Female Syrian golden hamsters aged 6-8wk, purchased from Harlan (Indianapolis, Indiana, USA) were housed in the specific pathogen-free unit of the Animal Resource Center at the Asan Institute for Life Sciences and Technology.

The KIGB-5 cell line, a gallbladder carcinoma cell line derived from Syrian golden hamsters, was established and kindly provided by Dr. Tajima (Nagasaki University, Nagasaki, Japan). This cell line was maintained in complete RPMI-1640 (GIBCO BRL, Grand Island, NY, USA) supplemented with 100 mL/L fetal bovine serum (FBS) (GIBCO BRL, Grand Island, NY, USA), 100 U/mL penicillin (Sigma, St. Louis, MO, USA), 0.1 mg/mL streptomycin (Sigma), and 10^-5 mol/L β-mercaptoethanol (Sigma)[17]. The 293 cell line, derived from human embryonic kidney, was maintained in high glucose DMEM (GIBCO BRL) and 100 mL/L FBS (GIBCO BRL).

Bone marrow cells were obtained by flushing the femurs and tibias of 6-8 wk old Syrian golden hamsters with RPMI-1640 medium (GIBCO BRL) using 2mL syringes with 23-guage needles. After a single-cell suspension was prepared, the cells were plated at a concentration of 5.0 ×10⁵ /mL in 75 cm² plastic tissue culture flasks, and cultured in McCoy’s 5A medium (Sigma) supplemented with 125 mL/L FBS, 125 mL/L horse serum (HS), 200mmol/L L-glutamine, 0.05 mg/mL hydrocortisone, 1 ng/mL recombinant human basic fibroblast growth factor (rh bFGF, Invitrogen Corporation, Groningen, Netherlands), and 0.5ng/mL rh IL-1α (Invitrogen) at 37 °C in 50 mL/L CO₂ and 950 mL/L air. The cultures were fed weekly by replacing 500 mL/L of the supernatant with fresh culture medium. When the cells became confluent, they were trypsinized and harvested for passage. Gene transduction was performed on cells after 5 passages.

Three replication-deficient adenoviral vectors were used, namely Ad/Lac-Z containing the β-galactosidase gene, Ad/∆E1 and Ad/hIL-2 harboring the human IL-2 gene. Each of these vectors was constructed from human adenovirus serotype 5 by homologous recombination. The expression of the inserted genes was driven by a CMV promoter. The recombinant adenoviruses were propagated into human 293 embryonic kidney cells, and the adenoviral titers were determined by a plaque-forming assay with these cells. Recombinant adenoviruses were diluted to a titer of 5.0 × 10⁸ PFU/mL in viral supernatant and stored at -70 °C.

Stromal cells (SCs) were seeded at a density of 1.0 × 10⁵ /mL in 75 cm² plastic tissue culture flasks and allowed to grow. The medium was removed, the cells were washed twice with PBS, and the modified adenovirus was added at various multiplicities of infection (MOI). The cells were cultured at 37°C for 24h in a humidified atmosphere of 50 mL/L CO₂.

The transduction efficiency of the recombinant adenoviruses was determined by an X-gal assay. BMSCs were transduced with Ad/Lac Z for 24h at 0, 10, 20, 50, 100 MOI. After one round of transfection the transduction efficiency was determined by the percentage of blue-stained cells, after subtraction of the percentage of non-specifically stained, uninfected BMSCs[7]. Stained positive cells were 5-10% at 10 MOI, 10-15% at 20 MOI, 40-50% at 50 MOI, 60-65% at 100 MOI (Figure 1).

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Figure 1 X-gal assay of hamster BMSCs transfected with Ad/ Lac Z at 0 (A), 10 (B), 20 (C), 50 (D), and 100 (E) MOI.

From the results of the preliminary experiments we decided to use 50 MOI for further experiments.

BMSCs were seeded in 12-well plates and transfected 24 h later with Ad/Lac Z. The cells were cultured for 24 h at 37 °C in a humidified atmosphere containing 50 mL/L CO₂. The media were removed and the cells were washed with PBS. Fixing solution was added to the transfected cells at room temperature for 5 min and removed. The cells were stained with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside(X-gal) overnight at 37°C in a humidified atmosphere containing 50 mL/L CO₂.

To test for homing of gene-transduced stromal cells, 6-8 wk old Syrian gold hamsters were infused intraperitoneally (IP) or injected via the femoral vein with transduced stromal cells mixed in 0.1 mL PBS at a dose of 2.5×10⁶ cells per animal. After one week, the hamsters were sacrificed and the bone marrow, spleen, liver, kidneys, and lungs were obtained. Single cells prepared from these organs using collagenase (GIBCO BRL) were cultured for four days and stained with X-gal to test for homing of the transfected cells.

