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Oncolytic viruses (OVs) represent a novel class of immunotherapeutics under development for the treatment of cancers. OVs that express a cognate or transgenic fusion protein is particularly promising as their enhanced intratumoral spread via syncytia formation can be a potent mechanism for tumor lysis and induction of antitumor immune responses. Rapid and efficient fusion of infected cells results in cell death before high titers are reached. Although this is an attractive safety feature, it also presents unique challenges for large-scale clinical-grade manufacture of OVs. Here we evaluate the use of four different suspension cell lines for the production of a novel fusogenic hybrid of vesicular stomatitis virus and Newcastle disease virus (rVSV-NDV). The candidate cell lines were screened for growth, metabolism, and virus productivity. Permissivity was evaluated based on extracellular infectious virus titers and cell-specific virus yields (CSVYs). For additional process optimizations, virus adaptation and multiplicity of infection (MOI) screenings were performed and confirmed in a 1 L bioreactor. BHK-21 and HEK293SF cells infected at concentrations of 2 × 106cells/mL were identified as promising candidates for rVSV-NDV production, leading to infectious titers of 3.0 × 108TCID50/mL and 7.5 × 107TCID50/mL, and CSVYs of 153 and 9, respectively. Compared to the AGE1.CR.pIX reference produced in adherent cultures, oncolytic potency was not affected by production in suspension cultures and possibly even increased in cultures of HEK293SF and AGE1.CR.pIX. Our study describes promising suspension cell-based processes for efficient large-scale manufacturing of rVSV-NDV.Key points•Cell contact-dependent oncolytic virus (OV) replicates in suspension cells.•Oncolytic potency is not encompassed during suspension cultivation.•Media composition, cell line, and MOI are critical process parameters for OV production.•The designed process is scalable and shows great promise for manufacturing clinical-grade material.

Supplementary tables S1-3 and figure legends. Supplementary Table S1. Antibodies and antibody dilution used for immunohistochemical analyses. Supplementary Table S2. Characterization of human HCC Supplementary Table S3. Number of tumors per analyzed animal of DEN and McA model.

Supplementary Fig. S4. Tumor vascularization and necrosis in human HCC.

The in vivo assessment of tissue metabolism represents a novel strategy for the evaluation of oncologic disease. Hepatocellular carcinoma (HCC) is a high-prevalence, high-mortality tumor entity often discovered at a late stage. Recent evidence indicates that survival differences depend on metabolic alterations in tumor tissue, with particular focus on glucose metabolism and lactate production. Here, we present an in vivo imaging technique for metabolic tumor phenotyping in rat models of HCC. Endogenous HCC was induced in Wistar rats by oral diethyl-nitrosamine administration. Peak lactate-to-alanine signal ratios (L/A) were assessed with hyperpolarized magnetic resonance spectroscopic imaging (HPMRSI) after [1

Virotherapy research involves the development, exploration, and application of oncolytic viruses that combine direct killing of cancer cells by viral infection, replication, and spread (oncolysis) with indirect killing by induction of anti-tumor immune responses. Oncolytic viruses can also be engineered to genetically deliver therapeutic proteins for direct or indirect cancer cell killing. In this review—as part of the special edition on “State-of-the-Art Viral Vector Gene Therapy in Germany”—the German community of virotherapists provides an overview of their recent research activities that cover endeavors from screening and engineering viruses as oncolytic cancer therapeutics to their clinical translation in investigator-initiated and sponsored multi-center trials. Preclinical research explores multiple viral platforms, including new isolates, serotypes, or fitness mutants, and pursues unique approaches to engineer them towards increased safety, shielded or targeted delivery, selective or enhanced replication, improved immune activation, delivery of therapeutic proteins or RNA, and redirecting antiviral immunity for cancer cell killing. Moreover, several oncolytic virus-based combination therapies are under investigation. Clinical trials in Germany explore the safety and potency of virotherapeutics based on parvo-, vaccinia, herpes, measles, reo-, adeno-, vesicular stomatitis, and coxsackie viruses, including viruses encoding therapeutic proteins or combinations with immune checkpoint inhibitors. These research advances represent exciting vantage points for future endeavors of the German virotherapy community collectively aimed at the implementation of effective virotherapeutics in clinical oncology., publishedVersion

Oncolytic viruses have made headlines owing to increasing numbers of reports of clinical success. Compelling data now indicate that small anticancer molecules can serve as potent sensitizers of tumour cells in hepatocellular carcinoma to enhance the oncolytic potential of systemically applied viruses.

Vesicular stomatitis virus (VSV) represents an attractive oncolytic virotherapy platform because of its potent tumor cell-killing and immune-stimulating properties; yet the clinical translation of VSV faces numerous challenges, such as inefficient systemic delivery and severe side effects such as neurotoxicity. We hypothesized that we could overcome these limitations and simultaneously enhance the therapy, by combining VSV with adoptively transferred T cell receptor (TCR) transgenic T cells as carrier cells. We show that CD8+ T central memory cells (CD8+ T cm) can be efficiently loaded with VSV, they support intracellular virus production, and they can efficiently transfer VSV to tumor cells without compromising their own viability or antitumor reactivity. Loading VSV onto CD8+ T cm not only improves the safety compared with systemic administration of naked virus, but this approach also allows for an effective delivery of virus to its tumor target, resulting in an effective combination therapy in NSG mice bearing subcutaneous human acute myeloid leukemia (AML) tumors. We conclude that the combination of potent tumor debulking provided by the oncolytic VSV with the added effector functions afforded by the cytotoxic immune carrier cells results in a potent and safer immunotherapeutic, which can be further developed for clinical translation. Keywords: adoptive T cell therapy, vesicular stomatitis virus, oncolytic virotherapy, immunotherapy, acute myeloid leukemia

