Fig. 1. Effect of complex composition and charge ratio (+/−) on luciferase gene expression in TSA cells. Cells were covered with 0.3 ml of DMEM-HG prior to the addition of cationic liposome/DNA complexes. EPOPC:Chol liposomes pre-incubated or not with HSA (32 μg/μg of DNA), were complexed with 1 μg of pCMVluc at the indicated lipid/DNA charge ratios. After 4 h incubation, the medium was replaced with DMEM-HG and the cells were further incubated for 48 h. The level of luciferase gene expression was evaluated as described in Materials and methods. The data are expressed as RLU of luciferase per mg of total cell protein (mean ± standard deviation obtained from triplicates), and are representative of at least three independent experiments.

Fig. 2. Effect of ganciclovir concentration on the viability of TSA cells. Cells were covered with 0.3 ml of DMEM-HG before the addition of 200 μl of HSA-EPOPC:Chol/DNA (+/−) (4/1) complexes (transfected cells-T(HSA): solid line) or 200 μl of EPOPC:Chol/DNA (+/−) (4/1) complexes (transfected cells-T: broken line), containing the pCMVtk plasmid, or 200 μl of HBS (non-transfected cells: dot line). After 4 h incubation, the medium was replaced with DMEM-HG (control cells and transfected cells non-treated with GCV) or with DMEM-HG containing different concentrations of ganciclovir (1, 25, 50 or 100 μM) and the cells were further incubated for 6 days. The medium with or without GCV was replaced daily and cell viability was assessed by the Alamar Blue assay, as described in Materials and methods. Cell viability (as a percentage of control cells) was calculated according to the formula (A₅₇₀ − A₆₀₀) of treated cells × 100 / (A₅₇₀ − A₆₀₀) of control cells.

Fig. 3. Effect of the in vitro pre-treatment of TSA cells on the subcutaneous tumor growth in BALB/c mice. TSA cells were transfected in vitro with the HSA-EPOPC:Chol/DNA (+/−) (4/1) complexes, containing the different plasmids (pCMVIL-12, pCMVtk or pCMVluc), or incubated with HBS. After 4 h incubation, the medium was replaced with DMEM-HG containing 10% FBS and the cells were further incubated for 24 h. Cells were then resuspended in PBS saline buffer, in order to obtain a final cell density of 500 × 10³ cells/ml, and 200 μl of the cell suspension were immediately injected subcutaneously in the left flank of female 8-week-old BALB/c mice. Animals injected with cells transfected with the complexes containing the pCMVtk plasmid were submitted to seven intraperitonial administrations of GCV (75 mg/kg), performed from the day of cell injection during 7 consecutive days. Tumor growth was monitored every 5 days by measuring two perpendicular tumor diameters with a calliper. The data are expressed as mean tumor volume (cm³) and represent the mean ± standard deviation obtained from six animals. Mice were sacrificed when the tumor volume reached approximately 1 cm³.

Fig. 4. Antitumoral effect of different gene therapy strategies against subcutaneous tumors induced with TSA cells in BALB/c mice. Animals were treated with HSA-EPOPC:Chol/DNA (+/−) (4/1) complexes containing: (A) 40 μg of plasmid DNA or (B) different amounts of plasmid DNA (10, 20 or 40 μg). 200 μl of TSA cells (100 × 10³ cells) were injected subcutaneously into the left flank of female 8-week-old BALB/c mice. When tumor volume reached approximately 0.02 cm³, usually 9 days after cell injection, the animals were submitted to different treatments, which consisted of four intratumoral administrations of 50 μl of HSA-EPOPC:Chol/DNA (+/−) (4/1) complexes or 50 μl of HBS (non-treated) performed on days 0, 3, 5 and 7. The therapeutic complexes contained different amounts of the pCMVIL-12 plasmid (IL-12) or pCMVtk plasmid (HSV-tk), while control complexes contained pCMVluc plasmid. Animals treated with the complexes containing the pCMVtk plasmid were submitted to seven intraperitonial administrations of GCV (75 mg/kg), performed from day 3 after the first gene treatment during 7 consecutive days. Tumor growth was monitored every 5 days by measuring two perpendicular tumor diameters with a calliper. The data are expressed as mean tumor volume (cm³) and represent the mean ± standard deviation obtained from six animals. Mice were sacrificed when the tumor volume reached approximately 1 cm³.

Fig. 5. Effect of different gene therapy strategies on tumor T lymphocyte infiltration. (A) Representative images of T-lymphocyte tumor infiltration obtained by fluorescence microscopy (original magnification: × 400); (B) number of immunostained cells counted under a × 400 microscopic field (mean ± standard deviation obtained from 10 fields). (a) and (NT) tumor from non-treated animal; (b) and (Control) tumor from animal treated with complexes containing pCMVluc plasmid; (c) and (IL-12) tumor from animal treated with complexes containing pCMVIL-12 plasmid; (d) and (HSV-tk) tumor from animal treated with complexes prepared with pCMVtk plasmid. Induction of tumor and treatment was performed as described in the legend to Fig. 4. Twelve days after the first of gene therapy treatment, animals were sacrificed, tumors were removed and 6 μm cryostat sections were incubated with a rat antimouse FITC-labeled mAb against CD3 protein.

Fig. 6. Effect of different gene therapy strategies on the tumor histology. Representative images: (a) tumor from non-treated animal; (b) tumor from animal treated with complexes prepared with pCMVluc plasmid; (c) tumor from animal treated with complexes containing pCMVIL-12 plasmid; (d) tumor from animal treated with complexes prepared with pCMVtk plasmid. Induction of tumor and treatment was performed as described in the legend to Fig. 4. Twelve days after the first gene therapy treatment, animals were sacrificed, tumors were removed and 6 μm cryostat sections were stained with hematoxylin and eosin (H&E).