genome-wide analysis of organ-preferential metastasis of ... · lung metastases versus all 15...

Genome-Wide Analysis of Organ-Preferential Metastasis ofHuman 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 TokushimaSchool of Medicine, Tokushima, Japan

AbstractAlthough a number of molecules have been implicated in

the process of cancer metastasis, the organ-selective

nature of cancer cells is still poorly understood. To

investigate this issue, we established a metastasis model

in mice with multiple organ dissemination by i.v. injection

of human small cell lung cancer (SBC-5) cells. We

analyzed gene-expression profiles of 25 metastatic

lesions from four organs (lung, liver, kidney, and bone)

using a cDNA microarray representing 23,040 genes and

extracted 435 genes that seemed to reflect the organ

specificity of the metastatic cells and the cross-talk

between cancer cells and microenvironment. Further-

more, we discovered 105 genes that might be involved in

the incipient stage of secondary-tumor formation by

comparing the gene-expression profiles of metastatic

lesions classified according to size (<1 or >2 mm) as

either ‘‘micrometastases’’ or ‘‘macrometastases.’’ This

genome-wide analysis should contribute to a greater

understanding of molecular aspects of the metastatic

process in different microenvironments, and provide

indicators for new strategies to predict and prevent

metastasis.

IntroductionMetastasis is the major cause of death due to cancer. Because

no absolutely effective methods for curing metastatic tumors are

available at present, novel strategies for prevention of metastasis

are urgently needed to improve the prognosis and quality of life

for cancer patients. Metastases occur in sequential steps that

include invasion of cancer cells from the primary site to blood

vessels or the lymphatic system, survival in the circulation,

intravascular transfer to distant organs, attachment to endothe-

lial cells, extravasation into the parenchyma, and outgrowth into

a secondary tumor with neovascularization (1–4).

Molecular interactions between cancer cells and their

microenvironment(s) play important roles throughout the

multiple steps of metastasis (5). Blood flow and other

environmental factors influence the dissemination of cancer

cells to specific organs (6). However, the organ specificity of

metastasis (i.e. , some organs preferentially permit migration,

invasion, and growth of specific cancer cells, but others do not)

is a crucial determinant of metastatic outcome, and proteins

involved in the metastatic process are considered to be

promising therapeutic targets.

More than a century ago, Stephen Paget suggested that the

distribution of metastases was not determined by chance, but

rather that certain tumor cells (‘‘seed’’) are likely to have an

affinity for the microenvironment of specific organs (‘‘soil’’)

and that metastases occur only when the seed and soil are

compatible (7). Various molecules such as adhesion molecules,

cytokines, chemokines, hormones, and hormone receptors play

important roles in preferential metastasis (1, 8–10), but the

precise mechanisms determining seed and soil compatibility

remain unsolved.

To examine the cellular and molecular bases of organ-

specific metastasis, we have established models of metastasis to

multiple organs by i.v. injection of eight different human lung

cancer cell lines to severe combined immune deficiency (SCID)

mice devoid of natural killer (NK) cells (11, 12). In the work

reported here, by means of a cDNA microarray consisting of

23,040 genes, we analyzed the gene-expression profiles of 25

metastatic lesions present in murine lung, liver, kidney, and bone

following i.v. injection of human small cell lung cancer (SCLC)

(SBC-5) cells. In the process, we identified candidate genes that

may affect or determine organ specificity of the metastatic cells,

as well as genes involved in progression frommicrometastasis to

macrometastasis. Genes in both categories represent potential

molecular targets for prevention of metastasis in humans.

ResultsMetastasis of Human SCLC Cell Line SBC-5 inNK Cell-Depleted SCID Mice

As we reported previously, i.v. injection of SBC-5 cells into

SCID mice lacking NK cells caused metastases to multiple

organs (11, 13). To compare gene-expression profiles of 25

selected metastatic lesions (10 in lung, 5 in liver, 5 in kidney, and

5 in bone), we collected pure populations of cancer cells by

laser-capture microdissection. Histopathological features of

each of the 25 lesions are shown in Fig. 1, A–D, and in

Table 1. Three of the 10 lung foci developed in subpleura, and 5

were accompanied by intravascular embolization. The distribu-

Received 11/12/02; revised 03/17/03; accepted 04/04/03.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Grant support: ‘‘Research for the Future’’ Program Grant of The Japan Societyfor the Promotion of Science (no. 00L01402) to Y.N.Requests for reprints: Yusuke Nakamura, Laboratory of Molecular Medicine,Human Genome Center, Institute of Medical Science, The University of Tokyo,4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81-3-5449-5372;Fax: 81-3-5449-5433. E-mail: [email protected] D 2003 American Association for Cancer Research.

