title: Ex vivo infection of live tissue with oncolytic viruses.
abstract: Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells(1). Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway(2-5). One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging(6,7). This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy(8). To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection(9). It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.
authors: Center for Innovative Cancer Research, Ottawa Hospital Research Institute (OHRI).
authors: Jean-Simon
authors: JS
authors: Diallo
authors: Dominic
authors: D
authors: Roy
authors: Hesham
authors: H
authors: Abdelbary
authors: Naomi
authors: N
authors: De Silva
authors: John C
authors: JC
authors: Bell
mesh_terms: D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods; D009369:Neoplasms / Q000628:therapy / Q000821:virology*; D050130:Oncolytic Virotherapy / Q000379:methods*; D050504:Oncolytic Viruses / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019076:Transgenes; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D010948:Viral Plaque Assay / Q000379:methods
chemical_list: D049452:Green Fluorescent Proteins; D049409:Luciferases, Firefly