Total RNA was extracted using Trizol (Life Technologies, Invitrogen) following the manufacturer’s instructions. The extracted RNA was resuspended in diethylpyrocarbonate-treated water at a concentration of 0.5 μg/μL. Two ug of each total RNA preparation was reverse-transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (Boeringer, Manheim, Germany) in 20 μL reaction mixture containing 50 mmol/L DTT, 1 μg oligo (dT) and 0.125 mmol/L dNTPs, as described by the manufacturer. The cDNAs were stored at -20°C or directly used for subsequent amplification. Amplification of human IL-2 mRNA was performed using primers: 5’-TTGCATTGCACTAAGTCTTGC-3’ (forward) and 5’-CAATGGTTGCTGTCTCATCAG-3’ (reverse), whereas amplification of GAPDH mRNA was performed using primers: 5’-ACCACAGTCCATGCCATCAC-3’ (forward) and 5’-TCCACCACCCTGTTGCTGTA-3’ (reverse). PCR was performed in reaction mixtures containing 10 mmol/L Tris-HCl (pH 8.3), 1.5 mmol/L MgCl₂, 50 mmol/L KCl, 200 μmol/L of each dNTP, 1 μmol/L of each primer, 0.5 units of Taq polymerase, and 1×concentration of PCR buffer, in a total volume of 50 μL. The amplification protocol consisted of 32 cycles of denaturation at 94°C for 30 s, annealing at 55°C (IL-2) or at 65°C (GAPDH) for 30 s, and extension at 72°C for 40 s (IL-2) or 80 s (GAPDH), using a Perkin-Elmer 9600 thermal cycler. The PCR products were separated on 15 g/L agarose gels stained with ethidium bromide.

BMSCs were seeded at a concentration of 2.5 × 10⁵ cells/mL and transduced with Ad/hIL-2 for 1, 2, 3, or 5 d .The supernatants were collected each day, filtered and tested for the presence of IL-2 by ELISA (Endogen, Woburn, MA, USA).

KIGB-5 cells were cultured for 6-7 passages, washed three times and resuspended in PBS. Each hamster was infused through the femoral vein with 5.0×10⁵ KIGB-5 cells. After seven days, the tumor-bearing hamsters were divided into four groups and injected intraperitoneally with PBS (n = 5), unmodified BMSCs (n = 5), BMSCs+Ad/hIL-2 (n = 5) or BMSCs+Ad/∆E1 (n = 5). Hamsters were sacrificed 4, 8, and 12 wk after tumor inoculation, and the number of metastatic lesions in each was assessed.

Following transduction of BMSCs with Ad/LacZ at 50 MOI for 24 h, 2.5×10⁶ cells were transplanted into each hamster. After one week, the hamsters were sacrificed and various organs including bone marrow, spleen, kidneys, liver, and lungs, were obtained. Single cell preparations were made from each of these organs and examined by X-gal assay. We found that the BMSCs made in our laboratory homed primarily into the bone marrow, spleen and liver, with fewer in the lungs and kidneys (Figure 2).

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Figure 2 X-gal assay of post -transfection in BM (A), spleen (B), kidney (C), lung (D), and liver (E).

The Ad/hIL-2 construct was designed to express human IL-2 protein in BMSCs. To confirm hIL-2 expression in BMSCs transduced with Ad/hIL-2, we tested these cells by RT-PCR and ELISA. Both showed IL-2 expression in transduced BMSCs (Figure 3).

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Figure 3 Expression and quantitative analysis of human IL-2. A: RT-PCR of in vitro cultured BMSC. Lane 1: 1x10⁶ stromal cells; lane 2: 1x10⁶ stromal cells containing 20 MOI Ad/∆E1; lane 3: 1x10⁶ stromal cells containing 20 MOI Ad/hIL-2; B: ELISA assay of IL-2 secretion from in vitro cultured BMSCs (2.5 X 10⁵/mL) of Syrian golden hamster after transduction with Ad/hIL-2 at MOI of 0(◆), 25(■), 50(▲), and 100 (●). Culture supernatants were harvested on different days and tested for hIL-2 secretion levels by ELISA.

Following injection of KIGB-5 cells into hamsters, we observed gross metastatic lung lesions after 8 wk (Figure 4).

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Figure 4 Gross findings of metastatic lung lesions in Syrian hamsters 8 wk after injection of KIGB-5 tumor cells in RPMI control (A), BMSCs (2. 5 x 10⁶ cells/d) (B), BMSCs containing 50 MOI Ad/∆E1 (2.5 x 10⁶ cells/day) (C), and BMSCs containing 50 MOI Ad/hIL-2 (2.5 x 10⁶ cells/d) (D). Hamsters injected with RPMI, BMSCs, or BMSCs containing Ad/∆E1 showed multiple metastatic lung lesions 8 wk after tumor injection, whereas hamsters injected with BMSCs containing Ad/IL-2 showed no evidence of disease.

Hamsters subsequently injected with RPMI, unmodified BMSCs, or BMSCs containing Ad/∆E1 had multiple metastatic lung lesions 8 and 12 wk after tumor cell injection, but no metastatic lesions were observed in the liver or other organs. In contrast, no evidence of metastatic tumors was observed in hamsters treated with BMSCs containing Ad/hIL-2. These findings were further confirmed by microscopic examination (Figure 5).

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Figure 5 Microscopic findings in Syrian hamsters 8 wk after injection of KIGB-5 tumor cells in RPMI control (A), BMSCs (2. 5 x 10⁶ cells/d) (B), BMSCs containing 50 MOI Ad/∆E1 (2.5 x 10⁶ cells/d) (C), and BMSCs containing 50 MOI Ad/hIL-2 (2.5 x 10⁶ cells/d) (D). Hamsters injected with RPMI, BMSCs, or BMSCs containing Ad/∆E1 showed multiple metastatic lesions in both lungs, whereas hamsters injected with BMSCs containing Ad/IL-2 showed no evidence of disease.