The Combination of 5-FU, Leucovorin and CPT-11 (FOLFIRI) Prolongs Survival through Inhibition of Metastasis in an Orthotopic Model of Colon Cancer

Materials and Methods

Drug administration. 5-FU, LV and CPT-11 were either purchased from or provided by Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan), Wyeth K. K. (Tokyo, Japan), and Yakult Honsha Co., Ltd. (Tokyo, Japan) respectively. These agents were adjusted to the administered doses with saline immediately before use. The dose and schedule of 5-FU and LV administration were preliminarily tested based on reports by Tschmelitsch et al. (8) and Behr et al. (9). The mice were randomly divided into four groups: Group 1: saline control; Group 2: 30 mg/kg 5-FU + 90 mg/kg LV; Group 3: 5-FU + LV + 16 mg/kg CPT-11; and Group 4: 5-FU + LV + 24 mg/kg CPT-11. Each group consisted of 7-9 mice. LV was intraperitoneally administered at 50 mg/kg on days 11, 16 and 21 after tumor transplantation, followed one hour later by intravenous administration of 5-FU at 30 mg/kg. CPT-11 was administered intravenously on days 12, 18 and 22 after tumor transplantation at a dose of either 18 mg/kg or 24 mg/kg. In the control group, saline was administered in the same manner, at a dose of 0.2 ml/20 g weight.

Green fluorescent protein (GFP) gene transduction of HCT-116 human colon cancer cells.

Production of GFP retrovirus. The pLEIN retroviral vector (Clontech Laboratories, Inc., Palo Alto, CA, USA), expressing GFP and the neomycin resistance gene on the same bicistronic message, was used as a GFP expression vector. PT67, an NIH3T3-derived packaging cell line, expressing the 10 Al viral envelope, was purchased from Clontech Laboratories, Inc. PT67 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Irvine Scientific, Santa Ana, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gemini Bio-products, Calabasas, CA, USA). For vector production, packaging cells (PT67) at 70% confluence were incubated with a precipitated mixture of DOTAP Liposomal Transfection Reagent N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) reagent (Boehringer Mannheim, Indianapolis, IN, USA). Saturating amounts of pLEIN plasmid were added for 18 h. Fresh medium was replenished at this time. The cells were examined by fluorescence microscopy 48 h after transduction. For selection, the cells were cultured in the presence of 500-2000 μg/ml G418 (Life Technologies, Inc., Grand Island, NY, USA) for 7 days to select for a clone producing high amounts of GFP retroviral vector (PT67-GFP) (10).

GFP gene transduction of cancer cells. For GFP gene transduction, 70% confluent cultures of cancer lines were used. Cancer cells were incubated for 72 h with a 1:1 precipitated mixture of retroviral supernatants of PT67-GFP cells and RPMI-1640 (Fisher Scientific, Santa Ana, CA, USA) containing 10% FBS. Fresh medium was replenished at this time. Cells were harvested with trypsin/EDTA 72 h after transduction and subcultured at a ratio of 1:15 in selective medium, which contained 200 μg/ml G418. The level of G418 was increased stepwise up to 800 μg/ml. GFP-expressing cancer cells were isolated with cloning cylinders (Bel-Art Products, Pequannock, NJ, USA) using trypsin/EDTA and amplified by conventional culture methods (11).

Orthotopic transplantation. Nude mice, 6 to 8 weeks old, were used. The mice were anesthetized prior to surgery with a ketamine mixture (10 μl ketamine HCl, 7.6 μl xylazine, 2.4 μl of acepromazine maleate and 10 μl H₂O) via s.c. injection. Fifty microliters containing 2×10⁶ HCT-116-GFP cells per mouse were injected into the subcutaneous tissue using a 0.5 ml 28 G latex-free syringe (TYCO Health Group LP, Mansfield, MA, USA). After subcutaneous tumor growth, tumor fragments (1 mm³), stably expressing GFP, harvested from the resulting s.c. tumors in the nude mice, were implanted by surgical orthotopic implantation on the colon of nude mice. After proper exposure of the colon through a lower-left abdominal incision, 8-0 surgical sutures were used to attach the tumor pieces under the serosa of the ascending colon (7). The incision in the abdominal wall was closed with a 6-0 surgical suture in one layer (12).

Animal care. The mice were housed in polycarbonate cages (L 22 cm × H 132 cm × H 135 cm) with sterilized sawdust on the floor and allowed free access to CE-2 feed (Oriental Yeast Co., Ltd., Tokyo, Japan) and sterilized tap water. Each cage contained 3-5 mice. The environment was kept at 25±2°C, with 55±10% humidity, and a luminous intensity of 5 lx, with a light cycle from 8:00 AM to 8:00 PM under laminar air flow. The weight of the transplanted animals was checked regularly. Autopsy was performed to determine and record metastatic status, as well as cause of death.

Whole-body imaging of tumor growth and metastasis. On days 35 and 64 after tumor transplantation, whole-body GFP imaging was performed on the mice, under pentobarbital anesthesia, with the FluorVivo™ small animal imaging system (INDEC BioSystems, Capitola, CA, USA) in order to determine the extent of the primary tumor and metastasis (13).

Pathological evaluation of tumor status in liver. After the mice were sacrificed, the liver was fixed with IHC Zinc Fixative solution (BD Biosciences, San Diego, CA, USA). Tissue specimens were then prepared by conventional procedures and stained with H & E. Evaluation was carried out according to Shimosato et al. (14).

Oncogene expression in mesenteric lymph nodes. Expression of the human enhancer of the zeste homolog 2 (hEZH2) and human keratin 19 (hKRT19) genes in the mesenteric lymph nodes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). EZH2 is a histone methyltransferase that regulates the survival and metastasis of cancer cells (15, 16), while KRT19 is a structural protein found in epidermal cells. We first confirmed expression of both genes in HCT-116 GFP cells. RNA was prepared in RNA-Later solution (Ambion, Austin, TX, USA). Table I shows the primers used in this experiment (15, 16). Mouse β-actin (mβ-actin) was used as the loading control for the tumor-transplanted mice, and human GAPDH (hGAPDH) for the HCT-116 cell lines. RT-PCR employed 35 cycles for hEZH2 and hKRT19; 25 cycles for mβ-actin and hGAPDH.

Statistical analysis of data. Data were expressed as average values and standard deviations (SD). The Kaplan-Meier survival curve was analyzed by the log-rank and Wilcoxon tests, and the pathological results by an ANOVA, with a p-value of less than 0.05 being considered statistically significant.