1. Engineered Newcastle Disease Virus as an Improved Oncolytic Agent Against Hepatocellular Carcinoma 
Jennifer Altomonte, Sabrina Marozin, Roland M Schmid, Oliver Ebert 
Molecular Therapy 
Volume 18, Issue 2, Pages (February 2010)
DOI: /mt
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
rNDV/F3aa(L289A) is sensitive to the inhibitory effects of interferon (IFN). (a) Primary human hepatocytes (PHHs), two human hepatocellular carcinoma (HCC) cell lines [(b) Huh7 and (c) HepG2], and (d) a rat HCC cell line (McA-RH7777) were untreated or treated overnight with increasing concentrations of universal type I interferon (IFN) (10–1,000 IU/ml) in 24-well dishes. Triplicate wells were infected with rVSV-LacZ, rNDV/F3aa, or rNDV/F3aa(L289A) at a multiplicity of infection of 1. Viral titers were determined by TCID50 of conditioned media harvested 24 hours postinfection. Values represent the mean ± SD. NDV, Newcastle disease virus; TCID50, 50% tissue culture infectious dose; VSV, vesicular stomatitis virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
rNDV/F3aa(L289A) induces interferon (IFN) signaling in primary human hepatocytes (PHHs). PHHs, two human hepatocellular carcinoma (HCC) cell lines (Huh7 and HepG2), and a rat HCC cell line (McA-RH7777) were co-transfected with (a) pIFN-β-Luciferase or (b) pISRE-Luciferase and pRL-Luciferase in 24-well dishes. At 24 hours post-transfection, cells were mock-treated, stimulated with polyinosinic:polycytidylic acid (pI:C) (2.5 mg/ml) or IFN (1,000 IU/ml), or infected with rVSV-LacZ, rNDV/F3aa, or rNDV/F3aa(L289A) at a multiplicity of infection of 1.0 and incubated overnight. Firefly-luciferase activity was normalized to renilla activity to control for transfection efficiency. Fold-induction of the promoters was calculated as a function of luciferase activity in stimulated versus mock-treated cells. Data are presented as the mean ± SD of triplicate measurements. NDV, Newcastle disease virus; VSV, vesicular stomatitis virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
F(L289A) modification results in enhanced fusogenic activity of Newcastle disease virus (NDV). Human (Huh7 and HepG2) and rat (McA-RH7777) cells were mock infected or infected with rNDV/F3aa or rNDV/F3aa(L289A) at a multiplicity of infection of 0.1. At 48 hours postinfection, cells (a) were stained for β-gal expression or (b) analyzed for syncytial index. The syncytial index was calculated as the number of nuclei per syncytia divided by the number of nuclei per field of view. Each cell line was analyzed in triplicate, and data are expressed as the mean ± SD.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
NDV causes transient body weight loss and elevation of liver enzymes in rats. Male non-tumor-bearing Buffalo rats were treated with PBS, or 108 TCID50 of rNDV/F3aa or rNDV/F3aa(L289A) by hepatic arterial infusion (N = 5). (a) Body weight was recorded daily, and (b) blood was drawn at the indicated time points for measurement of serum chemistries (right panel). Values are expressed as mean ± SD. BUN, blood urea nitrogen; GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; NDV, Newcastle disease virus; PBS, phosphate-buffered saline; TCID50, 50% tissue culture infectious dose.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
NDV/F3aa(L289A) treatment results in tumor-specific necrosis and virus replication in hepatocellular carcinoma (HCC)–bearing rats. Male Buffalo rats bearing multifocal orthotopic HCC nodules were treated with rNDV/F3aa(L289A) (108 TCID50) by hepatic arterial infusion, and killed at the indicated time points post-treatment (n = 3). (a) Tumor-containing liver sections were subjected to hematoxylin and eosin staining and analyzed for tumor necrosis and liver toxicity. Representative sections are shown at ×5 (overview) and ×20 (magnification of tumor and liver sections) magnification. Morphometric analysis of tumor necrosis was performed using ImageJ software (National Institutes of Health), and is shown as the mean ± standard deviation at each time point. (b) Tumor-containing liver sections were subjected to β-gal staining. Representative sections are shown at ×5 (overview) and ×20 (magnification of tumor and liver sections) magnification. Viral titers were quantified by TCID50 analysis of liver and tumor lysate, and are expressed as the mean ± SD. NDV, Newcastle disease virus; TCID50, 50% tissue culture infectious dose.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
Infiltration of natural killer (NK) and myeloperoxidase (MPO) cells to NDV/F3aa(L289A) infected hepatocellular carcinoma (HCC) tumors. Rats bearing multifocal HCC lesions were treated by transarterial infusion of NDV/F3aa(L289A) and killed at 30 minutes, and day 1, 3, 5, and 7 after treatment. Tumor-containing liver sections were processed for immunohistochemical staining for (a) NK cell marker and (b) MPO. Positive stained cells were quantified using ImageJ software (NIH), and the mean ± SD are shown. Representative immunohistochemical sections are presented in (c) ×5 and (d) ×20 magnification. NDV, Newcastle disease virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
rNDV/F3aa(L289A) therapy results in significant survival prolongation hepatocellular carcinoma (HCC)–bearing rats. Male Buffalo rats bearing multifocal HCC lesions were treated by hepatic arterial infusion of phosphate-buffered saline (PBS) (n = 10), rNDV/F3aa (n = 14), or rNDV/F3aa(L289A) (n = 13) and followed for survival. Animals were monitored daily, and survival was plotted as a Kaplan–Meier survival curve. P values for PBS versus rNDV/F3aa is <0.02, for rNDV/F3aa versus rNDV/F3aa(L289A) is <0.04. Statistical significance was determined by log-rank test. NDV, Newcastle disease virus.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2010 The American Society of Gene & Cell Therapy Terms and Conditions