Article Figures & Data
Figures
- Fig. 1.
Toxicity of vectors at varying levels of infected cells. Ovcar-5 (A and B) or CCD (C and D) cell lines were infected at varying MOI (20, 80, and 160) using the Ad-CMV-CD (B and D) or Ad-LP-CD (A and C) adenoviral vectors. The infected cells and noninfected cells were mixed in varying ratios to generate 0, 5, 10, 20, 30, 40, 50, 60, and 100% infected cells. Then cells were seeded in six-well plates and incubated for 5 days in 500 μm 5FC. Then the cells were trypsinized, and surviving cells were counted by trypan blue exclusion.
- Fig. 2.
Study of the toxicity of the control Lac-Z vector versus the CD vector. Cells (2×105) were infected at 0, 5, 20, 40, 80 and 160 MOI of vector with Ad-CMV-CD (A), Ad-Lp-CD (B), Ad-CMV-LacZ (C), Ad-Lp-LacZ (D) vectors for 90 min. Then cells were seeded in six-well plates in duplicate and incubated in 500 μm 5FC for 5 days. The percentage of surviving cells was counted by trypan blue exclusion.
- Fig. 3.
Ovarian organ cultures. Normal ovarian tissue was obtained from patients undergoing abdominal surgical procedures. The tissues were cut into small pieces and cultured in DMEM:Ham’s F12 medium with 10% charcoal-stripped serum. Twenty-four to forty-eight h later, the tissues were infected with vectors for 90 min, washed with PBS, and then incubated for 48 h. Then the tissues were frozen in OCT and sectioned, after which the frozen sections were stained by the X-Gal reaction. Left, no vector; middle, Ad-CMV-LacZ vector; right, Ad-Lp-LacZ vector.
- Fig. 4.
Effect of in vivo injection of tumor nodules with adenoviral vectors. Ovcar-5 (A) or EJ (B) cells (5×106) were injected s.c. into nude mice. After 3 weeks, the tumor nodules were measured. Then, 108 pfu of the Ad-CMV-CD, 108 pfu of the Ad-Lp-CD, or 108 pfu of the Ad-CMV-LacZ vectors were injected into each tumor nodule, and 500 mg/kg of 5FC was given i.p. once a day for 5 days. Seven days later, the tumor nodules were measured again. □ shows tumor volume before viral particles and 5FC treatment;
shows the tumor volume 7 days after exposure to viral particles and 5FC treatment. - Fig. 5.
Vector toxicity to tumor cells and adjacent tissues. Ovcar-5 cells (5x106) were injected s.c. After 3 weeks, 108 pfu of Ad-Lp-CD (right), 108 pfu of Ad-CMV-CD (left), or 108 pfu of Ad-CMV-LacZ (middle) vector were injected into each tumor nodule, and 500 mg/kg 5FC was given i.p. once a day for 5 days. Right (Ad-Lp-CD), most of the tumor cells are necrotic, whereas the adjacent muscle cells have a normal structure. Left (Ad-CMV-CD), after injection with the Ad-CMV-CD vector, the tumor cells are necrotic. Middle (Ad-CMV-LacZ), after injection of the Ad-CMV-LacZ vector, neither the muscle nor the tumor are necrotic.
