Genome-Wide Analysis of Organ-Preferential Metastasis of Human Small Cell Lung Cancer in Mice
Total Page：16
File Type：pdf, Size：1020Kb
Vol. 1, 485–499, May 2003 Molecular Cancer Research 485 Genome-Wide Analysis of Organ-Preferential Metastasis of Human Small Cell Lung Cancer in Mice Soji Kakiuchi,1 Yataro Daigo,1 Tatsuhiko Tsunoda,2 Seiji Yano,3 Saburo Sone,3 and Yusuke Nakamura1 1Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 2Laboratory for Medical Informatics, SNP Research Center, Riken (Institute of Physical and Chemical Research), Tokyo, Japan; and 3Department of Internal Medicine and Molecular Therapeutics, The University of Tokushima School of Medicine, Tokushima, Japan Abstract Molecular interactions between cancer cells and their Although a number of molecules have been implicated in microenvironment(s) play important roles throughout the the process of cancer metastasis, the organ-selective multiple steps of metastasis (5). Blood flow and other nature of cancer cells is still poorly understood. To environmental factors influence the dissemination of cancer investigate this issue, we established a metastasis model cells to specific organs (6). However, the organ specificity of in mice with multiple organ dissemination by i.v. injection metastasis (i.e., some organs preferentially permit migration, of human small cell lung cancer (SBC-5) cells. We invasion, and growth of specific cancer cells, but others do not) analyzed gene-expression profiles of 25 metastatic is a crucial determinant of metastatic outcome, and proteins lesions from four organs (lung, liver, kidney, and bone) involved in the metastatic process are considered to be using a cDNA microarray representing 23,040 genes and promising therapeutic targets. extracted 435 genes that seemed to reflect the organ More than a century ago, Stephen Paget suggested that the specificity of the metastatic cells and the cross-talk distribution of metastases was not determined by chance, but between cancer cells and microenvironment. Further- rather that certain tumor cells (‘‘seed’’) are likely to have an more, we discovered 105 genes that might be involved in affinity for the microenvironment of specific organs (‘‘soil’’) the incipient stage of secondary-tumor formation by and that metastases occur only when the seed and soil are comparing the gene-expression profiles of metastatic compatible (7). Various molecules such as adhesion molecules, lesions classified according to size (<1 or >2 mm) as cytokines, chemokines, hormones, and hormone receptors play either ‘‘micrometastases’’ or ‘‘macrometastases.’’ This important roles in preferential metastasis (1, 8–10), but the genome-wide analysis should contribute to a greater precise mechanisms determining seed and soil compatibility understanding of molecular aspects of the metastatic remain unsolved. process in different microenvironments, and provide To examine the cellular and molecular bases of organ- indicators for new strategies to predict and prevent specific metastasis, we have established models of metastasis to metastasis. multiple organs by i.v. injection of eight different human lung cancer cell lines to severe combined immune deficiency (SCID) Introduction mice devoid of natural killer (NK) cells (11, 12). In the work Metastasis is the major cause of death due to cancer. Because reported here, by means of a cDNA microarray consisting of no absolutely effective methods for curing metastatic tumors are 23,040 genes, we analyzed the gene-expression profiles of 25 available at present, novel strategies for prevention of metastasis metastatic lesions present in murine lung, liver, kidney, and bone are urgently needed to improve the prognosis and quality of life following i.v. injection of human small cell lung cancer (SCLC) for cancer patients. Metastases occur in sequential steps that (SBC-5) cells. In the process, we identified candidate genes that include invasion of cancer cells from the primary site to blood may affect or determine organ specificity of the metastatic cells, vessels or the lymphatic system, survival in the circulation, as well as genes involved in progression from micrometastasis to intravascular transfer to distant organs, attachment to endothe- macrometastasis. Genes in both categories represent potential lial cells, extravasation into the parenchyma, and outgrowth into molecular targets for prevention of metastasis in humans. a secondary tumor with neovascularization (1–4). Results Metastasis of Human SCLC Cell Line SBC-5 in NK Cell-Depleted SCID Mice As we reported previously, i.v. injection of SBC-5 cells into Received 11/12/02; revised 03/17/03; accepted 04/04/03. SCID mice