Genome-Wide Analysis of Organ-Preferential Metastasis of Human Small Cell Lung Cancer in Mice

Vol. 1, 485–499, May 2003 Molecular Cancer Research 485

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-

FIGURE 1. Histopathology of metastatic lesions (H&E stain). a. Lung (100). b. Liver (100). c. Kidney (100). d. Bone (40). e. Cancer cells arrested in the pulmonary artery not by the size restriction but by adhesive interactions (100). 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 (100). 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).

Discussion express adhesion molecules that can attach the cancer cells. A considerable body of evidence supports the concept that Each of these hypotheses suggests that interaction between organ specificity of metastasis is influenced not only by seed cancer cells and microenvironment influences the direction of and soil compatibility but also by physiological factors such metastatic organs. as patterns of blood flow and the size of cancer cells relative For the elucidation of the cross-talk between cancer cells to the diameter of capillaries (1, 2, 6). Considering the and microenvironment in each organ and a comprehensive sequential multisteps of metastasis, organ selectivity of survey of the factors regulating organ-specific metastasis, we metastasis seems to be determined after extravasation of performed cDNA-microarray analyses of the metastatic foci of cancer cells from primary site. Therefore, our experimental human SCLC (SBC-5) developed in four different murine metastasis model was likely to reflect the later steps. Actually, organs (lung, liver, kidney, and bone) and compared gene- although cancer cells were injected intravenously, some cells expression profiles among 25 of these lesions. The expression formed metastatic nodules not in the lungs, the first capillary patterns fell into four categories, each of which reflected a beds encountered, but in other organs (11). The distribution of specific organ. The 435 genes that distinguished these four tumor cells in the mice reproduced very well the distribution groups were extracted by statistical analysis and classified into patterns of human metastatic lung cancers (in humans, e.g., 12 categories on the basis of known biological functions SCLC cells can form metastases in multiple organs, mainly (Table 2). Among them, genes belonging to the cell-cell systemic lymph nodes and liver, whereas lung adenocarcino- signaling category included growth-factor receptors, cytokines, mas produce metastatic foci mainly in the lungs). This and chemokines. FGFR1 and FST were highly expressed in evidence suggests that seed and soil compatibility does cells metastasized in bone. FGFR1 is a receptor for fibroblast contribute to the directional migration and invasion of tumor growth factors (FGFs) and its downstream signals influence cells into specific organs, and also confirms that our model is mitogenesis and differentiation. Because FGFs are expressed valuable for investigation of the relevant mechanism(s) abundantly in bone tissue (16), the microenvironment of bone including the tumor-host interaction in microenvironment. is likely to be suitable for survival and proliferation of cancer Several hypotheses concerning the organ specificity of cells that express FGFR1. In SCLCs, metastatic cells in bone metastasis have been proposed (3, 8, 10, 14, 15). They are predominantly osteolytic. FST, an activin antagonist that include: (a) tumor cells that extravasate into secondary sites can inhibit bone formation (17), might promote the bone can survive and proliferate only in those organs that absorption caused by metastatic cells and contribute to the have appropriate growth factors; (b) chemoattractants home release of the growth factors such as FGFs that are stored the cancer cells toward specific organs by means of in bone tissue. This bidirectional interaction between tumor concentration gradients; and/or (c) secondary tumors can cells and the bone microenvironment seems to be important develop in certain organs, the endothelial cells of which for developing bone metastasis. PTHLH (alias PTHrP: parathyroid hormone related-peptide), a key mediator of osteolytic metastasis (16), was expressed by the tumor cells in all four metastatic sites we examined. Expression levels of PTHLH in bone metastases tended to be higher than in other Table 1. Size and Histopathological Features of 25 Metastatic Lesions affected organs, but the difference was not statistically significant (P = 0.057). Metastatic Organ ID The Major Histopathological Chemokines, secreted peptides that control the homing of Axis (mm) Feature leukocytes, are considered to contribute to the directional migration of cancer cells that express chemokine receptors on Lung 1 2.51 subpleura 2 2.20 subpleura their surfaces (1, 8). Three chemokine receptors, CCR4, 3 0.38 with embolization CCR5, and CCR9, were expressed in all 25 metastatic lesions 4 0.65 (data not shown), with no significant differences of expression 5 0.42 with embolization 6 2.00 levels. In the gene-expression database of 24 normal human 7 0.92 with embolization adult tissues we reported recently, SCYA4 (CCL4), one of the 8 1.30 subpleura ligands of CCR5, was expressed preferentially in lung, liver, 9 2.80 with embolization 10 0.62 with embolization lymph nodes, bone marrow, adipose tissue, and spleen (18). Liver 1 3.80 Because lung, liver, lymph nodes, and bone marrow are major 2 2.70 3 4.00 targets for metastasis of SCLC, CCL4/CCR5 interaction 4 1.82 may contribute to the organ-preferential metastasis of the 5 2.01 SCLC cells. Kidney 1 0.62 2 0.26 Adhesion, detachment, and aggregation of tumor cells 3 1.48 seem to play important roles in achieving metastasis. 4 0.49 Although most circulating cancer cells are arrested in 5 0.22 Bone 1 > 4.00 osteolytic capillary beds because of size restrictions (3), we observed 2 > 4.00 osteolytic adherence to the walls of pre-capillary vessels that were 3 > 4.00 osteolytic 4 > 4.00 osteolytic much larger in diameter than the cancer cells (Fig. 1E). A 5 > 4.00 osteolytic number of molecules involved in cell-cell or cell-matrix adhesion, such as integrins and selectins, appear to mediate

Table 2. Genes Predominantly Expressed in Metastasis in Each of the Four Organs: a. Lung; b. Kidney; c. Bone; d. Liver