Oncolytic viruses represent an exciting new aspect of the evolving field of cancer immunotherapy. We have engineered a novel hybrid vector comprising vesicular stomatitis virus (VSV) and Newcastle disease virus (NDV), named recombinant VSV-NDV (rVSV-NDV), wherein the VSV backbone is conserved but its glycoprotein has been replaced by the hemagglutinin-neuraminidase (HN) and the modified, hyperfusogenic fusion (F) envelope proteins of recombinant NDV. In comparison to wild-type VSV, which kills cells through a classical cytopathic effect, the recombinant virus is able to induce tumor-specific syncytium formation, allowing efficient cell-to-cell spread of the virus and a rapid onset of immunogenic cell death. Furthermore, the glycoprotein exchange substantially abrogates the off-target effects in brain and liver tissue associated with wild-type VSV, resulting in a markedly enhanced safety profile, even in immune-deficient NOD.CB17-prkdc(scid)/NCrCrl (NOD-SCID) mice, which are highly susceptible to wild-type VSV. Although NDV causes severe pathogenicity in its natural avian hosts, the incorporation of the envelope proteins in the chimeric rVSV-NDV vector is avirulent in embryonated chicken eggs. Finally, systemic administration of rVSV-NDV in orthotopic hepatocellular carcinoma (HCC)-bearing immune-competent mice resulted in significant survival prolongation. This strategy, therefore, combines the beneficial properties of the rapidly replicating VSV platform with the highly efficient spread and immunogenic cell death of a fusogenic virus without risking the safety and environmental threats associated with either parental vector. Taking the data together, rVSV-NDV represents an attractive vector platform for clinical translation as a safe and effective oncolytic virus. IMPORTANCE The therapeutic efficacy of oncolytic viral therapy often comes as a tradeoff with safety, such that potent vectors are often associated with toxicity, while safer viruses tend to have attenuated therapeutic effects. Despite promising preclinical data, the development of VSV as a clinical agent has been substantially hampered by the fact that severe neurotoxicity and hepatotoxicity have been observed in rodents and nonhuman primates in response to treatment with wild-type VSV. Although NDV has been shown to have an attractive safety profile in humans and to have promising oncolytic effects, its further development has been severely restricted due to the environmental risks that it poses. The hybrid rVSV-NDV vector, therefore, represents an extremely promising vector platform in that it has been rationally designed to be safe, with respect to both the recipient and the environment, while being simultaneously effective, both through its direct oncolytic actions and through induction of immunogenic cell death.

Oncolytic viruses are under intense development and have earned their place among the novel class of cancer immunotherapeutics that are changing the face of cancer therapy. Their ability to specifically infect and efficiently kill tumor cells, while breaking immune tolerance and mediating immune responses directed against the tumor, make oncolytic viruses highly attractive candidates for immunotherapy. Increasing evidence indicates that a subclass of oncolytic viruses, which encodes for fusion proteins, could outperform non-fusogenic viruses, both in their direct oncolytic potential, as well as their immune-stimulatory properties. Tumor cell infection with these viruses leads to characteristic syncytia formation and cell death due to fusion, as infected cells become fused with neighboring cells, which promotes intratumoral spread of the infection and releases additional immunogenic signals. In this review, we discuss the potential of fusogenic oncolytic viruses as optimal candidates to enhance immunotherapy and initiate broad antitumor responses. We provide an overview of the cytopathic mechanism of syncytia formation through viral-mediated expression of fusion proteins, either endogenous or engineered, and their benefits for cancer therapy. Growing evidence indicates that fusogenicity could be an important feature to consider in the design of optimal oncolytic virus platforms for combinatorial oncolytic immunotherapy.

Hepatocellular carcinoma (HCC) is a refractory malignancy with a high mortality and increasing worldwide incidence rates, including the United States and central Europe. In this study, we demonstrate that a specific inhibitor of signal transducer and activator of transcription 3 (STAT3), NSC74859, efficiently reduces HCC cell proliferation and can be successfully combined with oncolytic virotherapy using vesicular stomatitis virus (VSV). The potential benefits of this combination treatment are strengthened by the ability of NSC74859 to protect primary hepatocytes and nervous system cells against virus-induced cytotoxicity, with an elevation of the VSV maximum tolerated dose in mice. Hereby we propose a strategy for improving the current regimen for HCC treatment and seek to further explore the molecular mechanisms underlying selective oncolytic specificity of VSV.

