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Establishment of a Human Lung Cancer Cell Line with High Metastatic

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Establishment of a Human Lung Cancer Cell Line with High Metastatic ONCOLOGY REPORTS 28: 1727-1735, 2012 Establishment of a human lung cancer cell line with high metastatic potential to multiple organs: Gene expression associated with metastatic potential in human lung cancer TETSUHIRO NAKANO1, KIMIHIRO SHIMIZU1, OSAMU KAWASHIMA1, MITSUHIRO KAMIYOSHIHARA1, SEIICHI KAKEGAWA1, MASAYUKI SUGANO1, TAKASHI IBE1, TOSHITERU NAGASHIMA1, KYOICHI KAIRA2, NORIAKI SUNAGA2, YOUICHI OHTAKI1, JUN ATSUMI1 and IZUMI TAKEYOSHI1 Departments of 1Thoracic and Visceral Organ Surgery and 2Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma 371-8511, Japan Received January 18, 2012; Accepted March 7, 2012 DOI: 10.3892/or.2012.1972 Abstract. Convenient and reliable multiple organ metas- metastasis of lung cancer. This model system may provide tasis model systems might contribute to understanding the insights into the key genetic determinants of widespread mechanism(s) of metastasis of lung cancer, which may lead metastasis of lung cancer. to overcoming metastasis and improvement in the treatment outcome of lung cancer. We isolated a highly metastatic subline, Introduction PC14HM, from the human pulmonary adenocarcinoma cell line, PC14, using an in vivo selection method. The expression Lung cancer is a leading cause of cancer-related death world- of 34,580 genes was compared between PC14HM and parental wide (1). The 5-year survival rate for patients with non-small PC14 by cDNA microarray analysis. Among the differentially cell lung cancer (NSCLC) is less than 50% (2). The develop- expressed genes, expression of four genes in human lung ment of widespread distant metastases after initial surgery is cancer tissues and adjacent normal lung tissues were compared the main reason for cancer-related deaths in operable NSCLC. using real-time reverse transcription polymerase chain reac- Overcoming widespread metastases might contribute to a tion. Although BALB/c nude mice inoculated with parental marked improvement in the treatment outcome of lung cancer. PC14 cells had few metastases, almost all mice inoculated Understanding the mechanism(s) of metastasis of lung cancer with PC14HM cells developed metastases in multiple organs, is indispensable for the development of novel treatment strate- including the lung, bone and adrenal gland, the same progres- gies. sion seen in human lung cancer. cDNA microarray analysis It is thought that development of distant metastases requires revealed that 981 genes were differentially (more than 3-fold) a multi-step process, such as invading the surrounding tissue, expressed between the two cell lines. Functional classification entering the lymphatic or blood vessels, surviving in the circu- revealed that many of those genes were associated with cell lation, extravasating into a new tissue, and growing in the new growth, cell communication, development and transcription. organ (3). Lung cancers can metastasize to a variety of organs, Expression of three upregulated genes (HRB-2, HS3ST3A1 but like other cancers, there is an organ preference for meta- and RAB7) was higher in human cancer tissue compared to static spread in lung cancer. The organ-preference patterns normal lung tissue, while expression of EDG1, which was of tumor metastasis have been proposed to be the product downregulated, was lower in the cancer tissue compared to the of favorable interactions between metastatic tumor cells the normal lung. These results suggest that the newly established ‘seed’ and the organ microenvironment the ‘soil’ (4). PC14HM cell line may provide a mouse model of widespread To date, part of the physics and biology of cancer metas- tasis has been examined and several molecules associated with metastasis have been identified and shown to play key roles in metastasis (5). However, the molecular mechanisms of Correspondence to: Dr Kimihiro Shimizu, Department of metastasis in lung cancer remain unresolved. Thoracic and Visceral Organ Surgery, Gunma University Graduate Precise analyses of the pathological, biological, molec- School of Medicine, 3-39-15 Showa-machi Maebashi, Gunma ular, and genetic aspects of tumor metastasis using tissue 371-8511, Japan specimens from metastatic organs might contribute to further E-mail: [email protected] understanding of the mechanism of metastasis and to the development of new treatment modalities. However, it is much Key words: lung cancer, mouse model, metastasis, multiple organ, more difficult to obtain tissue specimens from metastatic microarray organs than from primary tumors, not least because surgery is not usually the standard treatment for metastasis of various cancers. Furthermore, preclinical therapeutic studies on 1728 NAKANO et al: METASTASIS-ASSOCIATED GENE EXPRESSION IN LUNG CANCER new treatment modalities are needed before clinical trials in Visceral organs were removed and inspected for the presence patients with advanced metastatic disease. of metastases. Several mouse models of metastasis have been developed for various cancers (6-9). However, the majority of human Selection of the highly metastatic subline. A human lung lung cancer cell lines do not have the ability to metastasize cancer cell line with high metastatic potential to multiple to multiple organs beyond the lung in conventional athymic organs was established by a method previously described (7). nude mice, even though the models were developed by directly Briefly, mice were injected i.v. with the parental PC14 cells and injecting human cancer cells into the vessels of these nude sacrificed when they became moribund. Under sterile condi- mice (10). One way to address this is to establish human cancer tions, organs with macroscopic metastasis were removed and cell lines with a robust ability to metastasize to multiple organs a metastasized nodule was dissected free of connective tissue in conventional nude mice (7,8,11). Such highly metastatic and blood clots, and rinsed twice with medium. The nodule cell lines might provide mouse metastasis models without was finely minced using sterile scalpel blades in medium, and troublesome manipulation and would be particularly suitable the cells were seeded in tissue culture dishes. Then, 4-6 weeks for comparative studies on biological and molecular mecha- later, the cells were reinjected i.v. into nude mice. This process nisms of metastasis using subclones with differing metastatic was repeated three times to establish the highly metastatic potential. subline, PC14HM. PC14HM was derived from an adrenal Important data on the molecular mechanisms of metastasis metastasis, which is uncommon, after i.v. injection of parental in cancers have been produced from comparative studies PC14 cells. The metastasized cells in the bone after reinjection of biological behavior and genetic status among clonally of the first metastatic subline were reinjected again and cancer related cell lines with different metastatic potentials (11-15). cells in the metastatic nodule in the bone were selected as the Several genes associated with metastasis have been identi- highly metastatic subline, PC14HM. fied by comparing the gene expression in cells with different metastatic potentials, indicating that this strategy is useful Tumorigenicity in nude mice. Tumorigenicity was examined for determining the molecular mechanisms of metastasis by injecting 1x107 cells/0.2 ml into the subcutis of the dorsal (12,16,17). However, the metastatic potential of these cell lines flanks of nude mice. The tumor size was measured twice a is practically restricted to the lung in mouse models. Cell lines week. Tumor volume (V) was calculated using the formula V with high metastatic potential to multiple organs would be = 1/2 x length x (width)2. more appropriate for comparative studies, because such cell lines would more closely reflect the features of human cancer Cell proliferation assay. Cells were seeded in a 96-well plate metastasis. at a density of 1x104 cells/well, and the number of cells at In this study, to develop a convenient and reliable multiple each time point was estimated using the TetraColor One Cell organ metastasis model system, we established a highly Proliferation Assay System (Seikagaku Corp., Tokyo, Japan) metastatic subline, PC14HM, from the PC14 human lung according to the manufacturer's protocol. Doubling times of adenocarcinoma cell line using an in vivo method. The highly the cells were calculated from the logarithmic phase of the metastatic subline had the ability to metastasize to multiple growth curve. organs. To search for genetic determinants of metastasis in human lung cancer, we compared gene expression profiles cDNA microarray analysis. Total RNA was extracted using between the new subline and the parent cells using microarray the RNeasy mini-kit (Qiagen, Tokyo, Japan) according to the and real-time reverse transcription polymerase chain reaction supplier's protocol. Microarray analysis was performed using (RT-PCR) analyses. a Filgen Array Human 35K (Filgen, Inc., Nagoya, Japan), consisting of 34,580 cDNA fragments of human genes, 8 Materials and methods cDNA fragments of control human housekeeping genes, and 6 cDNA fragments derived from other species arrayed and Human lung cancer cell lines. Human lung cancer PC14 cells immobilized. The 34,580 70-mer probes represented 24,650 were maintained in RPMI-1640 medium (Sigma, St. Louis, genes and 37,123 gene transcripts. Fluorescent probes were MO), supplemented with 10% heat-inactivated fetal bovine synthesized by incorporating Cy3- or Cy5-dUTP (Amersham serum (Life Technologies,
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