Symbol Description P value Ratio

a. Lung Cell adhesion LGALS1 lectin, galactoside-binding, soluble, 1 (galectin 1) < 0.001 5.36 PCDHGC3 protocadherin g subfamily C, 3 < 0.001 3.41 ITGB4 integrin, h4 < 0.001 2.74 LGALS3BP lectin, galactoside-binding, soluble, 3 binding protein < 0.01 2.28 SDC1 syndecan 1 < 0.01 3.56 GJB2 gap junction protein, h2, Mr 26,000 (connexin 26) < 0.05 2.11 Cytoskeleton/cell motility TUBB2 tubulin, h, 2 < 0.001 2.92 FLNA filamin A, a (actin-binding protein-280) < 0.001 2.46 RHOC ras homologue gene family, member C < 0.001 3.02 ACTC actin, a, cardiac muscle < 0.001 4.84 ACTA1 actin, a1, skeletal muscle < 0.001 5.40 ACTA2 actin, a2, smooth muscle, aorta < 0.001 2.96 ACTB actin, h < 0.001 4.12 ACTG2 actin, g2, smooth muscle, enteric < 0.001 5.49 ARPC4 actin-related protein 2/3 complex, subunit 4 (Mr 20,000) < 0.001 4.06 PTK9L protein tyrosine kinase 9-like (A6-related protein) < 0.001 2.09 LMNA lamin A/C < 0.001 3.97 RPS29 ribosomal protein S29 < 0.001 2.02 PFN1 profilin 1 < 0.01 2.16 Extracellular matrix (ECM) remodeling HTF9C HpaII tiny fragments locus 9C < 0.001 2.87 FLJ11618 hypothetical protein FLJ11618 < 0.01 2.11 Cell-cell signaling (cytokine/chemokine) MIF macrophage migration inhibitory factor < 0.001 2.01 TNFRSF1A tumor necrosis factor receptor superfamily, member 1A < 0.001 2.22 SCYB13 small inducible cytokine B subfamily, member 13 < 0.001 2.30 DDT D-dopachrome tautomerase < 0.001 2.10 Signal transduction FKBP8 FK506-binding protein 8 (Mr 38,000) < 0.001 2.48 PDAP1 PDGFA associated protein 1 < 0.001 5.69 ITPK1 inositol 1,3,4-triphosphate 5/6 kinase < 0.001 2.21 TM4SF7 transmembrane 4 superfamily member 7 < 0.001 3.75 IFITM1 interferon induced transmembrane protein 1 (9 – 27) < 0.01 5.92 ILK integrin-linked kinase < 0.01 2.00 TRAF2 TNF receptor-associated factor 2 < 0.05 2.10 Immune response C3 complement component 3 < 0.001 2.58 BF B-factor, properdin < 0.001 2.13 HLA-A major histocompatibility complex, class I, A < 0.001 2.86 HLA-B major histocompatibility complex, class I, B < 0.001 2.77 HLA-C major histocompatibility complex, class I, C < 0.001 2.16 HLA-DQA1 major histocompatibility complex, class II, DQ a1 < 0.001 19.77 HLA-DQB1 major histocompatibility complex, class II, DQ h1 < 0.001 5.08 PSME2 proteasome (prosome, macropain) activator subunit 2 < 0.001 2.64 IFITM2 interferon induced transmembrane protein 2 (1-8D) < 0.001 2.82 Metabolism COX6B cytochrome c oxidase subunit VIb < 0.001 2.64 COX8 cytochrome c oxidase subunit VIII < 0.001 2.84 COX7A2 cytochrome c oxidase subunit VIIa polypeptide 2 (liver) < 0.001 2.16 COX5B cytochrome c oxidase subunit Vb < 0.001 2.18 GPX1 glutathione peroxidase 1 < 0.001 4.99 GPX4 glutathione peroxidase 4 (phospholipid hydroperoxidase) < 0.001 2.67 MT2A metallothionein 2A < 0.001 2.09 APOC1 apolipoprotein C-I < 0.001 6.44 FDXR ferredoxin reductase < 0.001 2.58 HSD11B2 hydroxysteroid (11-h) dehydrogenase 2 < 0.001 2.63 ALAS1 aminolevulinate, y-, synthase 1 < 0.001 2.25 Cell cycle/apoptosis/DNA repair PPP1CA protein phosphatase 1, catalytic subunit, a isoform < 0.001 2.90 ERH enhancer of rudimentary (Drosophila) homologue < 0.001 2.09 PCBP4 poly(rC)-binding protein 4 < 0.01 2.46 CDC20 CDC20 (cell division cycle 20, Saccharomyces cerevisiae, homologue) < 0.01 2.19 SFN stratifin < 0.01 2.40 NOL3 nucleolar protein 3 (apoptosis repressor with CARD domain) < 0.01 2.64 Transcription NFKBIA NF-nB inhibitor < 0.001 2.15 DRAP1 DR1-associated protein 1 (negative cofactor 2 a) < 0.001 2.55 GATA2 GATA-binding protein 2 < 0.001 2.92 MBD2 methyl-CpG binding domain protein 2 < 0.001 3.25

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Table 2. (continued )

Symbol Description P value Ratio

Protein synthesis/processing FAU FBR-MuSV ubiquitously expressed (fox derived) < 0.001 2.74 RPS10 ribosomal protein S10 < 0.001 2.12 RPLP2 ribosomal protein, large P2 < 0.001 2.01 RPL18 ribosomal protein L18 < 0.001 2.05 MRPL23 mitochondrial ribosomal protein L23 < 0.001 3.15 PSMB8 proteasome (prosome, macropain) subunit, h type, 8 < 0.001 3.22 RPS26 ribosomal protein S26 < 0.001 3.12 PMM2 phosphomannomutase 2 < 0.001 2.06 FBXO2 F-box only protein 2 < 0.001 2.22 EEF1D eukaryotic translation elongation factor 1 y < 0.001 2.24 Neurogenesis The others CALM3 calmodulin 3 (phosphorylase kinase, y) < 0.001 2.25 CALM1 calmodulin 1 (phosphorylase kinase, y) < 0.001 2.27 Unknown EST < 0.001 2.11 EST < 0.001 4.01 EST < 0.001 2.05 EST < 0.001 21.33 BCL7C B-cell CLL/lymphoma 7C < 0.001 2.67 EST < 0.001 3.10 PTD008 PTD008 protein < 0.001 2.18 EST < 0.001 2.72 Homo sapiens cDNA: FLJ22175 fis, clone HRC00773 < 0.001 2.22 EST < 0.001 2.08 LOC51181 carbonyl reductase < 0.001 2.20 EST < 0.001 2.18 EST00098 hypothetical protein EST00098 < 0.001 2.48 EST < 0.001 2.65 CGI-96 CGI-96 protein < 0.001 2.44 EST < 0.001 2.00 EST < 0.001 2.03 HRB2 HIV rev binding protein 2 < 0.001 2.19 EST < 0.001 2.42 SELENBP1 selenium binding protein 1 < 0.001 6.99 EST < 0.001 2.48 EST < 0.01 2.07 EST < 0.01 2.13 FLJ10829 hypothetical protein FLJ10829 < 0.01 2.26 EST < 0.05 2.35 b. Kidney Cell adhesion LGALS9 lectin, galactoside-binding, soluble, 9 (galectin 9) < 0.001 2.50 ENTPD2 ectonucleoside triphosphate diphosphohydrolase 2 < 0.01 2.79 CLDN17 claudin 17 < 0.05 2.62 Cytoskeleton/cell motility ACTR1A ARP1 (actin-related protein 1, yeast) homologue A < 0.001 2.77 DKFZP586N1922 DKFZP586N1922 protein < 0.001 2.09 CYLN2 cytoplasmic linker 2 < 0.001 2.07 EPLIN epithelial protein lost in neoplasm h < 0.001 3.43 CCT-7 HIV-1 Nef interacting protein < 0.01 2.35 CNN3 calponin 3, acidic < 0.05 3.84 ECM remodeling COL1A1 collagen, type I, a1 < 0.01 2.51 Cell-cell signaling (cytokine/chemokine) BMP6 bone morphogenetic protein 6 < 0.001 2.79 INHBA inhibin, hA (activin A, activin AB a polypeptide) < 0.001 2.35 Signal transduction DUSP10 dual specificity phosphatase 10 < 0.001 2.09 PRSS11 protease, serine, 11 (IGF binding) < 0.01 2.66 Immune response HLA-DMA major histocompatibility complex, class II, DM a < 0.01 2.48 TRB@ T cell receptor h locus < 0.01 8.66 C1S complement component 1, s subcomponent < 0.05 2.17 Metabolism GCK glucokinase (hexokinase 4, maturity onset diabetes of the young 2) < 0.001 2.52 GPX3 glutathione peroxidase 3 (plasma) < 0.001 11.30 HMGCL 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase < 0.01 3.45 Cell cycle/apoptosis/DNA repair Septin 6 < 0.001 2.83

