HISASHI ISHIKURA, KAZUYA KONDO, TAKANORI MIYOSHI, YUJI TAKAHASHI, HARUHIKO FUJINO and YASUMASA MONDEN

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1 Green Fluorescent Protein Expression and Visualization of Mediastinal Lymph Node Metastasis of Human Lung Cancer Cell Line Using Orthotopic Implantation HISASHI ISHIKURA, KAZUYA KONDO, TAKANORI MIYOSHI, YUJI TAKAHASHI, HARUHIKO FUJINO and YASUMASA MONDEN The Department of Oncological and Regenerative Surgery, School of Medicine, The University of Tokushima, Tokushima, Japan Abstract. Background: Suppression of lymphatic metastasis improves survival in lung cancer patients. We have reported a patient-like model of lung cancer metastasis based on orthotopic implantation in severe combined immunodeficiency (SCID) mice and demonstrated the lymphogenous spread histologically using human NSCLC cell lines. Materials and Methods: To visualize micrometastases, we transfected the Aequorea Victoria jellyfish green fluorescent protein (GFP) gene to the cancer cell line. Results: The tumor cell lines were able to stably express GFP at high levels both in vitro and in vivo. Fluorescent tumors and metastasis of the mediastinum could be visualized at microscopic levels after transplantation. Conclusion: This model demonstrated that GFP gene-transfected tumor cells represent a new tool for evaluating the metastatic process and are very useful for developing chemotherapy to inhibit metastasis. The extent of lymphatic metastasis is the most important factor influencing prognosis in non-small cell lung cancer (NSCLC). The 5-year survival rate after resection for patients with pn2 disease is only 23% (1). Therefore, suppression of lymphatic metastasis improves survival in lung cancer patients. However, the biology of mediastinal metastasis is poorly understood because of the lack of a mediastinal-metastasis animal model of lung cancer. We have reported a patient-like model of lung cancer metastasis based on orthotopic implantation in severe combined immunodeficiency (SCID) mice and have Correspondence to: Kazuya Kondo, The Department of Oncological and Regenerative Surgery, School of Medicine, The University of Tokushima, Kuramoto-cho, Tokushima city, Tokushima , Japan. Tel: , Fax: , kondo@clin.med.tokushima-u.ac.jp Key Words: GFP, orthotopic implantation, SCID mice, lung cancer, model. demonstrated the lymphogenous spread histologically using human NSCLC cell lines (2, 3). After sacrifice of the SCID mice, the extent of lymphogenous and hematogenous metastasis was detected by macroscopically visualizing tumors of the bilateral lung, mediastinal tissues, thoracic cavity, liver, spleen, kidney and adrenal glands. It is difficult, however, to detect tumor metastasis in its early stages. Use of the green fluorescent protein (GFP) gene, cloned from the Aequorea Victoria jellyfish, as a marker for localization of tumor cells in host tissues has previously been described in several studies with good results. To visualize micrometastases in our model, we transfected the GFP gene to lung cancer cell lines. The tumor cell lines were able to stably express GFP at high levels both in vitro and in vivo. We report here a new orthotopic implantation model of the human lung cancer cell line based on transfer of the GFP reporter gene to tumor cells, which enables visualization of fluorescent tumors and metastasis to the mediastinum at the microscopic level after transplantation. Materials and Methods Animals and cells. Male SCID mice (6 weeks of age), with a CB-17 genetic background, were purchased from CLEA Japan Inc., Tokyo, Japan. These mice had been raised from birth in a specific pathogen-free environment. The human lung cancer cell line Ma44 (squamous cell carcinoma) was kindly provided by Drs Masuda and Takata, Osaka Prefectual Habikino Hospital, Osaka, Japan (3). The Ma44-3 cell line was cloned by the limiting dilution method in our laboratory. The Ma44-3 cell line was cultured in RPMI-1640 medium with 10% heatinactivated fetal bovine serum (Bio.Whittaker, USA), and maintained at 37ÆC in a humidified incubator with 5% CO 2 in air. GFP expression vector, transfection, subcloning. For GFP labeling of the cancer cells, EGFP-N1 (Clonetech Laboratories Inc., Paolo Alto, CA, USA), an expression vector containing one of the GFP variants, was transfected into tumor cells using a lipofection procedure (LipofectAMINE PLUS Reagent: Life Technologies, Inc., Rockville, /2004 $