Oncolytic viruses are promising new agents in cancer therapy. Success of tumor lysis is often hampered by low intra-tumoral titers due to a strong anti-viral host immune response and insufficient tumor targeting. Previous work on the co-assembly of oncolytic virus particles (VPs) with magnetic nanoparticles (MNPs) was shown to provide shielding from inactivating immune response and improve targeting by external field gradients. In addition, MNPs are detected by magnet resonance imaging (MRI) enabling non-invasive therapy monitoring. In this study two selected core-shell type iron oxide MNPs were assembled with adenovirus (Ad) or vesicular stomatitis virus (VSV). The selected MNPs were characterized by high r2 and r2(*) relaxivities and thus could be quantified non-invasively by 1.5 and 3.0 tesla MRI with a detection limit below 0.001 mM iron in tissue-mimicking phantoms. Assembly and cell internalization of MNP-VP complexes resulted in 81 - 97 % reduction of r2 and 35 - 82 % increase of r2(*) compared to free MNPs. The relaxivity changes could be attributed to the clusterization of particles and complexes shown by transmission electron microscopy (TEM). In a proof-of-principle study the non-invasive detection of MNP-VPs by MRI was shown in vivo in an orthotopic rat hepatocellular carcinoma model. In conclusion, MNP assembly and compartmentalization have a major impact on relaxivities, therefore calibration measurements are required for the correct quantification in biodistribution studies. Furthermore, our study provides first evidence of the in vivo applicability of selected MNP-VPs in cancer therapy.

Oncolytic viruses have gained much attention in recent years, due, not only to their ability to selectively replicate in and lyse tumor cells, but to their potential to stimulate antitumor immune responses directed against the tumor. Vesicular stomatitis virus (VSV), a negative-strand RNA virus, is under intense development as an oncolytic virus due to a variety of favorable properties, including its rapid replication kinetics, inherent tumor specificity, and its potential to elicit a broad range of immunomodulatory responses to break immune tolerance in the tumor microenvironment. Based on this powerful platform, a multitude of strategies have been applied to further improve the immune-stimulating potential of VSV and synergize these responses with the direct oncolytic effect. These strategies include: 1. modification of endogenous virus genes to stimulate interferon induction; 2. virus-mediated expression of cytokines or immune-stimulatory molecules to enhance anti-tumor immune responses; 3. vaccination approaches to stimulate adaptive immune responses against a tumor antigen; 4. combination with adoptive immune cell therapy for potentially synergistic therapeutic responses. A summary of these approaches will be presented in this review.

Recombinant vesicular stomatitis virus (VSV) shows promise for the treatment of hepatocellular carcinoma (HCC), but its safety and efficacy when administered in a setting of hepatic fibrosis, which occurs in the majority of clinical cases, is unknown. We hypothesized that VSV could provide a novel benefit to the underlying fibrosis, due to its ability to replicate and cause cell death specifically in activated hepatic stellate cells. In addition to the ability of VSV to produce a significant oncolytic response in HCC-bearing rats in the background of thioacetamide-induced hepatic fibrosis without signs of hepatotoxicity, we observed a significant downgrading of fibrosis stage, a decrease in collagen content in the liver, and modulation of gene expression in favor of fibrotic regression. Together, this work suggests that VSV is not only safe and effective for the treatment of HCC with underlying fibrosis, but it could potentially be developed for clinical application as a novel antifibrotic agent.

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Vesicular stomatitis virus (VSV), a negative-sense single-stranded-RNA rhabdovirus, is an extremely promising oncolytic agent for cancer treatment. Since oncolytic virotherapy is moving closer to clinical application, potentially synergistic combinations of oncolytic viruses and molecularly targeted antitumor agents are becoming a meaningful strategy for cancer treatment. Mitogen-activated protein kinase (MAPK) inhibitors have been shown to impair liver cell proliferation and tumor development, suggesting their potential use as therapeutic agents for hepatocellular carcinoma (HCC). In this work, we show that the impairment of MAPK in vitro did not interfere with the oncolytic properties of VSV in HCC cell lines. Moreover, the administration of MAPK inhibitors did not restore the responsiveness of HCC cells to alpha/beta interferon (IFN-α/β). In contrast to previous reports, we show that JNK inhibition by the inhibitor SP600125 is not responsible for VSV attenuation in HCC cells and that this compound acts by causing a posttranslational modification of the viral glycoprotein.

Tumour-specific replicating (oncolytic) viruses are novel anticancer agents, currently under intense investigation in preclinical studies and phase I-III clinical trials. Until recently, most studies have focused on the direct antitumour properties of these viruses. There is now an increasing body of evidence indicating that host immune responses may be critical to the efficacy of oncolytic virotherapy. Although the immune response to oncolytic viruses can rapidly restrict viral replication, thereby limiting the efficacy of therapy, oncolytic virotherapy also has the potential to induce potent antitumoural immune effectors that destroy those cancer cells, which are not directly lysed by virus. In this review, we discuss the role of the immune system in terms of antiviral and antitumoural responses, as well as strategies to evade or promote these responses in favour of improved therapeutic potentials.