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Table 2. (continued )

Symbol Description P value Ratio

Transcription P84 nuclear matrix protein p84 < 0.001 2.27 NSAP1 NS1-associated protein 1 < 0.001 2.17 ZNF258 Zinc finger protein 258 < 0.001 2.12 GCN5L2 GCN5-like 2 < 0.01 2.98 HSPC157 HSPC157 protein < 0.01 2.56 RNASE6PL ribonuclease 6 precursor < 0.01 2.30 Protein synthesis/processing HUGT1 UDP-glucose:glycoprotein glucosyltransferase 1 < 0.001 2.10 UBE2N ubiquitin-conjugating enzyme E2N < 0.001 2.24 SCAMP2 secretory carrier membrane protein 2 < 0.01 2.35 Neurogenesis DCTN1 Dynactin 1 (p150, glued homologue, Drosophila) < 0.001 2.14 ITM2B integral membrane protein 2B < 0.01 2.21 EFNB3 ephrin-B3 < 0.01 2.49 The others ATP1B1 ATPase, Na+/K+ transporting, h1 polypeptide < 0.001 3.25 SRI sorcin < 0.001 3.44 H1F2 H1 histone family, member 2 < 0.001 2.15 SUT1 sulfate transporter 1 < 0.05 2.15 Unknown Homo sapiens cDNA FLJ11838 fis, clone HEMBA1006624 < 0.001 2.21 EST < 0.001 2.51 EST < 0.001 2.04 ISYNA1 myo-inositol 1-phosphate synthase A1 < 0.001 2.25 Homo sapiens cDNA FLJ13549 fis, clone PLACE1007097 < 0.001 2.01 EST < 0.001 2.11 EST < 0.001 2.09 KIAA0709 endocytic receptor (macrophage mannose receptor family) < 0.001 2.30 LOC51243 hypothetical protein < 0.001 3.08 FJX1 putative secreted ligand homologous to fjx1 < 0.001 2.29 EST < 0.001 2.42 KIAA0442 KIAA0442 protein < 0.001 2.42 FLJ10769 hypothetical protein FLJ10769 < 0.001 2.01 EST < 0.001 2.17 EST < 0.001 2.12 ATP5H ATP synthase, H+ transporting, mitochondrial F1F0, subunit d < 0.001 2.02 LOC55902 acetyl-CoA synthetase < 0.001 2.20 EST < 0.01 2.07 EST < 0.01 2.84 LOC51064 glutathione S-transferase subunit 13 homologue < 0.01 3.52 Homo sapiens mRNA; cDNA DKFZp586K2123 < 0.01 2.29 NBEA neurobeachin < 0.01 2.05 EST < 0.01 2.28 EST < 0.01 2.03 FLJ10846 hypothetical protein FLJ10846 < 0.01 2.17 EST < 0.01 2.04 BRD7 bromodomain-containing 7 < 0.01 2.07 D2S448 Melanoma associated gene < 0.05 2.23 EST < 0.05 2.12 EST < 0.05 2.69 Homo sapiens cDNA FLJ20144 fis, clone COL07809 < 0.05 2.20 EST < 0.05 2.02 c. Bone Cell adhesion CELSR1 cadherin, EGF LAG seven-pass G-type receptor 1 < 0.001 3.31 PLXNC1 plexin C1 < 0.001 2.71 NEO1 neogenin (chicken) homologue 1 < 0.001 2.06 PTPRM protein tyrosine phosphatase, receptor type, M < 0.001 2.67 Cytoskeleton/cell motility KIAA0855 golgin-67 < 0.001 3.48 MESDC1 Mesoderm development candidate 1 < 0.01 2.58 LIMK2 LIM domain kinase 2 < 0.01 2.38 ECM remodeling KERA keratocan < 0.001 2.05 COL3A1 collagen, type III, a1 < 0.01 3.29 Cell-cell signaling (cytokine/chemokine) FST follistatin < 0.001 3.97 MST1 macrophage stimulating 1 (hepatocyte growth factor-like) < 0.001 2.30 FAP fibroblast activation protein, a < 0.001 2.01 FGFR1 fibroblast growth factor receptor 1 < 0.001 2.07

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Table 2. (continued )

