Nanhai G. Chen

Insertion of the human sodium iodide symporter to facilitate deep tissue imaging does not alter oncolytic or replication capability of a novel vaccinia virus.

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.

Use of an oncolytic vaccinia virus for the treatment of canine breast cancer in nude mice: preclinical development of a therapeutic agent

Mammary cancers together with cancers of the skin account for about 60% of the total cancers occurring in dogs. The veterinary options for therapeutic management of canine mammary cancer are limited and prognosis for such patients is poor. In this study, we analyzed the functionality of the oncolytic vaccinia virus strain GLV-1h68 as a possible therapeutic agent for canine mammary cancer. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the canine mammary adenoma cell line ZMTH3. Furthermore, after systemic administration this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. The efficient tumor colonization process resulted in inhibition of tumor growth and drastic reduction of tumor size. This is the first report demonstrating that vaccinia virus is an effective tool for the therapy of canine mammary cancers, which might next be applied to dogs with breast tumors.

Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma

Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.

Preferential Replication of Systemically Delivered Oncolytic Vaccinia Virus in Focally Irradiated Glioma Xenografts

Purpose: Radiotherapy is part of the standard of care in high-grade gliomas but its outcomes remain poor. Integrating oncolytic viruses with standard anticancer therapies is an area of active investigation. The aim of this study was to determine how tumor-targeted ionizing radiation (IR) could be combined with systemically delivered oncolytic vaccinia virus. Experimental Design: U-87 glioma xenografts were grown subcutaneously or orthotopically. Oncolytic vaccinia viruses GLV-1h68 and LIVP 1.1.1 were injected systemically and IR was given focally to glioma xenografts. In a bilateral tumor model, glioma xenografts were grown in both flanks, oncolytic vaccinia was injected systemically and radiation was delivered specifically to the right flank tumor, whereas the left flank tumor was shielded. Viral replication and tumor regression, after systemic injection, was analyzed and compared in irradiated and nonirradiated glioma xenografts. Results: Systemically administered oncolytic vaccinia virus replicated to higher titers in preirradiated U-87 xenografts than in nonirradiated glioma xenografts. This increased oncolytic viral replication correlated with increased tumor xenograft regression and mouse survival in subcutaneous and orthotopic U-87 glioma models compared with monotherapies. The ability of focal IR to mediate selective replication of oncolytic vaccinia was shown in a bilateral glioma model in which systemically administered oncolytic vaccinia replicated preferentially in the irradiated tumor compared with the nonirradiated tumor in the same mouse. Conclusion: These findings show a potential clinical role of focal IR in sensitizing irradiated tumor sites for preferential vaccinia virus–mediated oncolysis. Clin Cancer Res; 18(9); 2579–90. ©2012 AACR.

Efficient colonization and therapy of human hepatocellular carcinoma (HCC) using the oncolytic vaccinia virus strain GLV-1h68.

Virotherapy using oncolytic vaccinia virus strains is one of the most promising new strategies for cancer therapy. In this study, we analyzed for the first time the therapeutic efficacy of the oncolytic vaccinia virus GLV-1h68 in two human hepatocellular carcinoma cell lines HuH7 and PLC/PRF/5 (PLC) in cell culture and in tumor xenograft models. By viral proliferation assays and cell survival tests, we demonstrated that GLV-1h68 efficiently colonized, replicated in, and did lyse these cancer cells in culture. Experiments with HuH7 and PLC xenografts have revealed that a single intravenous injection (i.v.) of mice with GLV-1h68 resulted in a significant reduction of primary tumor sizes compared to uninjected controls. In addition, replication of GLV-1h68 in tumor cells led to strong inflammatory and oncolytic effects resulting in intense infiltration of MHC class II-positive cells like neutrophils, macrophages, B cells and dendritic cells and in up-regulation of 13 pro-inflammatory cytokines. Furthermore, GLV-1h68 infection of PLC tumors inhibited the formation of hemorrhagic structures which occur naturally in PLC tumors. Interestingly, we found a strongly reduced vascular density in infected PLC tumors only, but not in the non-hemorrhagic HuH7 tumor model. These data demonstrate that the GLV-1h68 vaccinia virus may have an enormous potential for treatment of human hepatocellular carcinoma in man.

Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells

BackgroundRecent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy.MethodsCancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44+CD24+ESA+ cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models.ResultsWe demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44+CD24+ESA+ cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44+CD24-ESA+ cells.ConclusionsTaken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.