With the clinical approval of Talimogen laherparepvec (T-VEC), an oncolytic Herpes-simplex-Virus 1, in 2015, oncolytic viruses have gained much attention as a versatile and promising platform for cancer therapy. Vesicular stomatitis virus (VSV) represents an attractive oncolytic agent due to its inherent ability to preferentially infect and kill tumor cells, boost an antitumor immune response, and its flexibility to efficiently express transgenes. In order to overcome potential hurdles for the systemic administration of VSV, which include neutralization and nonspecific uptake, we aim to develop a combination therapy which employs T-cell receptor (TCR) transgenic T cells directed against an epitope of Myeloperoxidase (Klar et al. 2014, Leukemia). As genetically engineered T cells are currently undergoing clinical trials, they have the potential to provide an ideal vehicle for a combination therapy with VSV. In our work, we have shown that human CD8+ central memory T cells (CD8+ Tcm) not only transport and deliver infectious virus to their tumor target while eliciting their own potent cytotoxic effector functions, but they also support viral amplification in the absence of significantly decreased cell viability in culture after 24h of viral infection. Moreover, we demonstrate in coculture assays with ML2 leukemia cells, that TCR transgenic CD8+ Tcm profit from the additional virus-mediated tumor cell killing as compared to a monotherapy with TCR transgenic CD8+ Tcm alone. Although viral titers in coculture assays with TCR transgenic cells were slightly decreased compared to those achieved in cocultures with T cells not expressing the TCR, we consider this to be an additional benefit of the combination therapeutic approach to reduce VSV-mediated off-target side effects, as VSV is known for its neurotoxic and hepatotoxic effects in a dose dependent manner. This is supported by our findings that highly immune-deficient NSG mice tolerate similar amounts of virus better if applied together with CD8+ Tcm. Furthermore, we demonstrate that the combination therapy for tumor-bearing NSG mice leads to enhanced transduction efficiency of VSV compared to that achieved by delivery of naked virus, as well as faster tumor cell killing compared to monotherapies with TCR transgenic CD8+ T cm or VSV alone when applied at the maximum tolerated dose. We therefore conclude that the combination of these monotherapies is a crucial step toward a broader and safer option for cancer treatment, as each offers a complimentary approach to tumor cell killing while simultaneously reducing the weaknesses of the other treatment option. We speculate that immune evasion can be efficiently overcome by application of the combination therapy while providing an efficient targeting and delivery vehicle of systemically applied oncolytic VSV with a concomitant reduction of toxicity. As both, rVSV and transgenic T cells are already undergoing clinical testing, our proposed combination therapy has the potential for a seamless translation to clinical application. Citation Format: Michael Karl Melzer, Lisa Zeitlinger, Sabine Mall, Katja Steiger, Angela Krackhardt, Oliver Ebert, Jennifer Altomonte. TCR transgenic T cells improve the anticancer potential of oncolytic vesicular stomatitis virus as cell carriers and as synergistic therapeutics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B14.

The aim of this study was to assess the clinical value of [(11)C]methionine-PET (MET-PET) for detection and localization of parathyroid adenomas in patients without prior thyroidectomy.A retrospective analysis of patients with suspected parathyroid adenomas undergoing imaging with MET-PET was performed. Prior thyroidectomy was an exclusion criterion. Forty-one patients with a total of 49 MET-PET scans were included. MET-PET consisted of whole-body images obtained 15-20 min after injection of 430 +/- 81 MBq of MET using a dedicated PET scanner. Imaging findings were validated by histology or other imaging studies and clinical follow-up on a lesion, side, and location basis. Comparison of PET results to other imaging modalities including ultrasound, MIBI scintigraphy, and morphological imaging [computed tomography (CT) and/or magnetic resonance imaging] and subgroup analysis of primary vs. secondary hyperparathyroidism was performed.Twenty-three of 49 PET scans revealed pathologic findings, whereas 26 of 49 scans were negative. Validation of PET findings for detection and localization of parathyroid adenomas resulted in an overall sensitivity of MET-PET of 54%, 49%, and 35% on a lesion, side, and location basis, respectively. Sensitivity of MET-PET was inferior compared to ultrasonography (50% vs. 93%), MIBI scintigraphy (53% vs. 74%) and morphological imaging (52% vs. 74%). Subgroup analysis revealed higher sensitivity for MET-PET in secondary HPT (sHPT) than primary HPT (pHPT; 62% vs. 43%; side basis).In patients with initial diagnosis of hyperparathyroidism and no prior thyroidectomy, the sensitivity of MET-PET for detection and localization of parathyroid adenomas is markedly lower compared to previous reports. While performance was better in sHPT, we believe that MET-PET cannot be recommended for pHPT localization in this clinically relevant subcollective. The clinical value of MET/PET in patients with hyperparathyroidism should be further investigated in a prospective study utilizing anatometabolic imaging with a PET/CT device.

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). We have observed earlier that, in contrast to cultured human HCC cells, primary human hepatocytes (PHHs) are refractory to VSV infection. Impairment of the type I interferon (IFN) pathway in HCC cells has been suggested to be the mechanism by which these cells become susceptible to VSV infection. The goal of this study was to elucidate the nature of the IFN defect in human HCC. We demonstrate here that the defect in IFN-beta signaling in HCC cells results from a deregulated IFN regulatory factor-3 (IRF3) pathway. Expression of IRF3-spliced variant (IRF3-nirs3) was constitutively observed in HCC cells and, importantly, also in primary HCC samples. In contrast, IRF3 was readily activated in PHHs after stimulation with dsRNA or infection with VSV. In addition, overexpression of IRF3-nirs3 significantly abrogated the IFN-beta response to VSV infection and improved viral growth. Our data provide evidence that aberrant splicing of IRF3 in HCC contributes to the defect in IFN-mediated antiviral defenses. This work may provide a potential molecular basis for selecting HCC patients for oncolytic VSV therapy in future clinical trials.