Symbol Description P value Ratio

Signal transduction PRP4 serine/threonine-protein kinase PRP4 homologue < 0.001 2.13 PPP3CC protein phosphatase 3 (formerly 2B) < 0.001 2.12 PTPN1 protein tyrosine phosphatase, non-receptor type 1 < 0.001 2.12 AKT2 v-akt murine thymoma viral oncogene homologue 2 < 0.001 2.14 CAV1 caveolin 1, caveolae protein, M r 22,000 < 0.001 2.79 PRKAR1A tissue-specific extinguisher 1 < 0.05 2.01 Immune response Metabolism Cell cycle/apoptosis/DNA repair TIA1 TIA1 cytotoxic granule-associated RNA-binding protein < 0.001 2.25 PRKDC protein kinase, DNA-activated, catalytic polypeptide < 0.001 2.62 RAD51L3 RAD51 (Saccharomyces cerevisiae)-like 3 < 0.001 2.38 DDB1 damage-specific DNA binding protein 1 (M r 127,000) < 0.001 2.16 BAK1 BCL2-antagonist/killer 1 < 0.01 2.16 CDK3 Cyclin-dependent kinase 3 < 0.01 2.00 Transcription SFRS11 splicing factor, arginine/serine-rich 11 < 0.001 2.02 DKFZP434P0721 similar to mouse Xrn1/Dhm2 protein < 0.001 2.23 SIRT5 sir2-like 5 < 0.001 2.23 TCEB1L transcription elongation factor B (SIII), polypeptide 1-like < 0.001 2.46 EZH1 enhancer of zeste (Drosophila) homologue 1 < 0.001 2.02 HSF2 heat shock transcription factor 2 < 0.001 2.15 EGR4 early growth response 4 < 0.001 2.22 HNRPU heterogeneous nuclear ribonucleoprotein U < 0.01 3.33 GLI3 GLI-Kruppel family member GLI3 < 0.01 2.27 EGR3 early growth response 3 < 0.01 2.95 LZTR1 leucine-zipper-like transcriptional regulator, 1 < 0.01 2.09 SMARCC1 SWI/SNF complex Mr 155,000 subunit < 0.05 2.36 Protein synthesis/processing MTIF2 mitochondrial translational initiation factor 2 < 0.001 2.08 MTHFD2 cyclohydrolase, NAD(+)-dependent < 0.01 2.27 RPL37A ribosomal protein L37a < 0.01 2.04 SEC63L SEC63, endoplasmic reticulum translocon component like < 0.01 2.36 LOC54516 similar to prokaryotic-type class I peptide chain release factors < 0.01 2.13 Neurogenesis DPYSL2 dihydropyrimidinase-like 2 < 0.001 3.51 GPM6B glycoprotein M6B < 0.001 2.50 SLIT2 slit (Drosophila) homologue 2 < 0.01 6.01 PRPS1 phosphoribosyl pyrophosphate synthetase 1 < 0.01 2.10 The others ERF Ets2 repressor factor < 0.001 2.24 DYT1 dystonia 1, torsion (autosomal dominant; torsin A) < 0.001 2.13 SLC11A2 solute carrier family 11, member 2 < 0.001 2.37 MDM2 mouse double minute 2, human homologue of; p53-binding protein < 0.001 2.03 KTN1 kinectin 1 (kinesin receptor) < 0.001 2.45 POV1 prostate cancer overexpressed gene 1 < 0.001 3.19 STK15 serine/threonine kinase 15 < 0.001 2.36 INPPL1 inositol polyphosphate phosphatase-like 1 < 0.001 2.02 GSK3B glycogen synthase kinase 3 h < 0.001 2.42 KDELR3 KDEL endoplasmic reticulum protein retention receptor 3 < 0.001 2.39 TRF4-2 topoisomerase-related function protein 4-2 < 0.001 2.37 RAB2 RAB2, member RAS oncogene family < 0.01 2.16 KIAA0102 KIAA0102 gene product < 0.01 2.24 SMT3H1 SMT3 (suppressor of mif two 3, yeast) homologue 1 < 0.01 2.32 ENTPD5 ectonucleoside triphosphate diphosphohydrolase 5 < 0.01 2.02 KIAA0939 KIAA0939 protein < 0.01 5.81 FUS1 lung cancer candidate < 0.01 2.18 LLGL2 lethal giant larvae (Drosophila) homologue 2 < 0.01 2.11 MYB v-myb avian myeloblastosis viral oncogene homologue < 0.01 2.03 Unknown EST < 0.001 2.14 EST < 0.001 2.45 SEC31B-1 Secretory pathway component Sec31B-1 < 0.001 3.07 EST < 0.001 2.04 MAC30 hypothetical protein < 0.001 2.14 Homo sapiens mRNA; cDNA DKFZp434C136 < 0.001 2.55 EST < 0.001 2.42 EST < 0.001 3.07 EST < 0.001 2.83 Homo sapiens cDNA: FLJ22562 fis, clone HSI01814 < 0.001 2.57 EST < 0.001 5.12 EST < 0.001 2.53

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Table 2. (continued )

Symbol Description P value Ratio

KIAA0729 KIAA0729 protein < 0.001 2.14 EST < 0.001 2.24 EST < 0.001 2.41 EST < 0.001 3.62 FLJ23399 hypothetical protein FLJ23399 < 0.001 2.44 KIAA0765 putative brain nuclearly targeted protein < 0.001 2.42 EST < 0.001 2.28 EST < 0.001 2.81 KIAA0088 KIAA0088 protein < 0.001 2.32 PRO2463 PRO2463 protein < 0.001 2.02 KIAA0459 KIAA0459 protein < 0.001 2.19 EST < 0.001 2.02 KIAA1025 KIAA1025 protein < 0.001 2.44 AF15Q14 AF15q14 protein < 0.001 2.59 KIAA1238 Homo sapiens mRNA; cDNA DKFZp586I0521 < 0.001 2.03 KIAA1278 KIAA1278 protein < 0.001 2.27 FLJ20195 hypothetical protein FLJ20195 < 0.001 2.04 VMP1 Likely orthologue of rat vacuole membrane protein 1 < 0.001 2.11 EST < 0.001 2.42 DKFZP761H171 hypothetical GTP-binding protein DKFZp761H171 < 0.001 2.63 EST < 0.001 2.46 EST < 0.001 2.49 EST < 0.001 2.75 EST < 0.001 2.11 EST < 0.001 2.20 EST < 0.001 2.01 Homo sapiens cDNA FLJ11998 fis, clone HEMBB1001521 < 0.001 2.36 EST < 0.001 2.27 KIAA0986 KIAA0986 protein < 0.001 2.93 EST < 0.001 3.02 EST < 0.001 2.04 EST < 0.001 2.12 EST < 0.01 2.31 Homo sapiens cDNA: FLJ22530 fis, clone HRC12866 < 0.01 2.26 EST < 0.01 2.22 FLJ23293 hypothetical protein FLJ23293 < 0.01 2.16 EST < 0.01 2.28 EST < 0.01 2.31 EST < 0.01 2.63 EST < 0.01 2.02 EST < 0.01 2.85 Homo sapiens cDNA FLJ13213 fis, clone NT2RP4001126 < 0.01 2.18 KIAA0105 Wilms’ tumour 1-associating protein < 0.01 2.43 EST < 0.01 2.23 EST < 0.01 2.32 EST < 0.01 2.51 LOC51153 FT005 protein < 0.01 3.14 EST < 0.01 2.36 Homo sapiens clone FLB3024 PRO0756 mRNA, complete cds < 0.01 2.17 EST < 0.01 2.22 EST < 0.01 2.76 S164 S164 protein < 0.01 2.38 Human clone 23589 mRNA sequence < 0.01 2.15 LANO LAP (leucine-rich repeats and PDZ) and no PDZ protein < 0.01 2.16 Homo sapiens cDNA: FLJ23538 fis, clone LNG08010 < 0.01 2.13 EST < 0.05 2.07 EST < 0.05 2.11 KIAA0161 KIAA0161 gene product < 0.05 2.44 EST < 0.05 2.60 EST < 0.05 2.10 EST < 0.05 2.07 EST < 0.05 2.22 DKFZP564K247 DKFZP564K247 protein < 0.05 2.26 SBB103 hypothetical SBBI03 protein < 0.05 2.27 EST < 0.05 2.22 Homo sapiens cDNA: FLJ21693 fis, clone COL09609 < 0.05 2.78 EST < 0.05 2.64 d. Liver Cell adhesion IGFBP7 insulin-like growth factor binding protein 7 < 0.001 2.29 CDH2 cadherin 2, type 1, N-cadherin (neuronal) < 0.01 3.01

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Table 2. (continued )