2 Figure 1. Stable high-level expression of GFP transductants in vitro. The human non-small lung cancer cell line Ma44-3 was transduced with the EGFP-N1 that expresses enhanced GFP. The stable high-expression clone was selected in 600 g/ml of G418 sulfate. Figure 3. Early-stage lymph node metastasis of Ma44-3-GFP tumor cells. a) The thoracic organ under a light scope 4 days after GFP-Ma44-3 cell injection. The lymph node metastases cannot be visualized under brightfield microscopy. b) The same field and a high-power field of the square viewed under fluorescence microscopy with GFP. Small tumor colonies (tumor metastasis) are brightly visualized. Figure 2. a) The primary GFP-Ma44-3 lung tumor injected in the left lung and visualized under bright-field microscopy 7 days after injection. b) The same field viewed under fluorescence with GFP. Primary tumors are brightly visualized. MD, USA). Selection medium containing 600 g/ml of G418 sulfate (Life Technologies, Inc.) was used for 2 weeks after transfection. G418-resistant clones were isolated, expanded and analyzed. Clonal cells were visualized under fluorescence microscopy to detect the presence of green fluorescence. Orthotopic intrapulmonary implantation procedure. As shown in our previous studies (2,3), Ma44-3 cells were harvested for implantation at 70% to 80% confluence using 1 mmol/l ethylenediaminetetraacetic acid (Wako Pure Chemical Industries Ltd., Japan) in phosphate-buffered saline (Nissui Pharmaceutical Co.Ltd., Japan). The cells were washed in RPMI-1640 medium and resuspended to a final density of 2.0x10 6 cells/ml in RPMI-1640 containing 0.1% bovine serum albumin (Boehringer Mannheim, Germany). The mice were fully anesthetized by ether inhalation. The experimental mice were placed in the right lateral decubitus position with the four limbs restrained. A 1-cm transverse incision was made on the left lateral skin just below the inferior border of the scapula of the SCID mouse. Muscles were separated from the ribs by sharp dissection and intercostal muscles were exposed. The left lung was visible through the intercostal muscles. A 30-gauge needle was inserted approximately 5 mm into the lung through the intercostal muscle, and an inoculum of 2.0x10 6 tumor cells/ml with 10 mg/ml Matrigel (Collaborative Biomedical Products, Bedford, Canada) was then dispersed into the left lung in a final volume of 10 Ìl (2.0x10 4 cells) medium. The procedure required 720

3 Ishikura et al: Green Fluorescent Protein Expression Visualizes Mediastinal Lymph Node Metastasis approximately 1 minute for completion and was easily performed. The skin incision was closed with 3-0 silk. Twenty-five mice were inoculated in the lung and were killed on various days (from day 3 to 15) after tumor-cell implantation by ether inhalation and cervical dislocation. Major organs (bilateral lungs, heart, liver, kidneys, adrenal glands and mediastinal tissues) were removed and were explored under a fluorescence stereoscopic microscope (Olympus, ASZX-12). The Institutional Animal Care and Use Committee of the University of Tokushima School of Medicine, Japan, approved all experimental protocols in this study. Microscopy. Light and fluorescence stereoscopy were carried out using a Olympus microscope (SZX-12) equipped with a Xenon and mercury lamp power supply (SZX-RFL2, PM-10SP). GFP filter sets (SZX-GFP) were used for fluorescent analysis. Results Transfection of GFP expression vector into human lung cancer cell line-ma44-3. The selected G418-resistant Ma44-3-GFP cells had bright GFP fluorescence. Clonal cells were visualized under fluorescence microscopy to detect the presence of green fluorescence in vitro (Figure 1). All tumor cells had green fluorescence. Not only the cytoplasm but also the nuclei of tumor cells turned green during development. GFP expression to visualize tumors in the lung. To confirm the presence of tumor cell fluorescence during the study, lung and mediastinal tissue was excised when animals were sacrificed. The freshly excised organs were observed directly under fluorescence stereoscopy. Tumors in inoculated sites were visualized clearly as a hemisphere shape when tumor cells were inoculated in the lung parenchyma beneath the pleura. However, tumors were not