Oncolytic virotherapy is a promising strategy for safe and effective treatment of malignancy. We have reported previously that recombinant vesicular stomatitis virus (VSV) vectors are effective oncolytic agents that can be safely administered via the hepatic artery in immunocompetent rats to treat multifocal hepatocellular carcinoma (HCC), resulting in tumor necrosis and prolonged survival. Though the results were encouraging, complete tumor regression was not observed, which led us to explore alternative approaches to further enhance the efficacy of VSV treatment. Transarterial embolization techniques have been shown to improve the efficiency and tumor selectivity of anticancer treatments. Degradable starch microspheres (DSM) are one such embolic agent that provides transient embolization of the therapeautic agent before being degraded by serum amylases. Here we demonstrate via dynamic contrast-enhanced magnetic resonance imaging that in our rat model of multifocal HCC, DSM injection into the hepatic artery results in a substantial reduction in tumor perfusion of systemically applied contrast agent. VSV, when administered in combination with DSM, results in enhanced tumor necrosis and synergistically prolongs survival when compared with VSV or DSM monotherapy. Conclusion: This regimen of viroembolization represents an innovative therapeutic modality that can augment the future development of transarterial oncolytic virus therapy for patients with advanced HCC. (HEPATOLOGY 2008;48:1864-1873.)

Oncolytic vesicular stomatitis virus (VSV) is being developed as a novel therapeutic agent for cancer treatment, although it is toxic in animals when administered systemically at high doses. Its safety can be substantively improved by an M Delta 51 deletion in the viral genome, and yet VSV(M Delta 51) induces a much greater, robust cellular inflammatory response in the host than wild-type VSV, which severely attenuates its oncolytic potency. We have reported that the oncolytic potency of wild-type VSV can be enhanced by vector-mediated expression of a heterologous viral gene that suppresses cellular inflammatory responses in the lesions. To develop an effective and safe VSV vector for cancer treatment, we tested the hypothesis that the oncolytic potency of VSV(M Delta 51) can be substantively elevated by vector-mediated expression of M3, a broad-spectrum and high-affinity chemokine-binding protein from murine gammaherpesvirus-68. The recombinant vector rVSV(M Delta 51)-M3 was used to treat rats bearing multifocal lesions (1-10 mm in diameter) of hepatocellular carcinoma (HCC) in their liver by hepatic artery infusion. Treatment led to a significant reduction of neutrophil and natural killer cell accumulation in the lesions, a 2-log elevation of intratumoral viral titer, substantively enhanced tumor necrosis, and prolonged animal survival with a 50% cure rate. Importantly, there were no apparent systemic and organ toxicities in the treated animals. These results indicate that the robust cellular inflammatory responses induced by VSV(M Delta 51) in HCC lesions can be overcome by vector-mediated intratumoral M3 expression, and that rVSV(M Delta 51)-M3 can be developed as an effective and safe oncolytic agent to treat advanced HCC patients in the future.

Oncolytic virotherapy is a promising strategy for treatment of malignancy, although its effectiveness is hampered by host antiviral inflammatory responses. The efficacy of treatment of oncolytic vesicular stomatitis virus (VSV) in rats bearing multifocal hepatocellular carcinoma (HCC) can be substantially elevated by antibody-mediated depletion of natural killer (NK) cells. In order to test the hypothesis that the oncotyic potency of VSV can be exponentially elevated by evasion of inflammatory responses in vivo, we constructed a recombinant VSV vector expressing equine herpes virus-1 glycoprotein G, which is a broad-spectrum viral chemokine binding protein (rVSV-gG). Infusion of rVSV-gG via the hepatic artery into immune-competent rats bearing syngeneic and multifocal HCC in their livers, resulted in a reduction of NK and NKT cells in the tumors and a 1-log enhancement in intratumoral virus titer in comparison with a reference rVSV vector. The treatment led to increased tumor necrosis and substantially prolonged animal survival without toxicities. These results indicate that rVSV-gG has the potential to be developed as an effective and safe oncolytic agent to treat patients with advanced HCC. Furthermore, the novel concept that oncolytic potency can be substantially enhanced by vector-mediated suppression of host antiviral inflammatory responses could have general applicability in the field of oncolytic virotherapy for cancer.

Glucose disposal induces a signal that modulates the transcriptional regulation of genes involved in the glycolysis and lipogenesis pathways. To investigate the role of glucose metabolism on hepatic gene expression independently from insulin action, we overexpressed glucokinase, the limiting enzyme in the glycolysis pathway, in the liver of streptozotocin-induced type 1 diabetic rats. By microarray analysis, we observed that critical genes such as liver-type pyruvate kinase, malic enzyme, fatty acid synthase, and stearoyl-CoA desaturase 1 were enhanced multiple-fold, whereas genes involved in mitochondrial fatty acid oxidation and the Krebs cycle were downregulated. Despite the increase in expression of fatty acid synthesis genes and the presence of steatosis, no major alterations to the levels of genes involved in VLDL assembly and secretion, such as diacylglycerol acyltransferases 1 and 2 and microsomal triglyceride transfer protein, were observed. Overall, our data suggest that the gene expression pattern induced by glucose metabolism favors fatty acid storage in the liver rather than secretion into the circulation.

Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancer-related death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk–treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications.

