PC14PE6 cells, the human lung adenocarcinoma cell line, were maintained in MEM (Hyclone) supplemented with 10% heat-inactivated fetal bovine serum and penicillin/streptomycin. Cells were cultured in an incubator with a humidified atmosphere of 5% CO₂ in air at 37℃.

Male BALB/c nude mice weighing 20~25 g (Orient, Korea), were maintained at 25℃ with a standard diet and water freely at least 10 days before experiments. Animals were maintained under pathogen-free conditions during experiments. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources published by the National Institutes of Health and according to the Animal Experiment Guidelines of Samsung Biomedical Research Institute.

Intrathoracic injections were performed following previous protocol with minor modifications (8). Briefly, the PC14PE6 tumor cells were harvested from subconfluent culture dishes by mechanical pipetting. The cells were washed once in serum-free medium, resuspended in PBS, and floating cells were collected after centrifugation at 4,000×g for 5 min. Trypan blue staining was used to assess cell viability. Single-cell suspensions of >90% viability were used for injections. The cells were resuspended in HBSS (H4385, Sigma) as a half volume of inoculum and diluted with an equal volume of growth factor-reduced Matrigel (Becton Dickinson & Co., San Jose, CA). Matrigel, a basement membrane matrix preparation, was extracted from Engelbreth-Holm-Swarm mouse sarcoma. Matrigel rapidly generates a gel formation at room temperature or above and acts as an anchor to prevent tumor cells from diffusing and spreading in the thorax after intrathoracic injection. Cell suspensions and injection kit were kept on ice until injection. Three doses of tumor cell inoculums (0.5×10⁶/70 µl; 1×10⁶/70 µl; 2×10⁶/70 µl) were used in the experiment.

Mice were anesthetized with an intraperitoneal injection of an aqueous solution (5 ml/kg) of xilazine (8 mg/kg) and ketamine (120 mg/kg) and were placed in the left lateral decubitus position. One-ml tuberculin syringes (Becton Dickinson) with 30-gauge hypodermic needles were used to inject the cell inoculum percutaneously into the right lateral thorax, at the lateral dorsal axillary line, approximately 1.5 cm above the lower rib line just below the inferior border of the scapula. The needle was quickly advanced approximately 6 mm into the thorax and was quickly removed after the injection of 70 µl cell suspension. After tumor injection, the mouse was turned to the right lateral decubitus position. Animals were observed for 30 min until fully recovered.

Mice were sacrificed under anesthesia every 3 days beginning 4 days after inoculation. Following thoracotomy, thoracic organs were removed en bloc, including all of the lymph nodes and tumors. After dissection and removal of the heart, both lungs, including tumor mass, were fixed in formalin and embedded in paraffin. Other organs such as brain, liver, spleen, kidney, muscle, adrenal gland, and lymph node on knee were also removed, fixed in formalin and embedded in paraffin to inspect for the presence of metastases. Whole organs were serially sectioned into 2-mm thick slices and embedded in one paraffin block.

Paraffin-embedded tissues were stained with hematoxylin and eosin (H&E) for routine histological examination. Briefly, the sections were de-waxed in xylene, re-hydrated through descending concentrations of ethanol and stained with hematoxylin (5 min) and eosin (2 min). An optical microscope (BX50, Olympus) was used for imaging.

On day 25 after inoculation of PC14PE6 cells, the whole thorax was occupied by a large lung cancer mass with scarce normal lung tissues. The largest tumor nodule was more than 1 cm in size. Microscopic examination of the lung tumor tissues demonstrated that normal alveolar structures of the lung had been destroyed by invasion of lung cancer cells, and the alveoli were filled with malignant cells (Fig. 1). When thoracic lymph nodes were dissected and examined microscopically, PC14PE6 malignant cells had efficiently infiltrated the parenchyma of the lymph nodes (Fig. 2).

We assessed the life span of orthotopic lung cancer model and investigated the life span in correlation with inoculated cell dose. Three doses of tumor cell inoculums (0.5×10⁶/70 µl; 1×10⁶/70 µl; 2×10⁶/70 µl) were administered to the BALB/c mice by intrathoracic inoculation. Body weights of the animals were measured every day and an autopsy was performed immediately after natural death caused by lung cancer.

Nine mice in three groups (3 mice per group) developed lung to lung metastases and lung to intrathoracic lymph node metastases in the preliminary experiment. As shown in Fig. 3, the longest life span among three different cancer cell dosage-inoculated groups was 32 days with inocula of 0.5×10⁶ cells in average and the shortest one was only 14 days with inocula 2×10⁶ cells. These data indicated that the PC14PE6 cancer cells could induce rapid pathological propagation in the BALB/c nude mouse lung after inoculation. Despite rapid tumor development with heavy tumor burden in the animals, we could not detect any significant alterations in the body weight from most of animals before and after tumor inoculation. There were no statistical differences between control mice and the three tumor cell-inoculated groups (p≥0.05).

We further investigated the tumor development of two of the inoculation groups, 1×10⁶ and 0.5×10⁶. The first intrathoracic tumor was detected 7 days after inoculation in the 1×10⁶ cancer cells-inoculated group (Table 1) when there were few cyst-like soft translucent masses in one of the three mice. By 10 days after inoculation, numerous tumor nodules, which had metastasized to lymph nodes and lung, were found upon thoracotomy. The average life span after inoculation was 25 days (13~39 days) in the 1×10⁶ cancer cells inoculum group.

In the 0.5×10⁶ cells inoculated group (Table 2), the first intrathoracic tumor was detected by thoracotomy 9 days after inoculation, which was longer than the time to detection in the 1×10⁶ cancer cells-inoculated group. By 10 days after inoculation, the intrathoracic tumor mass was prominent with formation of solid mass and metastases to intrathoracic lymph nodes and lung. The life span was between 19 and 52 days with an average of 32 days. The representative tumor development after inoculation of PC14PE6 cancer cells is demonstrated in Fig. 4 and 5.

In order to investigate the pattern of spread to other distant organs, the brain, liver, kidney, muscle, spleen, adrenal gland, and lymph node on knee were removed and inspected with an optical microscope. None of the organs were invaded by PC14PE6 adenocarcinoma cells before death in this series. The body weights of the animals did not change significantly, and there were no statistical differences between the control and the experimental group (p≥0.05).