Novel therapy for anaplastic thyroid carcinoma cells using an oncolytic vaccinia virus carrying the human sodium iodide symporter

BACKGROUND Anaplastic thyroid carcinoma (ATC) is fatal with resistance to radiotherapy because of the loss of intrinsic human sodium iodine symporter (hNIS). We determined whether vaccinia virus carrying hNIS kills and induces hNIS reexpression in ATC cells, facilitating deep-tissue imaging. METHODS Vaccinia virus (GLV-1h153) carrying hNIS was tested against ATC lines for killing and replication via cytotoxicity and viral plaque assays. Cellular radiouptake was determined using radiouptake assays. GLV-1h153-infected ATC xenografts were imaged via (99m)Tc-pertechnetate. RESULTS GLV-1h153 infected, replicated in, and killed all ATC cell lines. GFP expression confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1.0, GLV-1h153 reached near 100% cytotoxicity in 8305c and FRO by day 5 and 70% in the least sensitive cell line, 8505c. GLV-1h153-infected ATC cells had a 14-fold increase of hNIS-specific radiouptake compared with uninfected control 24 hours after infection at an MOI of 1.0. In vivo, GLV-1h153 facilitated imaging of hNIS expression in 8505c tumors using (99m)Tc-pertechnetate. CONCLUSION GLV-1h153 is an effective oncolytic agent against ATC. The results show hNIS-specific radiouptake in infected ATC cells, facilitating deep-tissue imaging. GLV-1h153 is a promising candidate for treatment and imaging, and potentially enhancing susceptibility to radioiodine therapy by converting non-hNIS-expressing cells into hNIS-expressing ATC cells.

Combination of fractionated irradiation with anti-VEGF expressing vaccinia virus therapy enhances tumor control by simultaneous radiosensitization of tumor associated endothelium.

Oncolytic viruses are currently in clinical trials for a variety of tumors, including high grade gliomas. A characteristic feature of high grade gliomas is their high vascularity and treatment approaches targeting tumor endothelium are under investigation, including bevacizumab. The aim of this study was to improve oncolytic viral therapy by combining it with ionizing radiation and to radiosensitize tumor vasculature through a viral encoded anti‐angiogenic payload. Here, we show how vaccinia virus‐mediated expression of a single‐chain antibody targeting VEGF resulted in radiosensitization of the tumor‐associated vasculature. Cell culture experiments demonstrated that purified vaccinia virus encoded antibody targeting VEGF reversed VEGF‐induced radioresistance specifically in endothelial cells but not tumor cells. In a subcutaneous model of U‐87 glioma, systemically administered oncolytic vaccinia virus expressing anti‐VEGF antibody (GLV‐1h164) in combination with fractionated irradiation resulted in enhanced tumor growth inhibition when compared to nonanti‐VEGF expressing oncolytic virus (GLV‐1h68) and irradiation. Irradiation of tumor xenografts resulted in an increase in VACV replication of both GLV‐1h68 and GLV‐1h164. However, GLV‐1h164 in combination with irradiation resulted in a drastic decrease in intratumoral VEGF levels and tumor vessel numbers in comparison to GLV‐1h68 and irradiation. These findings demonstrate the incorporation of an oncolytic virus expressing an anti‐VEGF antibody (GLV‐1h164) into a fractionated radiation scheme to target tumor cells by enhanced VACV replication in irradiated tumors as well as to radiosensitize tumor endothelium which results in enhanced efficacy of combination therapy of human glioma xenografts.

A Vaccinia Virus Encoding the Human Sodium Iodide Symporter Facilitates Long-Term Image Monitoring of Virotherapy and Targeted Radiotherapy of Pancreatic Cancer

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 124I 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 γ-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 GLV-1h153 and 131I led to enhanced tumor kill when compared with virus or 131I 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.

Vaccinia virus GLV-1h153 in combination with 131I shows increased efficiency in treating triple-negative breast cancer

We investigated the therapeutic efficacy of a replication‐competent oncolytic vaccinia virus, GLV‐1h153, carrying human sodium iodide symporter (hNIS), in combination with radioiodine in an orthotopic triple‐negative breast cancer (TNBC) murine model. In vitro viral infection was confirmed by immunoblotting and radioiodine uptake assays. Orthotopic xenografts (MDA‐MB‐231 cells) received intratumoral injection of GLV‐1h153 or PBS. One week after viral injection, xenografts were randomized into 4 treatment groups: GLV‐1h153 alone, GLV‐1h153 and 131I (~5 mCi), 131I alone, or PBS, and followed for tumor growth. Kruskal‐Wallis and Wilcoxon tests were performed for statistical analysis. Radiouptake assay showed a 178‐fold increase of radioiodine uptake in hNIS‐expressing infected cells compared with PBS control. Systemic 131I‐iodide in combination with GLV‐1h153 resulted in a 6‐fold increase in tumor regression (24 compared to 146 mm3 for the virus‐only treatment group; P<0.05; d 40). We demonstrated that a novel vaccinia virus, GLV‐1h153, expresses hNIS, increases the expression of the symporter in TNBC cells, and serves both as a gene marker for noninvasive imaging of virus and as a vehicle for targeted radionuclide therapy with 131I.—Gholami, S., Chen, C‐H., Lou, E., Belin, L. J., Fujisawa, S., Longo, V. A. Chen, N. G., Gönen, M., Zanzonico, P. B., Szalay, A. A., Fong, Y. Vaccinia virus GLV‐1h153 in combination with 131I shows increased efficiency in treating triple‐negative breast cancer. FASEB J. 28, 676–682 (2014). www.fasebj.org