1. Effect of inhibition of multiple steps of angiogenesis in syngeneic murine pleural mesothelioma 
Robert E. Merritt, MD, Reiko E. Yamada, BA, Nabil Wasif, MD, Ronald G. Crystal, MD, Robert J. Korst, MD 
The Annals of Thoracic Surgery 
Volume 78, Issue 3, Pages (September 2004)
DOI: /j.athoracsur

2. Fig 1
The combination of intratumoral AdPEDF and Adsflt-1 inhibits tumor growth and prolongs survival more effectively than either strategy alone in a flank tumor model of malignant pleural mesothelioma. Seven days after AB-12 flank tumor initiation, BALB/c mice were randomized to five groups: AdPEDF plus Adsflt-1 (♦, n = 8; 5 × 1010 particles of each vector); Adsflt-1 alone (○, n = 8); AdPEDF alone (▴, n = 8); a negative control vector (AdNull, □, n = 8); or phosphate-buffered saline (PBS, ■, n = 8). All animals received intratumoral injections and the total vector dose per mouse was 1011 particles suspended in 100 μL PBS for all groups in which vectors were administered. The tumor area was assessed every 2 to 3 days with microcalipers, and statistical analysis was performed 25 days after tumor initiation. The mice were sacrificed when they appeared moribund or when the largest tumor diameter reached 15 mm. Arrows indicate time point of vector (or PBS) administration. (A) Tumor area for AB-12 mesothelioma flank tumors in BALB/c mice. The data points represent the mean tumor area ± SEM. (B) Survival for BALB/c mice bearing AB-12 mesothelioma flank tumors. (AdPEDF = adenovirus vector encoding the human pigment epithelium-derived factor; Adsflt-1 = adenovirus vector encoding the human soluble vascular endothelial growth factor receptor-1.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )

3. Fig 2
Immunohistochemical analysis of flank tumor blood vessel density after intratumoral administration of AdPEDF and Adsflt-1. Seven days after AB-12 flank tumor initiation, BALB/c mice were randomized to one of five groups: AdPEDF plus Adsflt-1; Adsflt-1 alone; AdPEDF alone; a negative control vector (AdNull); or phosphate-buffered saline (PBS). All animals received intratumoral injections and the total vector dose was 1011 particles suspended in 100 μL PBS for all groups in which vectors were administered. The tumors were harvested 10 days after vector therapy and frozen sections were stained immunohistochemically for CD31. The dark staining represents endothelial cells within the AB-12 flank tumors. (A) PBS (n = 8). (B) AdNull (n = 8). (C) Adsflt-1 (n = 8). (D) AdPEDF (n = 8). (E) Adsflt-1 plus AdPEDF (n = 8; 5 × 1010 particles of each vector). (AdPEDF = adenovirus vector encoding the human pigment epithelium-derived factor; Adsflt-1 = adenovirus vector encoding the human soluble vascular endothelial growth factor receptor-1.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )

4. Fig 3
Tumor blood vessel density is significantly diminished after the combined administration of AdPEDF and Adsflt-1. Metamorph digital image analysis of the tumor sections from Figure 2 was performed as described in the Methods section. Five random ×20 fields of AB-12 tumor sections were used to quantify tumor vessel density (number of vessels/μm2). The tumor vessel density is reported as the mean number of CD31-positive vessels/μm2 ± SEM. (AdNull = a negative control vector; AdPEDF = adenovirus vector encoding the human pigment epithelium-derived factor; Adsflt-1 = adenovirus vector encoding the human soluble vascular endothelial growth factor receptor-1; PBS = phosphate-buffered saline.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )

5. Fig 4
Thoracic tumor burden after intrapleural administration of AdPEDF and Adsflt-1 in an orthotopic model of malignant pleural mesothelioma. BALB/c mice underwent intrapleural instillation of AB-12 mesothelioma cells as well as phosphate-buffered saline (PBS), a negative control vector (AdNull), AdPEDF alone, Adsflt-1 alone, or AdPEDF plus Adsflt-1. Vectors were instilled into the pleural cavity at the same time as the tumor cells and the total vector dose was 1011 particles suspended in 100 μL PBS for all groups in which vectors were administered. On day 14, the animals were sacrificed and necropsies were performed to assess the presence of tumor in the pleural cavity. In the photographs, tumor deposits are circled. (A) PBS (n = 5). (B) AdNull (n = 4). (C) Adsflt-1 (n = 4). (D) AdPEDF (n = 4). (E) Adsflt-1 plus AdPEDF (n = 5; 5 × 1010 particles of each vector); this panel represents the only animal with grossly visible tumor in the pleural cavity in the combination group. (AdPEDF = adenovirus vector encoding the human pigment epithelium-derived factor; Adsflt-1 = adenovirus vector encoding the human soluble vascular endothelial growth factor receptor-1; Th = thymus; H = heart; L = lungs.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )

6. Fig 5
Quantitative assessment of thoracic tumor burden after intrapleural administration of AdPEDF and Adsflt-1 in an orthotopic model of malignant pleural mesothelioma. Tumor deposits were excised from the animals in the experiment described in Figure 4 and weighed. Each data point represents an individual mouse, whereas the horizontal bars depict the mean weights. (AdNull = a negative control vector; AdPEDF = adenovirus vector encoding the human pigment epithelium-derived factor; Adsflt-1 = adenovirus vector encoding the human soluble vascular endothelial growth factor receptor-1; PBS = phosphate-buffered saline.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )

7. Fig 6
The combination of intrapleural AdPEDF and Adsflt-1 enhanced survival of tumor-bearing mice compared with either vector alone in an orthotopic model of malignant pleural mesothelioma. BALB/c mice underwent intrapleural instillation of AB-12 mesothelioma cells as well as phosphate-buffered saline (PBS, ■, n = 6); a negative control vector (AdNull, □, n = 6); AdPEDF alone (▴, n = 6); Adsflt-1 (○, n = 6); or AdPEDF plus Adsflt-1 (♦, n = 6; 5 × 1010 particles of each vector). Vectors were instilled into the pleural cavity at the same time as the tumor cells and the total vector dose was 1011 particles suspended in 100 μL PBS for all groups in which vectors were administered. The mice were observed daily and survival was recorded. (AdPEDF = adenovirus vector encoding the human pigment epithelium-derived factor; Adsflt-1 = adenovirus vector encoding the human soluble vascular endothelial growth factor receptor-1.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )

8. Fig 7
Effect of intrapleural instillation of AdPEDF and Adsflt-1 on lung histology in tumor-free BALB/c mice. Phosphate-buffered saline (PBS), a negative control vector (AdNull), Adsflt-1, or AdPEDF were instilled into the right pleural space of normal BALB/c mice. The total vector dose was 1011 particles suspended in 100 μL PBS for all groups in which vectors were administered. After 21 days, the animals were sacrificed and the lungs were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with hematoxylin & eosin. A separate group of normal mice underwent intratracheal instillation of porcine pancreatic elastase (30 μg in 50 μL PBS) 28 days before sacrifice to serve as a positive control for the measurement of pulmonary emphysema. (A) PBS. (B) AdNull. (C) Adsflt-1. (D) AdPEDF. (E) Elastase. (AdPEDF = adenovirus vector encoding the human pigment epithelium-derived factor; Adsflt-1 = adenovirus vector encoding the human soluble vascular endothelial growth factor receptor-1.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )

9. Fig 8
Intrapleural instillation of AdPEDF or Adsflt-1 does not increase interalveolar septal distance in normal mice. As a measure of pulmonary emphysema, the mean linear intercept was calculated from the lung sections of the animals depicted in Figure 7 as described in Methods. The mean linear intercept is determined by dividing the total length of a line drawn across a lung section by the total number of alveolar intercepts encountered. Bars represent the mean of six random measurements. Higher mean linear intercept values reflect greater interalveolar septal distance, a microscopic characteristic of pulmonary emphysema. (AdNull = a negative control vector; AdPEDF= adenovirus vector encoding the human pigment epithelium-derived factor; Adsflt-1 = adenovirus vector encoding the human soluble vascular endothelial growth factor receptor-1; PBS = phosphate-buffered saline.)
The Annals of Thoracic Surgery  , DOI: ( /j.athoracsur )