Efficacy of Dietary Antioxidants Combined with a Chemotherapeutic Agent on Human Colon Cancer Progression in a Fluorescent Orthotopic Mouse Model

Materials and Methods

GFP expression vector. pLEIN was purchased from Clontech (Clontech Laboratories, Palo Alto, CA, USA). The vector expresses enhanced GFP and the neomycin resistance gene on the same bicistronic message that contains an internal ribosome entry site.

GFP packaging cell culture, vector production, transfection and subcloning. PT67, an NIH3T3-derived packaging cell line, expressing the 10 Al viral envelope, was purchased from Clontech. PT67 cells were cultured in DMEM supplemented with 10% fetal bovine serum. For vector production, packaging cells (PT67) at 70% confluence were incubated with a precipitated mixture of N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate reagent and saturating amounts of pLEIN plasmid for 18 h. Fresh medium was replenished at this time. The cells were examined by fluorescence microscopy 48 h post-transfection. For selection, the cells were cultured in the presence of 200-1000 μg/ml G418 for 7 days.

GFP gene transduction of COLO-205 cells. For GFP gene transduction, 20% confluent COLO-205 cells (CRL 1435; ATCC, Manassas, VA, USA) were incubated with a 1:1 precipitated mixture of retroviral supernatants of PT67 cells and Ham's F-12 K containing 7% fetal bovine serum for 72 h. Fresh medium was replenished at this time. COLO-205 cells were harvested by trypsin-EDTA 72 h post-transduction and subcultured at a ratio of 1:15 into selective medium that contained 200 μg/ml G418. The level of G418 was increased to 1000 μg/ml stepwise. The brightest COLO-205 cell clones expressing GFP were selected, combined, and then amplified and transferred by conventional culture methods.

Subcutaneous xenograft. Tumor stocks were made by subcutaneously (s.c.) injecting COLO-205 GFP cells at 5×10⁶ cells/200 μl into the flank of nude mice. The strong GFP expression of tumors grown in the subcutis of mice was verified before harvest. The tumor tissues harvested from s.c. growth were inspected and any grossly necrotic or suspected necrotic or non-GFP tumor tissues in mice were removed. Tumor tissues were subsequently cut into small fragments of approximately 2 mm³.

Animals. Fifty male and 50 female athymic NCr nude mice, between 5 and 6 weeks of age, were used in this study, with an average weight of 20-25 g. The animals were bred and maintained in a HEPA-filtered environment with cages, food and bedding sterilized by autoclaving. The breeding pairs were obtained from Taconic Quality Laboratory Animals and Services for Research (Germantown, NY, USA). The animal diets were obtained from Ross Laboratories (Columbus, OH, USA). Hydrochloric acid at 0.005 mg/ml (w/v) was added to the autoclaved drinking water. The animals were examined daily for signs of illness. A total of 100 animals were used for this study. The orthotopically-transplanted animals used for the study were divided into 5 groups 3 days before treatment. Groups for each of the cohort conditions were randomly chosen.

Surgical orthotopic implantation (SOI) (14). The animals were transplanted by surgical orthotopic implantation (SOI) using the COLO-205 GFP tumor fragments harvested from the stock animals described above. The animals were anesthetized with the ketamine mixture (10 μl ketamine HCl, 7.6 μl xylazine, 2.4 μl acepromazine maleate, and 10 μl H₂O) via s.c. injection. The surgical area was sterilized using iodine and alcohol. An incision approximately 1.0 cm long was made along the midline in the lower abdomen of the nude mouse using a pair of surgical scissors. The serosa of the cecum near the ascending colon was removed, and then three or four 1 mm³ fragments of COLO-205 GFP tumor were transplanted onto the wall of the colon with 8-0 surgical sutures (nylon). The colon was then returned to the abdominal cavity. The abdomen was closed with 6-0 surgical sutures (silk). All procedures of the operation described above were performed with the aid of a 7 × magnification microscope (Olympus Corp., Tokyo, Japan) under HEPA filtered laminar flow hoods.