Advanced glycation end products (AGEs), known promoters of diabetic complications, form abundantly in heated foods and are ingested in bioreactive forms. To test whether dietary AGEs play a role in the progression of insulin resistance, C57/BL/KsJ db/db mice were randomly placed for 20 weeks on a diet with either a low AGE content (LAD) or a 3.4-fold higher content of AGE (high AGE diet [HAD]), including εN-carboxymethyllysine (CML) and methylglyoxal (MG). LAD-fed mice showed lower fasting plasma insulin levels throughout the study (P = 0.01). Body weight was reduced by ∼13% compared with HAD-fed mice (P = 0.04) despite equal food intake. LAD-fed mice exhibited significantly improved responses to both glucose (at 40 min, P = 0.003) and insulin (at 60 min, P = 0.007) tolerance tests, which correlated with a twofold higher glucose uptake by adipose tissue (P = 0.02). Compared with the severe hypertrophy and morphological disorganization of islets from HAD-fed mice, LAD-fed mice presented a better-preserved structure of the islets. LAD-fed mice demonstrated significantly increased plasma HDL concentrations (P < 0.0001). Consistent with these observations, LAD-fed mice exhibited twofold lower serum CML and MG concentrations compared with HAD-fed mice (P = 0.02). These results demonstrate that reduced AGE intake leads to lower levels of circulating AGE and to improved insulin sensitivity in db/db mice.

Purpose: Preclinical model systems should faithfully reflect the complexity of the human pathology. In hepatocellular carcinoma (HCC), the tumor vasculature is of particular interest in diagnosis and therapy. By comparing two commonly applied preclinical model systems, diethylnitrosamine induced (DEN) and orthotopically implanted (McA) rat HCC, we aimed to measure tumor biology noninvasively and identify differences between the models. Experimental Design: DEN and McA tumor development was monitored by MRI and PET. A slice-based correlation of imaging and histopathology was performed. Array CGH analyses were applied to determine genetic heterogeneity. Therapy response to sorafenib was tested in DEN and McA tumors. Results: Histologically and biochemically confirmed liver damage resulted in increased 18F-fluorodeoxyglucose (FDG) PET uptake and perfusion in DEN animals only. DEN tumors exhibited G1–3 grading compared with uniform G3 grading of McA tumors. Array comparative genomic hybridization revealed a highly variable chromosomal aberration pattern in DEN tumors. Heterogeneity of DEN tumors was reflected in more variable imaging parameter values. DEN tumors exhibited lower mean growth rates and FDG uptake and higher diffusion and perfusion values compared with McA tumors. To test the significance of these differences, the multikinase inhibitor sorafenib was administered, resulting in reduced volume growth kinetics and perfusion in the DEN group only. Conclusions: This work depicts the feasibility and importance of in depth preclinical tumor model characterization and suggests the DEN model as a promising model system of multifocal nodular HCC in future therapy studies. Clin Cancer Res; 21(19); 4440–50. ©2015 AACR. See related commentary by Weber et al., p. 4254

Oncolytic viral therapies have recently found their way into clinical application for hepatocellular carcinoma (HCC), a disease with limited treatment options and poor prognosis. Adding to the many intrinsic challenges of in vivo oncolytic viral therapy, is the complex microenvironment of the liver, which imposes unique limitations to the successful delivery and propagation of the virus. The normal liver milieu is characterized by an intricate network of hepatocytes and non-parenchymal cells including Kupffer cells, stellate cells, and sinusoidal endothelial cells, which can secrete antiviral cytokines, provide a platform for non-specific uptake, and form a barrier to efficient viral spread. In addition, NK cells are greatly enriched in the liver, contributing to the innate defense against viruses. The situation is further complicated when HCC arises in the setting of underlying hepatitis virus infection and/or hepatic cirrhosis, which occurs in more than 90% of clinical cases. These conditions pose further inhibitory effects on oncolytic virus therapy due to the presence of chronic inflammation, constitutive cytokine expression, altered hepatic blood flow, and extracellular matrix deposition. In addition, oncolytic viruses can modulate the hepatic microenvironment, resulting in a complex interplay between virus and host. The immune system undoubtedly plays a substantial role in the outcome of oncolytic virus therapy, both as an inhibitor of viral replication, and as a potent mechanism of virus-mediated tumor cell killing. This review will discuss the particular challenges of oncolytic viral therapy for HCC, as well as some potential strategies for modulating the immune system and synergizing with the hepatic microenvironment to improve therapeutic outcome.