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

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tion of metastases in lung was similar to that found in another

experimental model of metastasis published elsewhere (2). All

bone metastases were accompanied by the osteolytic changes

commonly observed in human SCLCs (11). As in human cases,

metastases in murine kidney were smaller than those arising in

other organs.

Cross-Hybridization of Mouse Messenger RNAAs the target-DNAs on our cDNA microarray mainly include

the 3Vuntranslated region of the human gene that is more specific

to individual genes and/or species, the incidence of cross-

hybridization between human and murine sequences was

supposed to be very low. Moreover, laser-capture micro-

dissection has surely reduced the contamination of normal

mouse cells. To assess the influence of contamination of normal

mouse mRNA and to consequently remove any experimental

noises in the statistical analysis, we performed laser-capture

microdissection of surrounding mouse normal tissues and

hybridized on the human cDNA microarrays. 2.56–3.07%

(590–707/23,040) of genes in each organ had the intensities

above cut-off value and these genes were excluded from the

further analysis.

Cluster Analysis of Gene-Expression Profiles of the 25Metastatic Lesions

To identify genes that were specifically expressed in each of

the four metastasized organs, we performed random permuta-

tion tests; this is an appropriate strategy for distinguishing two

known subgroups. We used the following combinations: 10

lung metastases versus all 15 others; 5 liver metastases versus

all 20 others; 5 kidney metastases versus all 20 others; and 5

bone metastases versus all 20 others. Table 2 lists 435 genes,

the median ratios of which between the two groups were >2

with P values <0.05, among the 23,040 genes examined on the

microarray. Hierarchical clustering of these 435 genes separated

the four organ-specific groups of metastatic lesions very clearly

(Fig. 2).

Genes Differentially Expressed Between‘‘Micrometastasis’’ and ‘‘Macrometastasis’’

Metastasis models in animals have a great advantage over the

clinical samples for examining the incipient stage of micro-

metastases (1, 2). The in vivo model used here also allowed us to

observe early events of the metastasis-developing process in

lung, i.e., arrest in the pulmonary artery (Fig. 1E) and

extravasation into lung tissue (Fig. 1F). Because searching for

genes differentially expressed between micrometastasis and

macrometastasis could be useful for understanding the biolog-

ical nature of secondary-tumor formation at the metastatic sites,

we applied a random permutation test to nine metastatic lesions

in the lung (five lesions were <1 mm and four were >2 mm) and

extracted 105 differentially expressed genes. Sixty-eight of the

genes were predominantly expressed in the smaller lesions, and

37 were predominant in the larger lesions (Table 3).

FIGURE 1. Histopathology of metastaticlesions (H&E stain). a. Lung (�100). b. Liver(�100). c. Kidney (�100). d. Bone (�40). e.Cancer cells arrested in the pulmonary artery notby the size restriction but by adhesive interactions(�100). f. Proliferation of multicellular tumoremboli in the pulmonary artery, initial minimalpenetration of cancer cells through the arteriolarwalls (arrow ), and invasion of the cancer cells tothe lung parenchyma (�100). A, artery; Al,alveolus; CV, central vein; Gl, glomerulus; T,renal tubule; B, bone; BM, basement membrane.

Genome-Wide Analysis of SCLC Metastasis486



DiscussionA considerable body of evidence supports the concept that

organ specificity of metastasis is influenced not only by seed

and soil compatibility but also by physiological factors such

as patterns of blood flow and the size of cancer cells relative

to the diameter of capillaries (1, 2, 6). Considering the

sequential multisteps of metastasis, organ selectivity of

metastasis seems to be determined after extravasation of

cancer cells from primary site. Therefore, our experimental

metastasis model was likely to reflect the later steps. Actually,

although cancer cells were injected intravenously, some cells

formed metastatic nodules not in the lungs, the first capillary

beds encountered, but in other organs (11). The distribution of

tumor cells in the mice reproduced very well the distribution

patterns of human metastatic lung cancers (in humans, e.g. ,

SCLC cells can form metastases in multiple organs, mainly

systemic lymph nodes and liver, whereas lung adenocarcino-

mas produce metastatic foci mainly in the lungs). This

evidence suggests that seed and soil compatibility does

contribute to the directional migration and invasion of tumor

cells into specific organs, and also confirms that our model is

valuable for investigation of the relevant mechanism(s)

including the tumor-host interaction in microenvironment.