Experimental design. The cohort study was grouped as follows for a total of 100 mice. Thirteen days after tumor tissue implantation (when SOI tumors reached an approximate mean caliper-measured size of 100-150 mm³), mice were grouped with five mice per cage, with four cages per group (10 males and 10 females). Each cage was provided with a feeding jar and a known quantity of food was supplied fresh each day. Cisplatin was administered intraperitoneally (i.p.) 3 days after changing diets. The food was replaced every 24 hours and weighed to determined intake (see Table I).

Feeding regimen. Mice had free access to food and water at all times. Antibiotics (sulfamethoxazole and trimethoprim) were added to the drinking water to prevent outbreak of bacterial infections. The feeding regimen is indicated in Table I. In all groups, mice were fed regular chow diet (AIN-93M-Diet B) before and during the implantation of the tumor. Two or three days before the commencement of chemotherapy, the diets were switched to the experimental diets. Mice were maintained on their respective diets until the end of the study. The study continued until a large number of animals started to die. Each cage was provided with specialized mouse feeding jars and a known quantity of food was supplied daily. Twenty-four-hour food intake was determined by measuring the food left over from the previous day. Per mouse food consumption was determined by dividing the total amount consumed by the number of mice per cage.

Diets. Diet A (modified AIN-93M mature rodent diet with 4% fish oil); Diet B [modified AIN-93M with antioxidants vitamin A, vitamin E, and selenium at levels present in the standard AIN-93M diet (control)]; Diet C (Diet A without added antioxidants vitamin A, vitamin E, or selenium); Diet D (Diet A with 5 times the amount of added antioxidants vitamin A, vitamin E, and selenium present in Diet B). The modified AIN-93M diet has the same composition as standard AIN-93M diet except that soybean oil is replaced by corn oil, fat content is 11g % not 5g % and + -BHQ is removed. All diets were prepared, weighed, and individually packaged at Abbott Laboratories-RPD (Columbus, Ohio, USA). The experimental diets were supplied as blinded diets. The individual diets were isocaloric and isonitrogenous. The pre-packaged diets were stored in the freezer until time of use.

Body weights. Thirteen days after tumor implantation, the body weight for each animal was followed on a weekly basis. An electronic balance was used to measure body weight.

Whole-body optical imaging of GFP-expressing tumors (15, 16). Seventeen days after implantation, whole-body optical imaging of GFP-expressing tumors was performed once a week. The time course of tumor progression was tracked by fluorescence imaging after transplantation. The time of tumor occurrence in different organs and the numbers of metastases were recorded in tumor-bearing animals. A Leica stereo fluorescence microscope model LZ2 equipped with a mercury lamp power supply was used. Selective excitation of GFP was produced through a D425/60 band-pass filter and 470 DCXR dichroic mirror. Emitted fluorescence was collected through a long-pass filter GG475 (Chroma Technology, Brattleboro, VT, USA) on a Hamamatsu C5810 3-chip cooled color CCD camera (Hamamatsu Photonics Systems, Bridgewater, NJ, USA). Experiments were controlled and images were processed for contrast and brightness and analyzed with the help of Image Pro Plus 3.1 software (Media Cybernetics, Silver Spring, MD, USA). High-resolution images were captured directly on the computer or continuously through video output on a high-resolution Sony VCR.

Direct open imaging of green fluorescent protein-expressing tumors (17). Mice were sacrificed, explored and images taken on day 44 after SOI when they appeared premorbid. GFP facilitated identification of tumors by fluorescence visualization.

Termination. The final tumor weights and the GFP tumor images were acquired after animals were sacrificed at the termination of the study, which was on day 44 after SOI.

Statistical methods used in efficacy evaluation. Final tumor weights of all 5 groups were analyzed using the Student's t-test and ANOVA test (with the Dunnett two-sided test) with an α=0.05.