IntroductionOncolytic viruses show promise for treating cancer. However, to assess therapeutic efficacy and potential toxicity, a noninvasive imaging modality is needed. This study aimed to determine if insertion of the human sodium iodide symporter (hNIS) cDNA as a marker for non-invasive imaging of virotherapy alters the replication and oncolytic capability of a novel vaccinia virus, GLV-1h153.MethodsGLV-1h153 was modified from parental vaccinia virus GLV-1h68 to carry hNIS via homologous recombination. GLV-1h153 was tested against human pancreatic cancer cell line PANC-1 for replication via viral plaque assays and flow cytometry. Expression and transportation of hNIS in infected cells was evaluated using Westernblot and immunofluorescence. Intracellular uptake of radioiodide was assessed using radiouptake assays. Viral cytotoxicity and tumor regression of treated PANC-1tumor xenografts in nude mice was also determined. Finally, tumor radiouptake in xenografts was assessed via positron emission tomography (PET) utilizing carrier-free 124I radiotracer.ResultsGLV-1h153 infected, replicated within, and killed PANC-1 cells as efficiently as GLV-1h68. GLV-1h153 provided dose-dependent levels of hNIS expression in infected cells. Immunofluorescence detected transport of the protein to the cell membrane prior to cell lysis, enhancing hNIS-specific radiouptake (P < 0.001). In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts (P < 0.001). Finally, intratumoral injection of GLV-1h153 facilitated imaging of virus replication in tumors via 124I-PET.ConclusionInsertion of the hNIS gene does not hinder replication or oncolytic capability of GLV-1h153, rendering this novel virus a promising new candidate for the noninvasive imaging and tracking of oncolytic viral therapy.

Despite advances in technology, the formidable challenge of treating cancer, especially if advanced, still remains with no significant improvement in survival rates, even with the most common forms of cancer. Oncolytic viral therapies have shown great promise for the treatment of various cancers, with the possible advantages of stronger treatment efficacy compared to conventional therapy due to higher tumor selectivity, and less toxicity. They are able to preferentially and selectively propagate in cancer cells, consequently destroying tumor tissue mainly via cell lysis, while leaving non-cancerous tissues unharmed. Several wild-type and genetically engineered vaccinia virus (VACV) strains have been tested in both preclinical and clinical trials with promising results. Greater understanding and advancements in molecular biology have enabled the generation of genetically engineered oncolytic viruses for safer and more efficacious treatment, including arming VACVs with cytokines and immunostimulatory molecules, anti-angiogenic agents, and enzyme prodrug therapy, in addition to combining VACVs with conventional external and systemic radiotherapy, chemotherapy, immunotherapy, and other virus strains. Furthermore, novel oncolytic vaccinia virus strains have been generated that express reporter genes for the tracking and imaging of viral therapy and monitoring of therapeutic response. Further study is needed to unlock VACVs’ full potential as part of the future of cancer therapy.

Oncolytic viruses have made their mark on the cancer world as a potential therapeutic option, with the possible advantages of reduced side effects and strengthened treatment efficacy due to higher tumor selectivity. Results have been so promising, that oncolytic viral treatments have now been approved for clinical trials in several countries. However, clinical studies may benefit from the ability to noninvasively and serially identify sites of viral targeting via molecular imaging in order to provide safety, efficacy, and toxicity information. Furthermore, molecular imaging of oncolytic viral therapy may provide a more sensitive and specific diagnostic technique to detect tumor origin and, more importantly, presence of metastases. Several strategies have been investigated for molecular imaging of viral replication broadly categorized into optical and deep tissue imaging, utilizing several reporter genes encoding for fluorescence proteins, conditional enzymes, and membrane protein and transporters. Various imaging methods facilitate molecular imaging, including computer tomography, magnetic resonance imaging, positron emission tomography, single photon emission CT, gamma-scintigraphy, and photoacoustic imaging. In addition, several molecular probes are used for medical imaging, which act as targeting moieties or signaling agents. This review will explore the preclinical and clinical use of in vivo molecular imaging of replication-competent oncolytic viral therapy.