Tables
- Table 1
Characterization of percentage of cells positive for the CAR, αvβ3, and αvβ5 receptors as measured by FACS analysis and study of infectivity of cells by Ad-CMV-LacZ Vector at 20 MOI as measured by β-galactosidase assay (X-Gal)
αvβ3, αvβ5, and CAR receptor levels were measured by mouse monoclonal antibodies, and the FITC-conjugated antimouse antibody was used to stain the cells. Then, FACS analysis was used to detect the percentage of the receptor-positive cells (n = 2). For infectivity, cells were exposed to virus in serum-free medium for 90 min at 20 MOI and incubated for 48 h in culture medium. Then, cells were stained by X-Gal analysis (n = 2). αvβ3 αvβ5 CAR β-Gal EJ 83 ± 8 82 ± 5 95 ± 8 95 ± 8 J82 56 ± 6 78 ± 7 80 ± 10 88 ± 10 Skov-3 64 ± 6 91 ± 8 87 ± 7 85 ± 11 Ovcar-5 48 ± 7 57 ± 5 88 ± 10 65 ± 8 Hey 81 ± 5 96 ± 10 0 10 ± 4 CCD 63 ± 8 93 ± 4 29 ± 5 70 ± 9 - Table 2
Comparison of β-galactosidase levels in cell line exposed to Ad-CMV-LacZ or Ad-Lp-LacZ (ONPG, OD)
Cells were exposed in serum-free conditions for 90 min at 20 MOI. After 48 h of incubation in culture medium, the level of the β-galactosidase (ONPG) in each cell line was measured by optical density, as outlined in “Materials and Methods” (n = 2). Ad-CMV-LacZ Ad-Lp-LacZ Ratio of CMV/Lp EJ 1.1 ± 0.2 0.9 ± 0.1 1.2 J82 1.0 ± 0.1 0.4 ± 0.1 2.5 Skov-3 0.9 ± 0.1 0.4 ± 0.1 2.2 Ovcar-5 0.9 ± 0.1 0.4 ± 0.1 2.2 CCD 0.9 ± 0.1 0.1 ± 0.01 9 - Table 3
Percentage of cells in explant cultures of ovarian cancer cells and normal peritoneal cells which score positive for β-galactosidase after exposure to the Ad-CMV-LacZ and the Ad-Lp-LacZ vectors
Samples of primary tumor, metastatic tumor, and normal peritoneum were cut into small pieces. These pieces were then digested with collagenase to produce tissue disaggregation, and the resulting cells were cultured in RPMI 1640 with 10% NBCS. All experiments were performed at 90% confluence. Samples of ascites were divided into the T25 flasks directly and washed to remove debris after cell attachment. Cells were infected in the flasks for 90 min, and after 48 h of incubation, the positive cells were measured by X-Gal staining or FACS. Ascites Primary tumor Metastatic tumor Normal peritoneum Ad-CMV-βgal X-Gal 50–80% 50–90% 45–85% 60–80% FACS 95% 94% 94% Ad-LP-βgal X-Gal 10–35% 15–60% 15–45% 1–4% FACS 39% 83% 38% CMV/LP ratio FACS 3/1 1/1 3/1 20–60/1 - Table 4
Cytotoxicity in monolayer culture of normal peritoneum and ovarian cancer cells after expression to Ad-Lp-CD and Ad-CMV-CD vectors and 5FC (percentage of cells killed)
In Ad-CMV-CD- and Ad-Lp-CD-infected samples, 500 μm 5-FC were added and incubated for 5 days, then the percentage of cells killed was estimated by comparing the percentage of cells which had died in the infected and uninfected control flasks. Ad-CMV-CD Ad-Lp-CD Ascites 98% 85% Metastatic tumor 85% 70% Primary tumor 90% 75% Normal peritoneum 95% 10% - Table 5
Tumor growth in animals injected with adenoviral LP vectors (percentage of animals found to be positive for tumors)
The SCID mice were injected with 40 million Ovcar-5 or Skov3 tumor cells, which had been infected previously in vitro with the Ad-Lp-LacZ vector or the Ad-Lp-CD vector. Starting on the second day, 500 mg/kg 5-FC was injected each day for 10 days. Animals were autopsied at 21 days after tumor cell injection, and the presence or absence of tumor nodules in the peritoneal cavity was assessed. Ad-Lp-LacZ-infected Ad-Lp-CD-infected Ovcar-5 (100 MOI) 10/10 (100%) 0/10 (0%) Skov3 (80 MOI) 5/5 (100%) 0/5 (0%)
Volume 61, Issue 11