visualized when tumor cells were inoculated in the lung parenchyma away from the pleura. Local tumors in the lung were visualized in the injected site as a bulge shape on day 7 under the light scope (Figure 2a), and this tumor displayed a very high fluorescence under fluorescence stereoscopy (Figure 2b). The fluorescence stereoscope could detect tumors located deep in the lung and was used to measure tumor size. GFP expression to visualize mediastinal metastasis. On day 4, we could detect the micrometastatic foci at the mediastinum under the fluorescence scope, although we could not detect them under the light scope (Figure 3a). Figure 3b shows an extended view of the white square of Figure 3a, which corresponds to lymphatic metastases around the esophagus on day 4. On day 7, hilar lymph node metastasis was seen (Figure 4a, c), and the metastatic foci of the mediastinum were seen clearly on day 11 under the fluorescence scope (Figure 4b,d). On day 14, all of the mediastinal lymph node swelling had increased, but we could not distinguish which lymph node contained the metastasis. However, in the GFP stereoscope we could clearly identify the metastatic lymph nodes in all five mice (Figure 5). All of the orthotopically implanted tumors metastasized to the mediastinal lymph node. Distant metastasis was not observed even under the fluorescence scope. Discussion The recently cloned (4) Aequorea Victoria green fluorescent protein (GFP) is a useful tool, functioning as an ideal fluorescent tag for the following reasons: 1) it is detectable without causing cytological damage; 2) it can label many cell types; and 3) it can be targeted to virtually any subcellular region. In addition, the GFP has proven to be a good marker for transduced cells in several in vivo studies (5-11). In the present investigation, the metastatic process for mediastinal lymph nodes in the lung cancer model was visualized by GFP directly under fluorescence microscopy. Our results provide compelling evidence that GFP fluorescence will facilitate our understanding of metastatic processes, including each step of the metastatic cascade. We have previously demonstrated the lymphogenous spread of human lung cancer in the SCID mouse using an orthotopic implantation model (6, 7). Our previous studies have microscopically clarified the process of lymphogenous metastasis, in which implanted tumor cells are anchored in the lung, migrate to the lymphatic vessels in the perivascular and peribronchial space and metastasize to the mediastinum. Our original model had the following advantages: 1) the metastatic form is very similar to that of clinical lung cancer; 2) the implantation procedure is simple and reproducible; and 3) this model can be produced in large quantities at one time for statistical analysis. However, in our model we could not detect an implanted tumor in the lung by the naked eye when tumor cells were inoculated in the lung parenchyma far from the visceral pleura. In addition, we did not detect micrometastasis of the mediastinum. In order to detect tumors in the lung and micrometastasis of the mediastium, bilateral lungs and mediastinal tissues were removed, fixed in formalin, and embedded in paraffin. Next, 5-Ìm histological sections were made from these blocks at 300-Ìm intervals. However, this work is very laborious. In the present study, we transfected the Aequorea Victoria jellyfish GFP gene to the Ma44-3 lung cancer cell line. We could detect small implanted tumors in the lung or tumors far from the visceral pleura under fluorescence microscopy. We could not, however, locate micrometastatic foci of the mediastinum stereoscopically on day 4. After day 7, we were able to see swelling of the mediastinal lymph node with the naked eye, although we could not determine whether the 721