Symbol Description P value Ratio

Cytoskeleton/cell motility CBX1 chromobox homologue 1 (Drosophila HP1 h) < 0.001 2.51 HECH heterochromatin-like protein 1 < 0.001 2.61 MYPT1 myosin phosphatase, target subunit 1 < 0.001 3.00 SDCBP syndecan binding protein (syntenin) < 0.001 2.26 CD2AP CD2-associated protein < 0.01 2.21 ECM remodeling CTSL2 cathepsin L2 < 0.01 2.06 P4HA1 prolyl 4-hydroxylase, a-1 subunit < 0.01 3.11 ADAM17 A disintegrin and metalloproteinase domain 17 < 0.01 2.04 Cell-cell signaling (cytokine/chemokine) LIF leukemia inhibitory factor < 0.001 3.49 IFN-g antagonist cytokine < 0.001 2.23 PBEF pre-B-cell colony-enhancing factor < 0.01 2.52 Signal transduction GNAS1 GNAS complex locus < 0.001 2.61 TIEG TGFB inducible early growth response < 0.001 2.11 RHEB2 Ras homologue enriched in brain 2 < 0.001 2.49 YWHAQ 14-3-3 protein H < 0.01 2.22 PTPN12 protein tyrosine phosphatase, non-receptor type 12 < 0.01 2.17 SSH3BP1 spectrin SH3 domain binding protein 1 < 0.05 2.10 LOC56990 non-kinase Cdc42 effector protein SPEC2 < 0.05 2.11 MTM1 myotubular myopathy 1 < 0.05 2.14 Immune response Metabolism PGK1 phosphoglycerate kinase 1 < 0.001 2.57 PDK1 pyruvate dehydrogenase kinase, isoenzyme 1 < 0.001 6.21 LDHB lactate dehydrogenase B < 0.001 3.37 ATQ1 antiquitin 1 < 0.001 2.22 AGL amylo, 6-glucosidase, 4-a-glucanotransferase < 0.001 2.18 PHKB phosphorylase kinase, h < 0.001 2.71 ACLY ATP citrate lyase < 0.05 2.40 Cell cycle/apoptosis/DNA repair CCNG1 cyclin G1 < 0.001 2.17 CAP-C chromosome-associated polypeptide C < 0.001 2.53 BNIP3L BCL2/adenovirus E1B 19kD-interacting protein 3-like < 0.01 2.15 REV3L REV3 (yeast homologue)-like < 0.01 2.45 Transcription TOP3 DNA topoisomerase III < 0.001 2.00 HNRPA2B1 heterogeneous nuclear ribonucleoprotein A2/B1 < 0.001 2.73 DDX15 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 15 < 0.001 3.26 HNRPA1 heterogeneous nuclear ribonucleoprotein A1 < 0.001 2.45 RBMX RNA binding motif protein, X chromosome < 0.001 2.21 HTATSF1 HIV TAT specific factor 1 < 0.001 2.31 TAF172 TBP-associated factor 172 < 0.001 2.18 TAF2B TBP-associated factor 2 < 0.001 2.64 LRRFIP1 leucine rich repeat (in FLII) interacting protein 1 < 0.001 2.24 BTF3 basic transcription factor 3 < 0.001 2.03 MYC v-myc avian myelocytomatosis viral oncogene homologue < 0.001 2.50 BHLHB2 basic helix-loop-helix domain containing, class B, 2 < 0.001 5.45 DBY DEAD/H box polypeptide, Y chromosome < 0.001 2.33 MXI1 MAX-interacting protein 1 < 0.01 2.86 ZNF9 zinc finger protein 9 < 0.01 2.62 BAZ1A bromodomain adjacent to zinc finger domain, 1A < 0.05 2.13 Protein synthesis/processing RABGGTB Rab geranylgeranyltransferase, h subunit < 0.001 2.76 RPL9 ribosomal protein L9 < 0.001 2.27 EIF3S6 eukaryotic translation initiation factor 3, subunit 6 (M r 48,000) < 0.001 2.11 RPL6 ribosomal protein L6 < 0.001 2.10 RPL7 ribosomal protein L7 < 0.001 2.40 RPS3A ribosomal protein S3A < 0.001 2.73 RPS4X ribosomal protein S4, X-linked < 0.001 2.16 EEF1A1 eukaryotic translation elongation factor 1 a1 < 0.001 2.57 LOC51280 golgi membrane protein GP73 < 0.01 2.16 Neurogenesis GPM6B glycoprotein M6B < 0.001 3.87 STMN2 Stathmin-like 2 < 0.001 3.78 GPI glucose phosphate isomerase < 0.001 2.44 The others CSPG6 chondroitin sulfate proteoglycan 6 (bamacan) < 0.001 2.97 ST13 suppression of tumorigenicity 13(Hsp70-interacting protein) < 0.001 2.29 BET1 Golgi vesicular membrane trafficking protein p18 < 0.001 2.15

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Table 2. (continued )

Symbol Description P value Ratio

SLC2A1 solute carrier family 2, member 1 < 0.001 3.11 SLC16A1 solute carrier family 16, member 1 < 0.001 2.35 RANBP2 RAN binding protein 2 < 0.001 2.33 ATP5A1 mitochondrial ATP synthetase, oligomycin-resistant < 0.001 2.01 P115 vesicle docking protein p115 < 0.01 2.54 SEC23B Sec23 (Saccharomyces cerevisiae) homologue B < 0.01 2.05 SCP2 sterol carrier protein 2 < 0.01 2.56 UBA2 SUMO activating enzyme subunit 2 < 0.05 2.50 SLC2A3 solute carrier family 2, member 3 < 0.05 2.96 SSFA2 sperm specific antigen 2 < 0.05 2.17 Unknown ARFGAP1 ADP-ribosylation factor GTPase activating protein 1 < 0.001 2.54 LOC51187 60S ribosomal protein L30 isolog < 0.001 2.26 KIAA1223 KIAA1223 protein < 0.001 2.00 LOC51606 CGI1 protein < 0.001 2.05 CL25022 hypothetical protein < 0.001 3.10 Homo sapiens CDA08 mRNA, complete cds < 0.001 2.39 Homo sapiens cDNA FLJ13018 fis, clone NT2RP3000685 < 0.001 2.77 KIAA0725 KIAA0725 protein < 0.001 2.04 EST < 0.001 2.04 KIAA0863 KIAA0863 protein < 0.001 2.34 EST < 0.001 2.34 Homo sapiens cDNA: FLJ22844 fis, clone KAIA5181 < 0.001 2.09 E2IG5 hypothetical protein, estradiol-induced < 0.001 2.20 EST < 0.001 2.14 PGK1 phosphoglycerate kinase 1 < 0.001 2.77 sequence 1 from US patent < 0.001 2.29 LIV LIV protein, estrogen regulated < 0.001 2.55 Homo sapiens cDNA FLJ14041 fis, clone HEMBA1005780 < 0.001 2.21 EST < 0.001 2.02 EST < 0.001 2.16 LOC56270 hypothetical protein 628 < 0.001 2.22 LOC51012 CGI07 protein < 0.001 3.03 RANBP6 RAN binding protein 6 < 0.001 2.47 KIAA1615 KIAA1615 protein < 0.001 2.03 EST < 0.001 2.39 EST < 0.01 2.45 KIAA0430 Human Chromosome 16 BAC clone CIT987SK-A-362G6 < 0.01 2.08 EST < 0.01 2.22 CH1 membrane protein CH1 < 0.01 2.75 EST < 0.01 2.15 EST < 0.01 2.35 KIAA0637 KIAA0637 gene product < 0.01 2.28 KIAA1014 KIAA1014 protein < 0.01 2.56 LCP host cell factor homologue < 0.01 2.46 FLJ10582 hypothetical protein FLJ10582 < 0.01 2.82 FHL1 four and a half LIM domains 1 < 0.01 2.34 Homo sapiens cDNA: FLJ23093 fis, clone LNG07264 < 0.01 2.34 DKFZP434N093 DKFZP434N093 protein < 0.01 2.13 LOC51030 CGI48 protein < 0.01 2.24 Homo sapiens mRNA; cDNA DKFZp586G2222 < 0.01 2.41 FLJ10461 hypothetical protein FLJ10461 < 0.01 2.80 KIAA0601 KIAA0601 protein < 0.05 2.44 FLJ10595 hypothetical protein FLJ10595 < 0.05 2.93 DKFZP564I052 DKFZP564I052 protein < 0.05 2.07 SNX2 sorting nexin 2 < 0.05 2.58 FLJ10326 hypothetical protein FLJ10326 < 0.05 2.29 Homo sapiens cDNA: FLJ21962 fis, clone HEP05564 < 0.05 2.94 SH3BGRL SH3 domain binding glutamic acid-rich protein like < 0.05 2.11 EST < 0.05 2.22 MPDU1 mannose-P-dolichol utilization defect 1 < 0.05 2.07