lacking NK cells caused metastases to multiple The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in organs (11, 13). To compare gene-expression profiles of 25 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. selected metastatic lesions (10 in lung, 5 in liver, 5 in kidney, and Grant support: ‘‘Research for the Future’’ Program Grant of The Japan Society 5 in bone), we collected pure populations of cancer cells by for the Promotion of Science (no. 00L01402) to Y.N. Requests for reprints: Yusuke Nakamura, Laboratory of Molecular Medicine, laser-capture microdissection. Histopathological features of Human Genome Center, Institute of Medical Science, The University of Tokyo, each of the 25 lesions are shown in Fig. 1, A–D, and in 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372; Fax: 81-3-5449-5433. E-mail: [email protected] Table 1. Three of the 10 lung foci developed in subpleura, and 5 Copyright D 2003 American Association for Cancer Research. were accompanied by intravascular embolization. The distribu- Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. 486 Genome-Wide Analysis of SCLC Metastasis FIGURE 1. Histopathology of metastatic lesions (H&E stain). a. Lung (Â100). b. Liver (Â100). c. Kidney (Â100). d. Bone (Â40). e. Cancer cells arrested in the pulmonary artery not by the size restriction but by adhesive interactions (Â100). f. Proliferation of multicellular tumor emboli in the pulmonary artery, initial minimal penetration of cancer cells through the arteriolar walls (arrow), and invasion of the cancer cells to the lung parenchyma (Â100). A, artery; Al, alveolus; CV, central vein; Gl, glomerulus; T, renal tubule; B, bone; BM, basement membrane. tion of metastases in lung was similar to that found in another tion tests; this is an appropriate strategy for distinguishing two experimental model of metastasis published elsewhere (2). All known subgroups. We used the following combinations: 10 bone metastases were accompanied by the osteolytic changes lung metastases versus all 15 others; 5 liver metastases versus commonly observed in human SCLCs (11). As in human cases, all 20 others; 5 kidney metastases versus all 20 others; and 5 metastases in murine kidney were smaller than those arising in bone metastases versus all 20 others. Table 2 lists 435 genes, other organs. the median ratios of which between the two groups were >2 with P values <0.05, among the 23,040 genes examined on the Cross-Hybridization of Mouse Messenger RNA microarray. Hierarchical clustering of these 435 genes separated As the target-DNAs on our cDNA microarray mainly include the four organ-specific groups of metastatic lesions very clearly the 3Vuntranslated region of the human gene that is more specific (Fig. 2). to individual genes and/or species, the incidence of cross- hybridization between human and murine sequences was Genes Differentially Expressed Between supposed to be very low. Moreover, laser-capture micro- ‘‘Micrometastasis’’ and ‘‘Macrometastasis’’ dissection has surely reduced the contamination of normal Metastasis models in animals have a great advantage over the mouse cells. To assess the influence of contamination of normal clinical samples for examining the incipient stage of micro- mouse mRNA and to consequently remove any experimental metastases (1, 2). The in vivo model used here also allowed us to noises in the statistical analysis, we performed laser-capture observe early events of the metastasis-developing process in microdissection of surrounding mouse normal tissues and lung, i.e., arrest in the pulmonary artery (Fig. 1E) and hybridized on the human cDNA microarrays. 2.56–3.07% extravasation into lung tissue (Fig. 1F). Because searching for (590–707/23,040) of genes in each organ had the intensities genes differentially expressed between micrometastasis and above cut-off value and these genes were excluded from the macrometastasis could be useful for understanding the biolog- further analysis. ical nature of secondary-tumor formation at the metastatic sites, we applied a random permutation test to nine metastatic lesions Cluster Analysis of Gene-Expression Profiles of the 25 in the lung (five lesions were <1 mm and four were >2 mm) and Metastatic Lesions extracted 105 differentially expressed genes. Sixty-eight of the To identify genes that were specifically expressed in each of genes were predominantly expressed in the smaller lesions, and the four metastasized organs, we performed random permuta- 37 were predominant in the larger lesions (Table 3). Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. Molecular Cancer Research 487 Discussion express adhesion molecules that can attach the cancer cells.