4 Figure 4. Superior mediastinal lymph nodes and hilar lymph nodes involved with GFP-Ma44-3 metastases visualized by GFP expression 11days after injection. a) Bright field microscopy in the superior mediastinum, b) the same field viewed under a fluorescence stereoscope with GFP, c) bright field microscopy in the hilum, d) the same field viewed under fluorescence stereoscope with GFP. A strong GFP-fluorescing metastasis could be detected in the mediastinum. Figure 5. Lymph nodes in the superior mediastinum and hilum massively involved with GFP-Ma44-3 metastasis visualized by GFP expression 14 days after injection. a) Bright field microscopy in the superior mediastinum and hilum, b) the same field viewed under a fluorescence stereoscope with GFP. swelling represented metastatic lesions or an inflammatory change. Our new model using GFP, however, could distinguish metastatic lymph node from inflammatory change. This model is thought to be applicable to the detection of distant metastasis. In our previous model, we needed to cut sections of liver, spleen and kidney at 2 to 3-mm intervals to identify the metastasis. If metastasis-like lesions are found, they must be confirmed microscopically. In such cases, we can visualize the metastasis in the new model under a fluorescence scope. Although Ma44-3 lines did not have distant metastasis, we have recently established lung cancer cell lines that metastasize to the lung (A549) and to the adrenal glands or ovary (Ma2) (data not shown). GFP gene transfection is useful for identifying distant metastasis for such cell lines. 722

5 Ishikura et al: Green Fluorescent Protein Expression Visualizes Mediastinal Lymph Node Metastasis We have recently reported that UFT and CDDP suppress mediastinal metastasis and prolong the life-span in our model (12). This model will now be more useful for evaluating the effects of chemotherapy because we can precisely quantify the implanted tumor (tumor size), the metastasis of the mediastinum and the distant metastasis stereoscopically. These results will provide us with exact information regarding the effects of chemotherapy. In addition, because each tumor cell has a copy of the GFP plasmid, the model will be more sensitive for detecting the presence of tumor cells by PCR methods(13). In conclusion, this model demonstrates that GFP genetransfected tumor cells represent a new tool for evaluating the metastatic process of lung cancer and is very useful for the development of therapy for the metastasis of lung cancer. References 1 Mountain CF: Revisions in the International system for staging lung cancer. Chest 111: , Miyoshi T, Kondo K, Ishikura H, Kinoshita H, Matsumori Y and Monden Y: SCID mouse lymphogenous metastatic model of human lung cancer constructed using orthotopic inoculation of cancer cells. Anticancer Res 20: , Ishikura H, Kondo K, Miyoshi T, Kinoshita H, Hirose T and Monden Y: Artificial lymphogenous metastatic model using orthotopic implantation of human lung cancer. Ann Thorac Surg 69: , Prasher DC, Eckenrode VK, Ward WW, Prendergast FG and Cormier MJ: Primary structure of the Aequorea victoria greenfluorescent protein. Gene 111: , Chalfie M, Tu Y, Euskirchen G, Ward WW and Prasher DC: Green fluorescent protein as a marker for gene expression. Science 263: , Zolotukhin S, Potter M, Hauswirth WW, Guy J and Muzyczka N: A "humanized" green fluorescent protein cdna adapted for high-level expression in mammalian cells. J Virol 70: , Chishima T, Miyagi Y, Wang X, Yamaoka H, Shimada H, Moossa AR and Hoffman RM: Cancer invasion and micrometastasis visualized in live tissue by green fluorescent protein expression. Cancer Res 57: , Kan Z and Liu TJ: Video microscopy of tumor metastasis: using the green fluorescent protein (GFP) gene as a cancer-celllabeling system. Clin Exp Metastasis 17: 49-55, Moore A, Sergeyev N, Bredow S and Weissleder R: A model system to quantitate tumor burden in locoregional lymph nodes during cancer spread. Invasion Metastasis 18: , Naumov GN, Wilson SM, MacDonald IC, Schmidt EE, Morris VL, Groom AC, Hoffman RM and Chambers AF: Cellular expression of green fluorescent protein, coupled with highresolution in vivo videomicroscopy, to monitor steps in tumor metastasis. J Cell Sci 112: , Yang M, Jiang P, Sun FX, Hasegawa S, Baranov E, Chishima T, Shimada H, Moossa AR and Hoffman RM: A fluorescent orthotopic bone metastasis model of human prostate cancer. Cancer Res 59: , Ishikura H, Kondo K, Miyoshi T, Kinoshita H, Takahashi Y, Fujino H and Monden Y: Suppression of mediastinal metastasis by Uracil-Tegafur or cis-diamminedichloroplatinum(2) using a lymphogenous metastatic model in a human lung cancer cell lines. Clin Cancer Res 7: , Norris PS and Haas M: A fluorescent p53gfp fusion protein facilitates its detection in mammalian cells while retaining the properties of wild-type p53. Oncogene 15: , Received December 8, 2003 Accepted February 16,