Several hypotheses concerning the organ specificity of

metastasis have been proposed (3, 8, 10, 14, 15). They

include: (a) tumor cells that extravasate into secondary sites

can survive and proliferate only in those organs that

have appropriate growth factors; (b) chemoattractants home

the cancer cells toward specific organs by means of

concentration gradients; and/or (c) secondary tumors can

develop in certain organs, the endothelial cells of which

express adhesion molecules that can attach the cancer cells.

Each of these hypotheses suggests that interaction between

cancer cells and microenvironment influences the direction of

metastatic organs.

For the elucidation of the cross-talk between cancer cells

and microenvironment in each organ and a comprehensive

survey of the factors regulating organ-specific metastasis, we

performed cDNA-microarray analyses of the metastatic foci of

human SCLC (SBC-5) developed in four different murine

organs (lung, liver, kidney, and bone) and compared gene-

expression profiles among 25 of these lesions. The expression

patterns fell into four categories, each of which reflected a

specific organ. The 435 genes that distinguished these four

groups were extracted by statistical analysis and classified into

12 categories on the basis of known biological functions

(Table 2). Among them, genes belonging to the cell-cell

signaling category included growth-factor receptors, cytokines,

and chemokines. FGFR1 and FST were highly expressed in

cells metastasized in bone. FGFR1 is a receptor for fibroblast

growth factors (FGFs) and its downstream signals influence

mitogenesis and differentiation. Because FGFs are expressed

abundantly in bone tissue (16), the microenvironment of bone

is likely to be suitable for survival and proliferation of cancer

cells that express FGFR1 . In SCLCs, metastatic cells in bone

are predominantly osteolytic. FST, an activin antagonist that

can inhibit bone formation (17), might promote the bone

absorption caused by metastatic cells and contribute to the

release of the growth factors such as FGFs that are stored

in bone tissue. This bidirectional interaction between tumor

cells and the bone microenvironment seems to be important

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

affected organs, but the difference was not statistically

significant (P = 0.057).

Chemokines, secreted peptides that control the homing of

leukocytes, are considered to contribute to the directional

migration of cancer cells that express chemokine receptors on

their surfaces (1, 8). Three chemokine receptors, CCR4,

CCR5 , and CCR9 , were expressed in all 25 metastatic lesions

(data not shown), with no significant differences of expression

levels. In the gene-expression database of 24 normal human

adult tissues we reported recently, SCYA4 (CCL4), one of the

ligands of CCR5 , was expressed preferentially in lung, liver,

lymph nodes, bone marrow, adipose tissue, and spleen (18).

Because lung, liver, lymph nodes, and bone marrow are major

targets for metastasis of SCLC, CCL4 /CCR5 interaction

may contribute to the organ-preferential metastasis of the

SCLC cells.

Adhesion, detachment, and aggregation of tumor cells

seem to play important roles in achieving metastasis.

Although most circulating cancer cells are arrested in

capillary beds because of size restrictions (3), we observed

adherence to the walls of pre-capillary vessels that were

much larger in diameter than the cancer cells (Fig. 1E). A

number of molecules involved in cell-cell or cell-matrix

adhesion, such as integrins and selectins, appear to mediate

Table 1. Size and Histopathological Features of 25Metastatic Lesions

Metastatic Organ ID The MajorAxis (mm)

HistopathologicalFeature

Lung 1 2.51 subpleura2 2.20 subpleura3 0.38 with embolization4 0.655 0.42 with embolization6 2.007 0.92 with embolization8 1.30 subpleura9 2.80 with embolization

10 0.62 with embolizationLiver 1 3.80

2 2.703 4.004 1.825 2.01

Kidney 1 0.622 0.263 1.484 0.495 0.22

Bone 1 >4.00 osteolytic2 >4.00 osteolytic3 >4.00 osteolytic4 >4.00 osteolytic5 >4.00 osteolytic