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BACKGROUND Reversible electroporation has long been used to transfer macromolecules into target cells in the laboratory by using an electric field to induce transient membrane permeability. Recently, the electric field has been modulated to produce permanent membrane permeability and cell death. This novel technique, irreversible electroporation (IRE), is being developed for nonthermal cancer ablation. We hypothesize that outside the central zone of IRE exists a peripheral zone of reversible electroporation where gene transfer may occur. METHODS IRE of the liver was performed in a Yorkshire pig model with administration of a plasmid expressing the marker gene green fluorescent protein (GFP) by bolus or primed infusion through the hepatic artery or portal vein. After six hours, the livers were harvested for fluorescent microscopy and histologic examination. RESULTS 31 out of 36 liver specimens treated with IRE and the GFP plasmid demonstrated strong green fluorescence. Liver ablation by IRE was clearly demarcated on histology. CONCLUSION IRE is a promising technique not only for surgical tissue ablation but also for gene therapy. As IRE ablation may leave behind intact tumor antigens, these findings encourage clinical studies of tumor ablation with delivery of immunostimulatory plasmids for combined local eradication and systemic immunotherapy.

To assess therapeutic response and potential toxicity of oncolytic virotherapy, a noninvasive, deep-tissue imaging modality is needed. This study aimed to assess the feasibility, parameters, and determining factors of serial imaging and long-term monitoring of virotherapy and the therapeutic response of pancreatic cancer xenografts treated with a vaccinia virus carrying the human sodium iodide symporter GLV-1h153. Methods: Pancreatic cancer xenografts (PANC-1) in nude mice were treated systemically or intratumorally with GLV-1h153 and serially imaged using 124 I PET at 1, 2, 3, and 5 wk after viral injection. Signal intensity was compared with tumor therapeutic response and optical imaging, and tumors were histologically analyzed for morphology and the presence of virus. Autoradiography was performed using technetium-pertechnetate and g-scintigraphy to assess determining factors for radiouptake in tumors. Finally, the enhanced therapeutic effect of combination therapy with GLV-1h153 and systemic radioiodine was assessed. Results: GLV-1h153 successfully facilitated serial long-term imaging of virotherapy, with PET signal intensity correlating to tumor response. GLV-1h153 colonization of tumors mediated radioiodine uptake at potentially therapeutic doses. Successful radiouptake required the presence of virus, adequate blood flow, and viable tissue, whereas loss of signal intensity was linked to tumor death and necrosis. Finally, combining systemically administered GLV1h153 and 131 I led to enhanced tumor kill when compared with virus or 131 I alone (P , 0.01). Conclusion: GLV-1h153 is a promising oncolytic agent for the treatment, long-term imaging, and monitoring of therapeutic response in a xenograft model of pancreatic cancer. GLV-1h153 provided insight into tumor biologic activity and facilitated enhanced tumor kill when combined with systemic targeted radiotherapy. These results warrant further investigation into parameters and potential synergistic effects of combination therapy.

IntroductionOncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS.MethodsGLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide 131I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via 124I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both 124I-PET and 99m-technecium gamma-scintigraphy.ResultsGLV-1h153 successfully facilitated time-dependent intracellular uptake of 131I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P<0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 109 plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean %ID/gm of 3.82±0.46 (P<0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV-1h153-infected tumors were detected via 124I-PET and 99m-technecium-scintigraphy.ConclusionGLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality.

BackgroundGastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with 99mTc pertechnetate scintigraphy and 124I positron emission tomography (PET).MethodsGLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. 99mTc pertechnetate scintigraphy and 124I microPET imaging were performed.ResultsGFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK- 45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by 99mTc pertechnetate scintigraphy and 124I microPET imaging 2 days after treatment.ConclusionsGLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings.