Herpes simplex virus-1 (HSV-1) oncolytic therapy and gene therapy are promising treatment modalities against cancer. NV1066, one such HSV-1 virus, carries a marker gene for enhanced green fluorescent protein (EGFP). The purpose of this study was to determine whether NV1066 is cytotoxic to lung cancer and whether EGFP is a detectable marker of viral infection in vitro and in vivo. We further investigated whether EGFP expression in infected cells can be used to localize the virus and to identify small metastatic tumor foci (<1 mm) in vivo by means of minimally invasive endoscopic systems equipped with fluorescent filters. In A549 human lung cancer cells, in vitro viral replication was determined by plaque assay, cell kill by LDH release assay, and EGFP expression by flow cytometry. In vivo, A549 cells were injected into the pleural cavity of athymic mice. Mice were treated with intrapleural injection of NV1066 or saline and examined for EGFP expression in tumor deposits using a stereomicroscope or a fluorescent thoracoscopic system. NV1066 replicated in, expressed EGFP in infected cells and killed tumor cells in vitro. In vivo, treatment with intrapleural NV1066 decreased pleural disease burden, as measured by chest wall nodule counts and organ weights. EGFP was easily visualized in tumor deposits, including microscopic foci, by fluorescent thoracoscopy. NV1066 has significant oncolytic activity against a human NSCLC cell line and is effective in limiting the progression of metastatic disease in an in vivo orthotopic model. By incorporating fluorescent filters into endoscopic systems, a minimally invasive means for diagnosing small metastatic pleural deposits and localization of viral therapy for thoracic malignancies may be developed using the EGFP marker gene inserted in oncolytic herpes simplex viruses.

Background: NV1066, a replication-competent oncolytic herpes simplex virus type 1 (HSV-1) attenuated by a deletion in the gene _134.5, preferentially replicates in and kills malignant cells. _134.5 encodes ICP34.5, a viral protein essential for productive replication, which has homology with mammalian stress response induced GADD34 (Growth Arrest and DNA Damage-Inducible Protein). We hypothesized that cisplatin upregulates GADD34 expression, which enhances NV1066 replication and oncolysis. Methods: Ten human malignant pleural mesothelioma (MPM) cell lines were infected with NV1066 at multiplicities of infection (MOI; ratio of viral particles per tumor cell) 0.005 to 0.8 in vitro, with and without cisplatin (1 to 4 µM). In the MPM cell line VAMT, viral replication was determined by plaque assay, cell kill by lactate dehydrogenase assay, and GADD34 induction by quantitative RT-PCR and Western blot. Synergistic efficacy was confirmed by the isobologram and combination index methods of Chou-Talalay. GADD34 upregulation by cisplatin was inhibited with GADD34 siRNA to further confirm the synergistic efficacy dependence with GADD34. Results: Combination therapy with NV1066 and cisplatin showed strong synergism in epithelioid (H-2452, H-Meso), sarcomatoid (H-2373, H-28), and biphasic (JMN, Meso-9, MSTO-211H) MPM cell lines, and an additive effect in others. In VAMT cells combination therapy enhanced viral replication 4 to11-fold (p < 0.01) and cell kill 2 to 3-fold (p < 0.01). Significant dose reductions for both agents (2 to 600-fold) were achieved over a wide range of therapeutic-effect levels (LD50 – LD99) without compromising cell kill. Synergistic cytotoxicity correlated with GADD34 upregulation (2 to 4-fold, p < 0.01) and was eliminated following transfection with GADD34 siRNA. Conclusion: Cisplatin-induced GADD34 expression selectively enhanced the cytotoxicity of the _134.5-deficient oncolytic virus, NV1066. This provides a cellular basis for combination therapy with cisplatin and NV1066 to treat MPM and achieve synergistic efficacy, while minimizing dosage and toxicity.

BACKGROUND:Replication-competent, tumor specific herpes simplex virus NV1066 expresses green fluorescent protein (GFP) in infected cancer cells. We sought to determine the feasibility of GFP-guided imaging technology in the intraoperative detection of small tumor nodules.METHODS:Human cancer cell lines were infected with NV1066 at multiplicities of infection of 0.01, 0.1 and 1. Cancer cell specific infectivity, vector spread and GFP signal intensity were measured by flow cytometry and time-lapse digital imaging (in vitro); and by use of a stereomicroscope and endoscope equipped with a fluorescent filter (in vivo).RESULTS:NV1066 infected all cancer cell lines and expressed GFP at all MOIs. GFP signal was significantly higher than the autofluorescence of normal cells. One single dose of NV1066 spread within and across body cavities and selectively infected tumor nodules sparing normal tissue. Tumor nodules undetectable by conventional thoracoscopy and laparoscopy were identified by GFP fluorescence.CONCLUSION:Virally-directed fluorescent imaging (VFI) is a real-time novel molecular imaging technology that has the potential to enhance the intraoperative detection of endoluminal or endocavitary tumor nodules.

PURPOSE:Although a variety of malignant tumors are susceptible to therapy with oncolytic herpes simplex viruses, the determinants of tumor sensitivity to these viruses are poorly understood. Nectin-1 is a cell surface adhesion molecule that is a component of intercellular adherens junctions and also functions as a herpes viral receptor. Because highly invasive cells may have decreased intercellular adhesion, we sought to determine if such cells might also have altered availability of cell surface nectin-1 to act as a herpes receptor.EXPERIMENTAL DESIGN AND RESULTS:A series of squamous cell carcinoma lines of increasing migratory and invasive potential, termed MG1-MG14, were selected by serial passages of murine SCC7 through Matrigel invasion chambers. Available cell surface nectin-1 was enhanced on the MG11 and MG14 cell lines in comparison to SCC7 as measured by cellular ELISA and immunofluorescence microscopy. A replication-competent, oncolytic herpes virus (NV1023) showed an increased ability to enter MG11 and MG14 cells as compared with SCC7 cells. Furthermore, MG11 and MG14 supported increased herpes viral replication and cytotoxicity over SCC7. For all three of the cell lines, viral entry assays revealed that the actively migrating cells were significantly more susceptible to herpes infection than the nonmigrating cells.CONCLUSIONS:These results show that malignant cells with highly migratory and invasive properties may exhibit increased cell surface nectin-1 availability, which may serve as a herpes viral receptor to enhance the efficacy of herpes oncolytic therapy. This finding has implications regarding patient selection for future clinical trials using these promising therapeutic vectors.

BACKGROUND:Basic science research (BSR) publications in general surgical (GS) journals are an important "translational bridge" for practicing surgeons and surgical trainees. The purpose of this study is to characterize the BSR publications in GS journals and to analyze their citation frequencies. METHODS:In 1996, all (224) BSR publications in the five highest rated U.S. GS journals (by impact factor) were reviewed, characterized, and their citation frequencies were compared to BSR publications in non-GS journals. RESULTS:On average, a BSR publication from these journals is cited 32 times (range 1-141, median 11). Half of the publications were cited more than 10 times in 6 years and 22% were cited twice, or more, within 1 year of publication. One in four publications were cited twice or more in journals with an impact factor greater than five. Citation frequencies of BSR publications in GS journals were related to the journal impact factor (P = 0.07), and to having a basic scientist (i.e., Ph.D.) as one of the authors (P < 0.01). Citation characteristics of BSR publications in GS journals were similar to those of BSR publications in non-GS journals with similar impact factors. CONCLUSIONS:We found that BSR publications in the U.S. GS journals studied had significant citation frequencies.

Purpose: Oncolytic herpes simplex viruses (HSVs) may have significant antitumor effects resulting from the direct lysis of cancer cells. HSVs may also be used to express inserted transgenes to exploit additional therapeutic strategies. The ability of an interleukin (IL)-12-expressing HSV to treat squamous cell carcinoma (SCC) by inhibition of tumor angiogenesis is investigated in this study. Experimental Design: A replication-competent, attenuated, oncolytic HSV carrying the murine IL-12 gene (NV1042), its non-cytokine-carrying analog (NV1023), or saline was used to treat established murine SCC flank tumors by intratumoral injection. The expression of secondary antiangiogenic mediators was measured. Angiogenesis inhibition was assessed by in vivo Matrigel plug assays, flank tumor subdermal vascularity, and in vitro endothelial cell tubule formation assay. Results: Intratumoral injections of NV1042 (2 × 107 plaque-forming units) into murine SCC VII flank tumors resulted in smaller tumor volumes as compared with NV1023 or saline. IL-12 and IFN-γ expression in tumors was 440 and 2.2 pg/mg, respectively, at 24 h after NV1042 injection, but both IL-12 and IFN-γ were undetectable (<0.2 pg/mg) after NV1023 or saline injections. Expression of two antiangiogenesis mediators, monokine induced by IFN-γ and IFN-inducible protein 10, was elevated after NV1042 treatment. Matrigel plug assays of NV1042-transfected SCC VII tumor cells demonstrated significantly decreased hemoglobin content and microvessel density as compared with NV1023 and PBS. Excised murine flank tumors treated with NV1042 had decreased subdermal vascularity as compared with NV1023 and PBS. Both splenocytes and IL-12 expression by NV1042 were required for in vitro inhibition of endothelial tubule formation. Conclusions: IL-12 expression by an oncolytic herpes virus enhances therapy of SCC through antiangiogenic mechanisms. Strategies combining HSV oncolysis with angiogenesis inhibition merit further investigation for potential clinical application.

Introduction: Replication-competent, attenuated herpes simplex virus type-1 (HSV-1) selectively infect and lyse cancer cells. The purpose of this study was to determine whether HSV-1 therapy was cytotoxic to malignant mesothelioma (MM) cell lines and improves survival in a murine model of MM.

Purpose: There currently is no therapy that enhances the survival of patients with distantly metastatic squamous cell carcinoma (SCC). Engineered herpes oncolytic viruses are effective therapeutic agents when delivered directly to tumors in animal models, but their efficacy in treating disseminated disease is poorly defined. Experimental Design: We treated disseminated pulmonary SCC in mice with an interleukin (IL)-12-expressing oncolytic herpes virus (NV1042) or with the parent oncolytic virus (NV1023, IL-12 deficient) by i.v. tail vein administration. Results: Lung IL-12 was 16.1 pg/mg and IFN-γ was 4.3 pg/mg at day 1 after a single dose of NV1042 (5 × 107 plaque-forming units); levels of both were undetectable for NV1023. 5-Bromo-4-chloro-3-indolyl-β-d-galactopyranoside histochemistry demonstrated viral infection of disseminated pulmonary tumor nodules by both vectors at day 1, with sparing of adjacent alveolar cells. NV1042-treated lungs showed no surface nodules at day 12, in contrast to NV1023-treated (92 ± 27 surface nodules) and PBS-treated (225 ± 9 surface nodules) lungs. Significantly enhanced survival was observed in NV1042-treated animals compared with NV1023- and PBS-treated animals (log rank < 0.05). In animals with a low tumor burden, 100% of NV1042-treated, 70% of NV1023-treated, and none of the control animals achieved long-term survival. NV1042 efficacy was similar to NV1023 efficacy in animals depleted of CD4/CD8 T lymphocytes, showing that IL-12 expression enhances oncolytic activity through immune effects. Histology showed no cytopathic effects in non-tumor-bearing lung, brain, spleen, liver, and pancreas after completion of viral therapy. No animals demonstrated any visible side effects attributable to viral therapy. Conclusions: The i.v. delivery of an oncolytic herpes virus may achieve effective infection, oncolysis, and transgene expression at distant tumor sites. This approach to systemic therapy combining oncolysis with IL-12 immune stimulation led to significantly improved survival in animals with disseminated SCC.