Abstract
Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.
Highlights
The development of the oncolytic virus therapy of cancer and its progress in clinical testing require the development of appropriate and reliable tools, such as reporter genes, to monitor beneficial outcomes as well as potential side effects
Reporter gene products that can be released from the infected cells, either by active secretion or due to tumor cell lysis, would allow easier detection of the biomarker protein in patient samples such as blood and/or other body fluids
We previously showed that tissue distribution of Recombinant vaccinia virus (rVACV) can be assessed in mice by reporter gene-mediated optical- [4,5], optoacoustic- [6], PET- [7,8], and MR-imaging [6]
Summary
The development of the oncolytic virus therapy of cancer and its progress in clinical testing require the development of appropriate and reliable tools, such as reporter genes, to monitor beneficial outcomes as well as potential side effects. Reporter gene products that can be released from the infected cells, either by active secretion or due to tumor cell lysis, would allow easier detection of the biomarker protein in patient samples such as blood and/or other body fluids. Such reporter proteins could be assessed repeatedly from the blood and would be a helpful indicator of viral presence in the body. If the product of the reporter gene is an enzyme, the activity of the enzyme may serve as a biomarker providing a quantifiable predictor of therapeutic performance of the oncolytic treatment
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