Objectives: The high sensitivity to motion artifacts is a major limiting factor for applying the dynamic 3D T1-weighted gradient-echo (3D T1w GRE) technique for dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) experiments in small rodents. Dynamic quantification of the relaxation rate R1 (1/T1) presents an alternative approach to reduce these motion artifacts. In this work, an optimized 2D single-shot Look-Locker based T1 mapping technique, named GOLD, applying radial sampling in the golden-angle view order and contrast-enhancing k-space filter was evaluated for its use in free-breathing quantitative DCE-MRI of rat liver on a clinical 1.5 T MRI system. Materials and Methods: In vitro measurements and initial in vivo experiments in healthy rats were performed to evaluate the accuracy and resilience of the GOLD technique to motion artifacts. Unifocal hepatocellular carcinoma (HCC) was established in 20 male Buffalo rats. Twelve days after tumor cell implantation, animals were screened for intrahepatic tumor nodules by high-resolution T2-weighted MRI. Quantitative DCE-MRI experiments applying bolus injected gadopentetate dimeglumine were performed in 11 HCC-bearing rats using the GOLD technique. For comparison, a standard 3D T1w GRE sequence was applied in 6 additional rats. Results: Phantom experiments showed good agreement for T1 values measured by the GOLD method and an inversion recovery spectroscopy measurement. The in vivo experiments in healthy rats confirmed the robustness of the GOLD method in T1 value determination and its resilience to motion artifacts. Gadopentetate dimeglumine concentration (CGd) time curves determined from free-breathing GOLD-based DCE-MRI experiments of HCC-bearing rats allowed reliable and robust pharmacokinetic modeling (Ktrans, ve, lag time Td, and slow washout rate rwo) of tumor, liver, and spinal muscle. In comparison to the dynamic 3D T1w GRE, the GOLD method showed less variation and jitter in the CGd time curves and significantly increased accuracy (in terms of the goodness of fit) in the pharmacokinetic modeling. Significant differences were detected for Ktrans and ve with the 3D T1w GRE method apparently underestimating those parameters. Conclusions: The GOLD technique allowed dynamic sampling of 2D axial T1 maps of the rat abdomen with 6-second temporal resolution enabling simultaneous and robust pharmacokinetic modeling of HCC, normal liver, and spinal muscle.

Background & Aims The hepatocellular carcinoma (HCC) exhibits varying degrees of vascularization with more poorly differentiated carcinoma commonly exhibiting high amounts of vascularization. Transcatheter arterial embolization (TAE) of HCC tumor nodules results in varying amounts of tumor necrosis. Reliable quantification of necrosis after TAE, would aid in treatment planning and testing of novel combinatorial treatment regimen. The aim of this work was to validate different imaging parameters as individual or combined predictors of tumor necrosis after TAE in an orthotopic rat HCC tumor model. Methods Unifocal rat HCC was imaged by T 2 -weighted MRI, quantitative dynamic contrast enhanced (DCE) MRI, diffusion weighted MRI (DWI) and [ 18 F]-FDG PET imaging before (day−1) and after (days 1 and 3) TAE. Univariate and multivariate regression analyses were carried out to analyze the ability of each imaging parameter to predict the percent residual vital tumor ( vtu ) and vital tissue ( vti ) as determined by quantitative histopathology. Results TAE induced a wide range of tumor necrosis. Tumor volume was the only parameter showing a correlation with vti ( r 2 =0.63) before TAE. After TAE, moderate correlations were found for FDG tracer uptake ( r 2 =0.56) and plasma tissue transfer constant ( r 2 =0.55). Correlations were higher for the extravascular extracellular volume fraction ( v e , r 2 =0.68) and highest for the apparent diffusion coefficient (ADC, r 2 =0.86). Multivariate analyses confirmed highest correlation of ADC and v e with vtu and vti . Conclusions DWI and DCE-MRI with the respective parameters ADC (day 3) and v e (day 1) were identified as the most promising imaging techniques for the prediction of necrosis. This study validates a preclinical platform allowing for the improved tumor stratification after TAE and thus the testing of novel combinatorial therapy approaches in HCC.

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). Identifying key regulators in diverse transduction pathways that define VSV oncolysis in cancer cells represents a fundamental prerequisite to engineering more effective oncolytic viral vectors and adjusting combination therapies. After having identified defects in the signalling cascade of type I interferon induction, responsible for attenuated antiviral responses in human HCC cell lines, we have now investigated the role of cell proliferation and translation initiation. Cell cycle progression and translation initiation factors eIF4E and eIF2Bepsilon have been recently identified as key regulators of VSV permissiveness in T-lymphocytes and immortalized mouse embryonic fibroblasts, respectively. Here, we show that in HCC, decrease of cell proliferation by cell cycle inhibitors or siRNA-mediated reduction of G(1) cyclin-dependent kinase activities (CDK4) or cyclin D1 protein expression, do not significantly alter viral growth. Additionally, we demonstrate that translation initiation factors eIF4E and eIF2Bepsilon are negligible in sustaining VSV replication in HCC. Taken together, these results indicate that cellular proliferation and the initiation phase of cellular protein synthesis are not essential for successful VSV oncolysis of HCC. Moreover, our observations indicate the importance of cell-type specificity for VSV oncolysis, an important aspect to be considered in virotherapy applications in the future.

Background/Aim: Ischemia/reperfusion (I/R) injury is a main cause of primary dysfunction or nonfunction after liver transplantation (LTx). Recent evidence indicates that an increase in nitric oxide (NO) production after LTx is associated with I/R injury. The aim of this study was to demonstrate that low-dose FK506 in combination with aminoguanidine (AGH), which leads to a reduction of NO levels, has a protective effect by reducing I/R associated injury after LTx. Materials and methods: Fortyone DA-(RT1av1) rats served as donors and recipients for syngenic orthotopic arterialised LTx. They were divided into 4 groups: controls without pre-/treatment (I), pre-/treatment with high-dose FK506 (II), pre-/treatment with AGH only (III), and pre-/treatment with low-dose FK506 in combination with AGH (IV). After LTx the laboratory parameters and liver biopsy were performed. Results: The levels of transaminase (ALT) in groups I, II and III were significantly higher on day 3 after LTx compared to group IV (p = 0.001, p = 0.001, p = 0.000). In group IV the I/R-associated liver necrosis rate was reduced significantly. Conclusion: Our results demonstrated that a combined dual pharmacological pretreatment (group IV) reduced I/R injury of the graft after LTx in a rat model.