Note: P value: P value of random permutation test; ratio: ratio of median values between two groups of random permutation test (see ‘‘Materials and Methods’’).

the adhesion of cancer cells to the vessel wall in specific molecular family, LGALS1, is highly expressed in pulmonary organs (10). Consequently, the adhesive interaction between metastases and another, LGALS9, in renal metastases. In cancer cells and endothelium is likely to be associated with the addition, LGALS3 has shown an association with pulmonary organ selectivity of metastasis. Our data suggest that lectin, metastasis of osteosarcoma (19), and its binding protein a family of h-galactoside-binding proteins implicated in LGALS3BP, an indicator of the metastatic propensity of lung modulating cell-cell and cell-matrix interactions, may play an cancer (20), was also highly expressed in pulmonary metastases important role in organ preference because one member of this in our murine model.

The gene expression of cancer cells could be influenced by the microenvironment of each organ where they metastasized. Consequently, this list includes genes, the expression of which was altered by the cross-talk between cancer cells and host microecology in secondary site. In this model, it is difficult to distinguish initial difference and post- metastatic alteration of gene expression; however, many of the genes listed here had already been associated with cancer invasion and metastasis, several with respect to metastasis to specific organs. For example, ITBG4, SDC1, C3, MT2A, and CALM, which were predominantly expressed in pulmonary metastases in our murine model, have been associated with pulmonary metastasis of neoplasms originating outside the lung (10, 19, 21, 23, 24). In vivo videomicroscopy studies have revealed that early phases of the metastatic process are completed quite efficiently through sequential steps, whereas growth phases of metastatic cells are very inefficient. Those observations suggest that regulators of tumor growth at secondary sites should be key targets for preventing metastasis (3, 25). To clarify the mechanism(s) operating later in the process of metastasis, we applied random permutation tests to compare lung-metastatic nodules classified according to the growth step from micrometastasis to macrometastasis (see ‘‘Materials and Methods’’). The 105 genes that were differentially expressed between the two groups were classified according to their function. A number of genes involved in the cell motility, cell adhesion, and ECM remodeling were predominantly expressed in micrometastasis. For example, HSPB3, ACTB, FIGURE 2. Cluster analysis of 25 metastatic lesions. Horizontal row, ACTA2, TMSB10, MYH7, FLNA, and ARPC4, the expres- single gene; vertical column, metastatic lesion. Red indicates a high level of expression relative to the mean; blue indicates a low level of expression sions of which were elevated in micrometastasis, coordin- relative to the mean. Highly expressed genes in lung metastases (a), in ately form lamellipodia and new adhesion sites at the kidney metastases (b), in bone metastases (c), and in liver metastases (d). leading edge of the invading cells, and move the cell The lists of the genes specifically expressed in each of the four metastasized organs (a – d) are shown in Table 2, a – d, respectively. forward by contraction of actomyosin-based cytoskeletal filaments (22). MMP1, which encodes a secreted enzyme that breaks down interstitial collagens (types I, II, and III), A number of genes associated with the cytoskeleton or was also up-regulated in the smaller lesions. On the other with cell motility were differentially expressed among the hand, none of the genes belonging to the categories four organ groups. Especially in pulmonary metastatic foci, documented above were highly expressed in the larger actin isoforms and related genes such as RHOC, ARPC4, lesions. Enhanced expression of these genes in the smaller PFN1, and PTK9L were expressed more strongly than in lesions might reflect active cellular movement and invasion metastatic lesions of the other three organs. RHOC is a of cancer cells in micrometastasis. Because the differential member of the Ras-related GTP-binding protein family and expression of 105 genes between the two groups might regulates reorganization of the actin cytoskeleton; its reflect differences in the biological features of these tumors, enhanced expression has been associated with pulmonary further investigations of nearly half of the genes of metastasis of melanoma cells (21). ARPC4 and PFN1 are unknown functions listed here should provide important implicated in directional movement of cells by promoting insights into the progression from micrometastasis to actin polymerization and controlling formation of filopodia macrometastasis. and lamellipodia (22). On the other hand, PTK9L is In summary, we identified dozens of molecules that might associated with actin depolymerization. Therefore, up-regulation be associated with organ-preferential metastasis or with tumor of these genes might reflect active cellular movement of progression from micrometastasis to macrometastasis. Our cancer cells. Because cellular movement is essential for results support the notion that metastasis is a complicated, migration and invasion of cancer cells, genes involved in the multistep phenomenon and that each step requires several key cellular cytoskeleton and motility may well contribute to molecules. Further analysis using clinical materials might help metastasis. In addition, a number of genes having various to clarify the mechanism of organ-specific metastasis and to functions such as remodeling of the ECM, or participating in further define the genes of importance. This should eventually immune responses or signal transduction, were differentially lead to molecular target-based chemotherapy and prevention expressed in each metastatic site. of metastasis.

