immuno-competent host and tumor cells syngeneic to the host so that the experimental 
glioma model more closely modeled a naturally-arising glioma. 
ADV-tk/GCV Treatment of 9L Experimental Tumors 
In vivo the 9L tumor cells exhibit morphology described as mixed glioblastoma 
multiforme and sarcoma, or gliosarcoma (Barker et al., 1 973; Weizsaecker et al., 1981) The 
9L glioma cells (kindly provided by P.J. Tofilon of M.D. Anderson Hospital, Houston, TX) 
were maintained in DMEM supplemented with 10% fetal bovine serum, penicillin (100 U/ml), 
and streptomycin (100 mg/ml) in 5% C0 2 at 37°C. The tumor cells were harvested for 
injection by treating the cells at 37°C with 0.25% trypsin in 1.0 mM EDTA for 5 minutes. The 
cells were collected in DMEM, washed, and resuspended in HBSS at a concentration of 2.0 
x 10 3 cells/ml. Cells were counted before and after concentrating and prior to injection with a 
hemacytometer. Following the implantation procedure the viability of the cell preparation 
was assessed by trypan blue exclusion analysis. 
Adult female Fischer 344 rats (155-175 grams) were used as host animals. Rats were 
anesthetized with an intramuscular injection (0.6 ml/kg) of an anesthetic consisting of 
ketamine (42.8 mg/ml), xylazine (8.6 mg/ml), and acepromazine (1.4 mg/ml) and were 
placed into a stereotaxic frame. A mid-line incision was made in the scalp, and a burr hole 
was made with a 0.9 mm drill bit 1.8 mm to the right and 2.5 mm anterior to the bregma. 
Using a 10 ml syringe fitted with a 26 gauge needle and connected to the manipulating arm 
of the stereotactic frame, 1 x 10 4 9L glioma cells suspended in 5 ml of HBSS were injected 
in 0.2 ml increments over 5 minutes into the right caudate nucleus at a depth of 4.5 mm 
from the dura. The needle was left in place for 3 minutes and then withdrawn slowly over 
another 3 minutes. The burr hole was closed with bone wax and the scalp wound was 
closed with clips. Tumors were 1 .65 ± 0.094 mm 2 (n=4) in diameter 9 days after tumor cell 
injection and, if left untreated, killed the hosts at a mean time of 20 days after implantation. 
Eight days after 9L tumor cell injection either ADV-tk or ADV-bgal was injected into the 
tumors using the same coordinates that were used for tumor implantation. Viral particles 
(1.2 x 1 0 9 ) in 6 ml of 10 mM Tris-HCI, pH 7.4, 10% glycerol and 1 mM MgCI 2 were injected 
at 6 sites within the tumor bed. Starting at a depth of 5.5 mm below the dural surface, 1 ml 
of virus was injected and the needle raised 0.5 mm where another 1ml was injected. This 
was repeated until a total of six 1 ml injections were made through the core of the tumor. 
Virus was injected over 5 minutes at each position and then the needle was withdrawn 
slowly over 5 minutes. Carbon particles (<30 mm) were placed on the shaft of the injection 
needle to mark the injection site. The wound was closed with clips. 
To test the effectiveness of ADV-tk and GCV treatment on experimental 9L tumors, 3 
treatment groups were established: 1) ADV-tk plus 100 mg/kg GCV (n=6); 2) ADV-bgal plus 
100 mg/kg GCV (n=4); 3) ADV-tk plus saline (n=7). Treatment began 12 hours after viral 
injection. The animals received intraperitoneal injections of GCV or of normal saline twice a 
day for 6 consecutive days. Twenty days after tumor cell injection or at death, the animals 
were perfused with fixative, the brains sectioned and stained, and the tumor size 
morphometrically determined. Animals that were treated with ADV-bgal and GCV 
(lOOmg/kg) or with ADV-tk and saline all had large tumors 20 days after tumor cell injection 
(Fig. 7A). The tumors were characterized by hypercellularity, nuclear pleomorphism, and 
mitoses without inflammatory cell infiltration. The tumors were generally well circumscribed 
and caused compression of adjacent brain tissue. However, focal peri-vascular glioma 
infiltration into adjacent brain was seen. In contrast, no tumor cells were seen in the brains 
Recombinant DNA Research, Volume 20 
