Abstract
Fluorescence in situ hybridization (FISH) is a specific, sensitive, accurate, and reliable technique widely applied in both research and clinic. Here we describe the detailed protocol of a FISH method established by us to serve the scientific purposes of the first oncolytic parvovirus clinical trial (ParvOryx01). This trial was launched in Germany in 2011. After trial completion in 2015, results were published in Molecular Therapy in 2017. The primary purpose of the trial was to evaluate the safety of an oncolytic parvovirus, H-1PV (ParvOryx), in recurrent glioblastoma patients. In addition, the efficiency of H-1PV tumor targeting after intratumoral or systemic virus administration was assessed by FISH detection of viral nucleic acids (genomic single-stranded DNA, mRNA and parvovirus double-stranded replicative forms) in formalin-fixed paraffin-embedded glioblastoma tissues resected at day 10 after ParvOryx treatment. The FISH method allowed the detection—for the first time in humans—of H-1PV replication markers in brain tumors of parvovirus-treated patients. A protocol combining mRNA FISH with simultaneous immunofluorescent staining for tumor and tumor microenvironment markers was also developed and is described here, in order to better characterize H-1PV cellular targets and H-1PV treatment-associated tumor microenvironment changes.
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Acknowledgments
Andreas Unterberg (principal investigator) and Karsten Geletneky conducted the first oncolytic parvovirus clinical trial (ParvOryx01) in the Department of Neurosurgery of Heidelberg University Hospital. ParvOryx01and the trial-accompanying laboratory research were sponsored by ORYX GmbH & Co.KG (Baldham/Munich, Germany). Assia Angelova and Alexandra Just were financially supported by ORYX funding. We thank Milena Barf for assistance with protein immunostaining. We acknowledge image analysis assistance by Felix Bestvater, Damir Crunic and Manuela Brom (Light Microscopy unit, DKFZ, Heidelberg). We are grateful to Jörg Schlehofer (Division of Tumor Virology, DKFZ, Heidelberg) and Andrii Domanskyi (Institute of Biotechnology, University of Helsinki) for helpful discussions.
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Kiprianova, I., Just, A., Leuchs, B., Rommelaere, J., Angelova, A.L. (2020). Fluorescence In Situ Hybridization (FISH) Detection of Viral Nucleic Acids in Oncolytic H-1 Parvovirus-Treated Human Brain Tumors. In: Engeland, C. (eds) Oncolytic Viruses. Methods in Molecular Biology, vol 2058. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9794-7_20
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DOI: https://doi.org/10.1007/978-1-4939-9794-7_20
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