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or a control adenovirus ADV/β-galactosidase at different multiplicity of infection (m.o.i.) in a total volume of 0.5 ml. After 2 hr, the viral supernatant was replaced with 2.5 ml of medium. Then, two days after, the supernatant was collected and tested for mIL-12 bioactivity. One ml of supernatant was added to 106 splenocytes from naive mice in a total volume of 2 ml. After 48 hr the supernatant was collected and analyzed for interferon (IFN)-γ release by ELISA (Endogen, Cambridge, MA).

Establishment and Treatment of Hepatic Metastasis Model of Colon Carcinoma. Metastatic colon carcinoma was induced in the liver by intrahepatic implantation of 5×104 MCA-26 cells at the tip of the left lateral liver lobe of 8- to 12-week-old syngeneic BALB/mice (Harlan-Sprague-Dawley). At day 7, various titers of recombinant adenoviral vectors were injected intratumorally in 50 μl of 10 mM Tris·HCL (pH 7.4)/1 mM MgCl2/10% (vol/vol) glycerol/Polybrene (20 μg/ml). All experiments were performed in accordance with the animal guidelines at Baylor College of Medicine.

Cytotoxic T-Lymphocyte (CTL) Assay. Viable splenocytes were isolated from various animal treatment groups at different time points after primary hepatic tumor inoculation. In vitro stimulation was performed for 5 days in 24-well plates, each well containing 6×106 splenocytes, recombinant mIL-2 (20 units/ml) and 5×105 MCA-26 cells that had received 15,000 rad (150 Gy) of radiation. Effector cells were co-incubated with Cr51 (150 μCi for 5×106) labeled target cells for 4 hr at 37°C in different effector and target cell ratios. Parental MCA-26 cells were used as target cells for the CTL assay. After incubation, the radioactivity of 100 μl of the supernatant was counted in a gamma counter. The percentage of specific cytolysis was calculated as (experimental release— spontaneous release)/(maximum release—spontaneous release)×100.

Total radioactivity present in target cells was analyzed by lysing the cells with 10% SDS. Data represent the mean of triplicate cultures and were analyzed by logistic regression.

Morphological and Histopathological Analyses of Hepatic Tumors. Fourteen days after various gene therapy treatments, the animals were sacrificed and tumor volume was calculated according to the formula V=A×B2 (A=largest diameter; B=smallest diameter). For histopathological analysis, livers from euthanized animals of various treatment groups were collected and cut in the middle at the site of the original tumor inoculation. The tissue was then fixed in 10% buffered formalin and stained with hematoxylin and eosin for histopathological analysis.

Long-Term Survival Analyses. The tumor-bearing animals were treated with various recombinant vectors and kept for observation. Days of death were recorded and the results were analyzed statistically using a logrank test (9).

RESULTS

Suicide Gene Therapy for Liver Metastases of Colon Cancer. The gene encoding herpes simplex virus type 1 thymidine kinase (HSV-tk) is the most widely investigated suicide gene for cancer therapy. Unlike the mammalian thymidine kinases, HSV-tk efficiently phosphorylates nucleosidic analogs such as acyclovir or ganciclovir (10). The monophosphate form is subsequently converted into the di- and triphosphate forms by cellular kinases. The triphosphate is the toxic form of the nucleosidic analog, as it can be incorporated into elongating DNA in the dividing cells that results in cell death (1113). This suicide gene therapy strategy for the treatment of hepatic metastases of colon cancer was investigated in rodent models. The regression of pre-established liver metastases after intratumoral injection of retrovirus-producer cells expressing HSV-tk followed by ganciclovir treatment has been reported (14). Infection of cancer cells after intratumoral injection of retroviral supernatant is low (15), although the grafting of virus producer-cells led to the transduction of up to 10% of the cells inside the tumor (1416).

To overcome the low in vivo transduction efficiency of the retrovirus, recombinant adenoviral vectors have been used to transfer the HSV-tk gene into tumor cells. This approach, evaluated in mice, was very efficient in mediating the regression of a variety of tumors (1720). In a mouse liver metastasis model of colon carcinoma, better than 80% of tumor regression was achieved after the suicide gene treatment (18). However, such extensive tumor destruction was not sufficient to yield significant survival benefit as compared with control vector treated mice (Fig. 1). Relapse of the hepatic tumors or the presence of disseminated metastases in other organs accounted for this lack of extended survival.

Combination Suicide and IL-2 Gene Therapy for Hepatic Metastases of Colon Carcinoma. Adenoviral mediated gene transfer of mIL-2 into the tumor was synergistical with HSV-tk and induced a systemic antitumor immunity that resulted in the further regression of the hepatic tumor as well as protection against distant site challenges of parental tumor cells (18). The antitumor immunity was attributed partly to the activation and proliferation of tumor specific CD8+ cytotoxic T lymphocytes. Animals treated with ADV/tk and ADV/mIL-2 lived significantly longer than those treated with ADV/tk or ADV/mIL-2 alone (Fig. 1). The control animals died between day 15 and day 30, while 60% of the animals were still alive at day 35 after combination treatment. The mean survival time has been increased from 22 days in the control-treated animals to 35 days in animals treated with both the HSV-tk and the mIL-2 vectors, and the results are statistically significant (P<0.03). In this model, ADV/mIL-2 treatment alone did not improve the long-term survival of the animal, as it did not cause regression of the hepatic tumors (10). After combination gene treatment, the antitumor immunity in the animals waned gradually over time (Fig. 2), resulting in the death of the animals due to tumor, recurrence in the liver and at distant sites. The antitumoral effect in the animals after combination treatment was mediated by a CTL response that could no longer be detected at day 38 (Fig. 2). To achieve long-term

FIG. 1. Long-term survival of animals after the combination treatment. ADV/mIL-2+ADV/tk. Seven days after intrahepatic cancer cells injection, the tumor -bearing animals were divided into four treatment groups: ◇) ADV/β-galactosidase (5×108 pfu), n=4; (●) ADV/mIL-2 (3×108 pfu), n=4; (Δ) ADV/tk (5×108 pfu), n= 5; and (□) ADV/tk (5×108 pfu)+ADV/mIL-2 (3×108 pfu), n= 5. All the animals received ganciclovir treatment 12 hr after virus injection and were observed for survival over time, (logrank test, P< 0.03).



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