Molecular Cancer Research 487



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. LungCell adhesionLGALS1 lectin, galactoside-binding, soluble, 1 (galectin 1) <0.001 5.36PCDHGC3 protocadherin g subfamily C, 3 <0.001 3.41ITGB4 integrin, h4 <0.001 2.74LGALS3BP lectin, galactoside-binding, soluble, 3 binding protein <0.01 2.28SDC1 syndecan 1 <0.01 3.56GJB2 gap junction protein, h2, M r 26,000 (connexin 26) <0.05 2.11Cytoskeleton/cell motilityTUBB2 tubulin, h, 2 < 0.001 2.92FLNA filamin A, a (actin-binding protein-280) <0.001 2.46RHOC ras homologue gene family, member C <0.001 3.02ACTC actin, a, cardiac muscle <0.001 4.84ACTA1 actin, a1, skeletal muscle <0.001 5.40ACTA2 actin, a2, smooth muscle, aorta <0.001 2.96ACTB actin, h <0.001 4.12ACTG2 actin, g2, smooth muscle, enteric <0.001 5.49ARPC4 actin-related protein 2/3 complex, subunit 4 (M r 20,000) <0.001 4.06PTK9L protein tyrosine kinase 9-like (A6-related protein) < 0.001 2.09LMNA lamin A/C <0.001 3.97RPS29 ribosomal protein S29 <0.001 2.02PFN1 profilin 1 <0.01 2.16Extracellular matrix (ECM) remodelingHTF9C Hpa II tiny fragments locus 9C <0.001 2.87FLJ11618 hypothetical protein FLJ11618 <0.01 2.11Cell-cell signaling (cytokine/chemokine)MIF macrophage migration inhibitory factor < 0.001 2.01TNFRSF1A tumor necrosis factor receptor superfamily, member 1A <0.001 2.22SCYB13 small inducible cytokine B subfamily, member 13 <0.001 2.30DDT D-dopachrome tautomerase <0.001 2.10Signal transductionFKBP8 FK506-binding protein 8 (M r 38,000) <0.001 2.48PDAP1 PDGFA associated protein 1 <0.001 5.69ITPK1 inositol 1,3,4-triphosphate 5/6 kinase <0.001 2.21TM4SF7 transmembrane 4 superfamily member 7 <0.001 3.75IFITM1 interferon induced transmembrane protein 1 (9 –27) <0.01 5.92ILK integrin-linked kinase <0.01 2.00TRAF2 TNF receptor-associated factor 2 <0.05 2.10Immune responseC3 complement component 3 <0.001 2.58BF B-factor, properdin <0.001 2.13HLA-A major histocompatibility complex, class I, A <0.001 2.86HLA-B major histocompatibility complex, class I, B <0.001 2.77HLA-C major histocompatibility complex, class I, C <0.001 2.16HLA-DQA1 major histocompatibility complex, class II, DQ a1 <0.001 19.77HLA-DQB1 major histocompatibility complex, class II, DQ h1 <0.001 5.08PSME2 proteasome (prosome, macropain) activator subunit 2 <0.001 2.64IFITM2 interferon induced transmembrane protein 2 (1-8D) <0.001 2.82MetabolismCOX6B cytochrome c oxidase subunit VIb <0.001 2.64COX8 cytochrome c oxidase subunit VIII < 0.001 2.84COX7A2 cytochrome c oxidase subunit VIIa polypeptide 2 (liver) < 0.001 2.16COX5B cytochrome c oxidase subunit Vb <0.001 2.18GPX1 glutathione peroxidase 1 <0.001 4.99GPX4 glutathione peroxidase 4 (phospholipid hydroperoxidase) < 0.001 2.67MT2A metallothionein 2A <0.001 2.09APOC1 apolipoprotein C-I <0.001 6.44FDXR ferredoxin reductase <0.001 2.58HSD11B2 hydroxysteroid (11-h) dehydrogenase 2 <0.001 2.63ALAS1 aminolevulinate, y-, synthase 1 <0.001 2.25Cell cycle/apoptosis/DNA repairPPP1CA protein phosphatase 1, catalytic subunit, a isoform <0.001 2.90ERH enhancer of rudimentary (Drosophila ) homologue <0.001 2.09PCBP4 poly(rC)-binding protein 4 <0.01 2.46CDC20 CDC20 (cell division cycle 20, Saccharomyces cerevisiae , homologue) <0.01 2.19SFN stratifin <0.01 2.40NOL3 nucleolar protein 3 (apoptosis repressor with CARD domain) <0.01 2.64TranscriptionNFKBIA NF-nB inhibitor < 0.001 2.15DRAP1 DR1-associated protein 1 (negative cofactor 2 a) < 0.001 2.55GATA2 GATA-binding protein 2 <0.001 2.92MBD2 methyl-CpG binding domain protein 2 <0.001 3.25

(continued on next page)