1 Department of Surgery, Klinikum Rechts der Isar, Technische Universitat of Munich, Munich, Germany 2 National Shalimov Institute of Surgery and Transplantology from Ukrainian Academy of Medical Sciences, Kiev, Ukraine 3 Institute of Pathology, Technische Universitat of Munich, Munich, Germany 4 Department of Medicine, Mount Sinai School of Medicine, NYU Langone Medical Center, New York, NY, USA 5 Institute of Clinical Forensic Medicine, Ludwig Maximilians University of Munich, Munich, Germany

Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, which is currently under investigation as a clinical oncolytic agent. Several clinical trials have reported NDV to be a safe and effective agent for cancer therapy; however, there remains a clear need for improvement in therapeutic outcome. The endogenous NDV fusion (F) protein directs membrane fusion, which is required for virus entry and cell–cell fusion. Here, we report a novel NDV vector harboring an L289A mutation within the F gene, which resulted in enhanced fusion and cytotoxicity of hepatocellular carcinoma (HCC) cells in vitro, as compared with the rNDV/F3aa control virus. In vivo administration of the recombinant vector, termed rNDV/F3aa(L289A), via hepatic arterial infusion in immune-competent Buffalo rats bearing multifocal, orthotopic liver tumors resulted in tumor-specific syncytia formation and necrosis, with no evidence of toxicity to the neighboring hepatic parenchyma. Furthermore, the improved oncolysis conferred by the L289A mutation translated to significantly prolonged survival compared with control NDV. Taken together, rNDV/F(L289A) represents a safe, yet more effective vector than wild-type NDV for the treatment of HCC, making it an ideal candidate for clinical application in HCC patients.

Recombinant oncolytic viruses represent a promising alternative option for the treatment of malignant cancers. We have reported earlier the safety and efficacy of recombinant vesicular stomatitis virus (VSV) vectors in a rat model of hepatocellular carcinoma (HCC). However, the full potential of VSV therapy is limited by a sudden decline in intratumoral virus replication observed early after viral administration, a phenomenon that coincides with an accumulation of inflammatory cells within infected lesions. To overcome the antiviral function of these cells, we present a recombinant virus, rVSV-UL141, which expresses a protein from human cytomegalovirus known to downregulate the natural killer (NK) cell-activating ligand CD155. The modified vector resulted in an inhibition of NK cell recruitment in vitro, as well as decreased intratumoral accumulations of NK and NKT cells in vivo. Administration of rVSV-UL141 through hepatic artery infusion in immune-competent Buffalo rats harboring orthotopic, multi-focal HCC lesions resulted in a one-log elevation of intratumoral virus replication over a control rVSV vector, which translated to enhance tumor necrosis and substantial prolongation of survival. Moreover, these results were achieved in the absence of apparent toxicities. The present study suggests the applicability of this strategy for the development of effective and safe oncolytic agents to treat multi-focal HCC, and potentially a multitude of other cancers, in the future.

Ischemia/reperfusion (I/R) injury is a main cause of primary dysfunction or non-function after liver transplantation (LTx). Recent evidence indicates that an increase in nitric oxide (NO) production after LTx is associated with I/R injury. The aim of this study was to demonstrate that low-dose FK506 in combination with aminoguanidine (AGH), which leads to a reduction of NO levels, has a protective effect by reducing I/R associated injury after LTx. Fortyone DA-(RT1av1) rats served as donors and recipients for syngenic orthotopic arterialised LTx. They were divided into 4 groups: controls without pre-/treatment (I), pre-/treatment with high-dose FK506 (II), pre-/treatment with AGH only (III), and pre-/treatment with low-dose FK506 in combination with AGH (IV). After LTx the laboratory parameters and liver biopsy were performed. The levels of transaminase (ALT) in groups I, II and III were significantly higher on day 3 after LTx compared to group IV (p = 0.001, p = 0.001, p = 0.000). In group IV the I/R-associated liver necrosis rate was reduced significantly. Our results demonstrated that a combined dual pharmacological pretreatment (group IV) reduced I/R injury of the graft after LTx in a rat model.

High-fructose consumption is associated with insulin resistance and diabetic dyslipidemia, but the underlying mechanism is unclear. We show in hamsters that high-fructose feeding stimulated forkhead box O1 (FoxO1) production and promoted its nuclear redistribution in liver, correlating with augmented apolipoprotein C-III (apoC-III) production and impaired triglyceride metabolism. High-fructose feeding upregulated peroxisome proliferator-activated receptor-γ coactivator-1β and sterol regulatory element binding protein-1c expression, accounting for increased fat infiltration in liver. High-fructose-fed hamsters developed hypertriglyceridemia, accompanied by hyperinsulinemia and glucose intolerance. These metabolic aberrations were reversible by fenofibrate, a commonly used anti-hypertriglyceridemia agent that is known to bind and activate peroxisome proliferator-activated receptor-α (PPARα). PPARα physically interacted with, but functionally antagonized, FoxO1 in hepatic apoC-III expression. These data underscore the importance of FoxO1 deregulation in the pathogenesis of hypertriglyceridemia in high-fructose-fed hamsters. Counterregulation of hepatic FoxO1 activity by PPARα constitutes an important mechanism by which fibrates act to curb apoC-III overproduction and ameliorate hypertriglyceridemia., This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-066301.