Table 3. Genes Predominantly Expressed in Micrometastasis and Macrometastasis

Symbol Description P value Ratio

a. Micrometastasis Cell adhesion GP110 Adhesion regulating molecule 1 < 0.001 2.00 SDC1 syndecan 1 < 0.01 2.30 STEAP six transmembrane epithelial antigen of the prostate < 0.01 2.03 CEACAM4 carcinoembryonic antigen-related cell adhesion molecule 4 < 0.05 2.25 PCDHGC3 protocadherin g subfamily C, 3 < 0.05 2.01 Cytoskeleton/cell motility HSPB3 heat shock Mr 27,000 protein 3 < 0.001 2.04 ARPC4 actin related protein 2/3 complex, subunit 4 (M r 20,000) < 0.001 2.03 ACTA2 actin, a2, smooth muscle, aorta < 0.01 3.08 ACTB actin, h < 0.01 2.42 MYH7 myosin, heavy polypeptide 7, cardiac muscle, h < 0.01 2.34 TMSB10 thymosin, h10 < 0.05 2.04 ECM remodeling MMP1 matrix metalloproteinase 1 (interstitial collagenase) < 0.05 2.55 HTF9C HpaII tiny fragments locus 9C (collagen type iii) < 0.05 2.43 Cell-cell signaling (cytokine/chemokine) FGF19 fibroblast growth factor 19 < 0.001 2.03 SCYB13 small inducible cytokine B subfamily, member 13 < 0.05 2.05 EGFR epidermal growth factor receptor < 0.05 5.79 Signal transduction NR1I3 nuclear receptor subfamily 1, group I, member 3 < 0.001 2.14 FSTL1 follistatin-like 1 < 0.001 2.03 SHC1 SHC (Src homology 2 domain-containing) transforming protein 1 < 0.01 2.04 IFITM1 interferon induced transmembrane protein 1 (9 – 27) < 0.05 2.76 Immune response MD-2 MD-2 protein < 0.001 2.00 IFITM2 interferon induced transmembrane protein 2 (1-8D) < 0.01 2.56 IGKC immunoglobulin n constant < 0.05 2.27 DC class II histocompatibility antigen a-chain < 0.05 3.45 Metabolism MAN1B1 mannosidase, a, class 1B, member 1 < 0.001 2.23 FBP2 fructose, 6-bisphosphatase 2 < 0.001 2.29 NUCB1 nucleobindin 1 < 0.01 2.39 PMM2 phosphomannomutase 2 < 0.05 2.09 Cell cycle/apoptosis/DNA repair Transcription EEF1E1 eukaryotic translation elongation factor 1 q1 < 0.001 2.00 NFX1 nuclear transcription factor, X-box binding 1 < 0.05 2.07 Protein synthesis/processing HUGT1 UDP-glucose:glycoprotein glucosyltransferase 1 < 0.05 2.14 PPP1CA protein phosphatase 1, catalytic subunit, a isoform < 0.05 2.45 Neurogenesis THY1 Thy cell surface antigen < 0.001 2.84 The others RAB32 RAB32, member RAS oncogene family < 0.001 2.00 GBF1 golgi-specific brefeldin A resistance factor 1 < 0.001 2.66 ATP1B1 ATPase, Na+/K+ transporting, h1 polypeptide < 0.01 4.31 FRZB frizzled-related protein < 0.05 6.47 KIAA1011 synaptic nuclei expressed gene 2 < 0.05 2.00 COPE coatomer protein complex, subunit q < 0.05 2.04 Unknown EST < 0.001 2.19 Homo sapiens mRNA for FLJ00116 protein, partial cds < 0.001 2.39 EST < 0.001 2.14 EST < 0.001 2.19 EST < 0.001 2.14 EST < 0.001 2.26 EST < 0.001 2.02 Homo sapiens cDNA FLJ13945 fis, clone Y79AA1000969 < 0.01 2.22 FLJ10846 hypothetical protein FLJ10846 < 0.01 2.50 LOC51690 U6 snRNA-associated Sm-like protein LSm7 < 0.01 2.10 EST < 0.01 2.07 EST00098 hypothetical protein EST00098 < 0.01 2.06 RGC32 RGC32 protein < 0.01 2.61 Homo sapiens cDNA FLJ12595 fis, clone NT2RM4001344 < 0.01 2.36 Homo sapiens mRNA; cDNA DKFZp586F1323 < 0.01 2.47 EST < 0.05 2.17 LOC55895 Mr 22,000 peroxisomal membrane protein-like < 0.05 2.30 Homo sapiens cDNA: FLJ21880 fis, clone HEP02743 < 0.05 2.67 EST < 0.05 2.16

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Table 3. (continued)

Symbol Description P value Ratio

HSPC157 HSPC157 protein < 0.05 3.24 EST < 0.05 2.06 EST < 0.05 2.25 EST < 0.05 2.40 EST < 0.05 2.09 EST < 0.05 2.03 EST < 0.05 2.57 EST < 0.05 3.55 Homo sapiens cDNA FLJ12425 fis, clone MAMMA1003104 < 0.05 2.32 EST < 0.05 2.41 b. Macrometastasis Cell adhesion Cytoskeleton/cell motility ECM remodeling Cell-cell signaling (cytokine/chemokine) PDGFRA platelet-derived growth factor receptor, a polypeptide < 0.05 2.08 TGFBR2 transforming growth factor, h receptor II (M r 70,000 – 80,000) < 0.05 2.72 Signal transduction RHEB2 Ras homologue enriched in brain 2 < 0.001 2.01 RalGPS1A Ral guanine nucleotide exchange factor RalGPS1A < 0.01 2.42 AKAP9 A kinase (PRKA) anchor protein (yotiao) 9 < 0.05 2.20 RAB2L RAB2, member RAS oncogene family-like < 0.05 2.02 GNAS1 G protein, a stimulating activity polypeptide 1 < 0.05 2.22 CD47 CD47 antigen (Rh-related antigen, integrin-associated signal transducer) < 0.05 2.31 Immune response Metabolism LDHA lactate dehydrogenase A < 0.01 2.51 Cell cycle/apoptosis/DNA repair BNIP3L BCL2/adenovirus E1B M r 19,000-interacting protein 3-like < 0.01 3.15 Transcription BTF3 basic transcription factor 3 < 0.01 2.22 ZNF277 zinc finger protein 277 < 0.05 2.34 SMARCE1 SWI/SNF related, matrix associated, actin-dependent regulator of chromatin, subfamily e, member 1 < 0.05 2.13 Protein synthesis/processing SMT3H1 SMT3 (suppressor of mif two 3, yeast) homologue 1 < 0.001 2.19 HUGT1 UDP-glucose:glycoprotein glucosyltransferase 1 < 0.05 2.41 Neurogenesis SYNGR3 synaptogyrin 3 < 0.001 2.01 The others NARF nuclear prelamin A recognition factor < 0.01 2.08 GMPS guanine monophosphate synthetase < 0.05 2.22 DNMT2 DNA (cytosine-5-)-methyltransferase 2 < 0.05 2.83 Unknown ESTs < 0.001 2.07 ESTs, highly similar to I38945 melanoma ubiquitous mutated protein < 0.001 2.18 Homo sapiens mRNA; cDNA DKFZp761K2024 < 0.001 2.60 ESTs < 0.001 2.09 PTD004 hypothetical protein < 0.01 2.48 KIAA1265 KIAA1265 protein < 0.01 2.33 KIAA0071 KIAA0071 protein < 0.01 2.18 CHAC Chorea acanthocytosis < 0.05 2.00 pseudo-keratin K16 type I < 0.05 2.13 KIAA1250 likely homologue of rat kinase D-interacting substance of M r 220,000 < 0.05 3.04 FLJ10120 hypothetical protein FLJ10120 < 0.05 2.11 Homo sapiens cDNA FLJ10533 fis, clone NT2RP2001056 < 0.05 2.11 SENP2 sentrin-specific protease < 0.05 2.07 MUL Mulibrey nanism < 0.05 2.21 ESTs < 0.05 2.12 ESTs < 0.05 2.34 FLJ13433 hypothetical protein FLJ13433 < 0.05 2.08 Homo sapiens cDNA FLJ14041 fis, clone HEMBA1005780 < 0.05 3.00

Note: P value: P value of random permutation test; ratio: ratio of median values between two groups of random permutation test (see ‘‘Materials and Methods’’)

Materials and Methods Reagent Cell Line Anti-mouse interleukin 2 receptor h-chain monoclonal Human SCLC cell line SBC-5 was kindly provided by Dr. K. antibody, TM-h1 (IgG2b), was supplied by Drs. M. Miyasaka Hiraki (Okayama University, Okayama, Japan). The SBC-5 cells and T. Tanaka (Osaka University, Osaka, Japan) (26). None of were maintained in Eagle’s MEM supplemented with 10% fetal this material contained endotoxins, as judged by the Limulus bovine serum (Life Technologies, Inc., Grand Island, NY), amoebocyte assay (Seikagaku Kogyo, Tokyo, Japan; minimum gentamycin (Schering-Plough, Osaka, Japan), and 4 mM HEPES. detection level 0.1 ng/ml).

Animals Cross-Hybridization of Mouse Messenger RNA Male SEB-17/Icr-scid mice, 6–8 weeks old, were obtained To assess the influence of contamination of normal mouse from Charles River Laboratories (Yokohama, Japan) and mRNA, we microdissected normal mouse cells in individual maintained under specific pathogen-free conditions throughout organs and hybridized on the human cDNA microarrays by the the experiment. Experiments were performed following the same method as described above. ethical guidelines of our university. Cluster Analysis of Gene-Expression Profiles Experimental Metastasis of SBC-5 in Mice Lacking To identify genes that were expressed differently among the NK Cells four types of metastatic tissue, we applied random-permutation To facilitate the metastasis of human SBC-5, NK cells were tests to estimate the ability of each gene to distinguish between depleted in SCID mice by i.p. injection of TM-h1Ab (1 mg/1 ml two groups (each organ-specific metastasis versus the mixture in PBS/mouse) 2 days before inoculation of tumor cells (12). The of metastases in the other three organs). The comparative tumor cells were first harvested and washed with Ca2+- and combinations were as follows: (a) 10 lung metastases versus 15 Mg2+-free PBS; cell viability was determined by the trypan blue metastases in three other organs; (b) 5 liver metastases versus exclusion test, and only cell suspensions showing >90% viability 20 others; (c) 5 kidney metastases versus 20 others; and (d)5 were used. We injected 0.3 ml of tumor-cell suspension (1–5 bone metastases versus 20 others. Mean (l) and standard (r) 106 cells) into the lateral tail vein of non-anesthetized mice. The deviations were calculated from the log-transformed relative mice were sacrificed 35 days after inoculation, and four organs expression ratios of each gene in both groups. A discrimination (lung, liver, kidney, and bone tissues) containing macroscopic score (DS) for each gene was defined as follows: lesions were excised, embedded in TissueTek OCT medium (Sakura, Tokyo, Japan), and snap frozen at 80jC. DS ¼ðl1 l2Þ=ðr1 þ r2Þ

Laser-Capture Microdissection The samples were randomly permutated 10,000 times for We prepared 8-Am-thick frozen sections, which were fixed each pair of groups. Because the DS data set of each gene in 70% ethanol for 30 s, stained with H&E, and dehydrated first showed a normal distribution, we calculated a P value for the with 99.5% ethanol for 5 min and then with xylene for 1 min. user-defined grouping (29). The stained tissues were observed microscopically; 25 A hierarchical clustering analysis was applied to the 25 metastatic lesions (10 lung, 5 liver, 5 kidney, and 5 bone metastatic loci and the 435 genes extracted by the random tumors) were selected for laser-capture microdissection with a permutation tests, using Web-available software (‘‘Cluster’’ and PixCell II LCM system, according to the manufacturer’s ‘‘TreeView’’) written by M. Eisen. (http://genome-www5. protocols (Arcturus Engineering, Mountain View, CA). stanford.edu/MicroArray/SMD/restech.html).

RNA Extraction and T7-Based RNA Amplification Identification of Genes Differentially Expressed Between Total RNA was extracted from each captured cancer tissue Micrometastasis and Macrometastasis using the RNeasy mini kit (Qiagen, Valencia, CA) and RNase- To compare gene-expression profiles between small and free DNase according to the manufacturer’s protocols. Total large metastatic foci, we also applied a random permutation test RNAs extracted from each of the 25 metastatic lesions were to 9 of the 10 lesions in lung: 5 that were <1 mm (average size: subjected to T7-based RNA amplification, as described 0.60 mm, SD: 0.22); and 4 that were >2 mm (average: 2.40 mm, previously (27). Two rounds of amplification yielded 40–200 SD: 0.32) (29). To determine the size of the tumors, we first A Ag of aRNA (over 100,000-fold) from each sample. Aliquots sliced each lesion from the top to the bottom (8 m thick) and (2.5 Ag) of a RNA from individual lesions (test probes) and measured the maximum axis of the largest tumor section. The from mixture of aRNAs from all 25 lesions (a control probe) Union Internationale Centre le Cancer advocates the use of the were labeled respectively with Cy5-dCTP or Cy3-dCTP. term micrometastasis to denote in humans a metastatic lesion smaller than or equal to 2 mm in diameter (30, 31). As there is no definition of this term for the mouse, we more strictly defined cDNA Microarrays micrometastasis as a lesion smaller than 1 mm in diameter. Our ‘‘genome-wide’’ cDNA microarray system contains 23,040 cDNAs selected from the UniGene database of the National Center for Biotechnology Information (28). Fabrica- References 1. Chambers, A. F., Groom, A. C., and MacDonald, I. C. Dissemination and tion of the microarray, hybridization, washing, and detection of growth of cancer cells in metastatic sites. Nat. Rev. Cancer, 2: 563 – 572, 2002. signal intensities were described previously (27). To normalize 2. Cameron, M. D., Schmidt, E. E., Kerkvliet, N., Nadkarni, K. V., Morris, V. L., the amount of mRNA between tumors and controls, the Cy5/ Groom, A. C., Chambers, A. F., and MacDonald, I. C. Temporal progression of Cy3 ratio for each gene’s expression was adjusted so that the metastasis in lung: cell survival, dormancy, and location dependence of metastatic inefficiency. Cancer Res., 60: 2541 – 2546, 2000. averaged Cy5/Cy3 ratio of 52 housekeeping genes was equal to 3. Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., and 1. We assigned a cut-off value to each microarray slide, using a Muschel, R. J. Intravascular origin of metastasis from the proliferation of variance analysis. Genes, the Cy3 or Cy5 signal intensities of endothelium-attached tumor cells: a new model for metastasis. Nat. Med., 6: which were lower than the cut-off values, were excluded from 100 – 102, 2000. 4. Fidler, I. J., Yano, S., Zhang, R. D., Fujimaki, T., and Bucana, C. D. The further investigation. We also excluded data from genes where seed and soil hypothesis: vascularisation and brain metastases. Lancet Oncol., 3: the signal/noise ratio was <3. 53 – 57, 2002.

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Soji Kakiuchi, Yataro Daigo, Tatsuhiko Tsunoda, et al.

Mol Cancer Res 2003;1:485-499.

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