolated from can be cultured. 2.  Primary canine cancer cells will be susceptible to myxoma virus infection and lysis.

Limitations •  We have yet to prove that the cells cultured from our

canine tumor explants are truly neoplastic. •  We have yet to determine what antiviral defense

mechanisms have been lost in our cell cultures rendering them susceptible to MYVX infection and lysis.

Recommendations and Future Plans •  Perform additional cell viability assays for

quantification of cell death for all cell cultures. •  Determine if antiviral mechanisms are lacking in the

primary canine cancer cell cultures (e.g. evaluate type I interferon RNA levels and TNF-alpha production in response to MYXV infection).

•  Perform genomic analysis to determine if cultured cells are unique canine cell lines.

•  Test cells for the ability to form tumors in nude mice •  Evaluate the effects of the recombinant myxoma virus

in tumors grown in nude mice.

The molecular weight of the protein detected in infected cells (IN) is consistent with that of the MYXV protein, m130R, expressed at late time points of viral replication. Numbers to the left are molecular weight in kilodaltons.

For each cell line images on left are

control MOCK-infected samples, images on

right are MYVX infected samples.

Cytopathic changes and decreased confluency were observed in all

MYXV infected samples.

MYXV-RFP was

detected in all samples incubated with virus.

Cell viability was significantly decreased in MYXV-infected samples (MOI = 1).

* P values = 0.0286

1.  OSA Holly 2.  OSA King 3.  OSA 4.  STS Moose 5.  STS Scarlett 6.  Mesothelium

Nuclear stain DAPI (blue) Cytokeratin (green)

Vimentin (red)

All canine tumor explants were vimentin positive

and cytokeratin negative indicating they are from

mesenchymal origin.

The control cells (mesothelium) are both

vimentin and cytokeratin positive.

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