Protein synthesis/processingFAU FBR-MuSV ubiquitously expressed (fox derived) <0.001 2.74RPS10 ribosomal protein S10 <0.001 2.12RPLP2 ribosomal protein, large P2 <0.001 2.01RPL18 ribosomal protein L18 <0.001 2.05MRPL23 mitochondrial ribosomal protein L23 <0.001 3.15PSMB8 proteasome (prosome, macropain) subunit, h type, 8 <0.001 3.22RPS26 ribosomal protein S26 <0.001 3.12PMM2 phosphomannomutase 2 <0.001 2.06FBXO2 F-box only protein 2 <0.001 2.22EEF1D eukaryotic translation elongation factor 1 y <0.001 2.24NeurogenesisThe othersCALM3 calmodulin 3 (phosphorylase kinase, y) < 0.001 2.25CALM1 calmodulin 1 (phosphorylase kinase, y) < 0.001 2.27Unknown

EST <0.001 2.11EST <0.001 4.01EST <0.001 2.05EST <0.001 21.33

BCL7C B-cell CLL/lymphoma 7C <0.001 2.67EST <0.001 3.10

PTD008 PTD008 protein <0.001 2.18EST <0.001 2.72Homo sapiens cDNA: FLJ22175 fis, clone HRC00773 <0.001 2.22EST <0.001 2.08

LOC51181 carbonyl reductase <0.001 2.20EST <0.001 2.18

EST00098 hypothetical protein EST00098 <0.001 2.48EST <0.001 2.65

CGI-96 CGI-96 protein <0.001 2.44EST <0.001 2.00EST <0.001 2.03

HRB2 HIV rev binding protein 2 <0.001 2.19EST <0.001 2.42

SELENBP1 selenium binding protein 1 <0.001 6.99EST <0.001 2.48EST <0.01 2.07EST <0.01 2.13

FLJ10829 hypothetical protein FLJ10829 <0.01 2.26EST <0.05 2.35

b. KidneyCell adhesionLGALS9 lectin, galactoside-binding, soluble, 9 (galectin 9) <0.001 2.50ENTPD2 ectonucleoside triphosphate diphosphohydrolase 2 <0.01 2.79CLDN17 claudin 17 <0.05 2.62Cytoskeleton/cell motilityACTR1A ARP1 (actin-related protein 1, yeast) homologue A <0.001 2.77DKFZP586N1922 DKFZP586N1922 protein <0.001 2.09CYLN2 cytoplasmic linker 2 <0.001 2.07EPLIN epithelial protein lost in neoplasm h <0.001 3.43CCT-7 HIV-1 Nef interacting protein <0.01 2.35CNN3 calponin 3, acidic <0.05 3.84ECM remodelingCOL1A1 collagen, type I, a1 <0.01 2.51Cell-cell signaling (cytokine/chemokine)BMP6 bone morphogenetic protein 6 <0.001 2.79INHBA inhibin, hA (activin A, activin AB a polypeptide) < 0.001 2.35Signal transductionDUSP10 dual specificity phosphatase 10 <0.001 2.09PRSS11 protease, serine, 11 (IGF binding) <0.01 2.66Immune responseHLA-DMA major histocompatibility complex, class II, DM a <0.01 2.48TRB@ T cell receptor h locus <0.01 8.66C1S complement component 1, s subcomponent <0.05 2.17MetabolismGCK glucokinase (hexokinase 4, maturity onset diabetes of the young 2) <0.001 2.52GPX3 glutathione peroxidase 3 (plasma) <0.001 11.30HMGCL 3-hydroxymethyl-3-methylglutaryl-Coenzyme A lyase <0.01 3.45Cell cycle/apoptosis/DNA repair

Septin 6 <0.001 2.83


Table 2. (continued )





TranscriptionP84 nuclear matrix protein p84 <0.001 2.27NSAP1 NS1-associated protein 1 <0.001 2.17ZNF258 Zinc finger protein 258 <0.001 2.12GCN5L2 GCN5-like 2 <0.01 2.98HSPC157 HSPC157 protein <0.01 2.56RNASE6PL ribonuclease 6 precursor <0.01 2.30Protein synthesis/processingHUGT1 UDP-glucose:glycoprotein glucosyltransferase 1 <0.001 2.10UBE2N ubiquitin-conjugating enzyme E2N <0.001 2.24SCAMP2 secretory carrier membrane protein 2 <0.01 2.35NeurogenesisDCTN1 Dynactin 1 (p150, glued homologue, Drosophila ) < 0.001 2.14ITM2B integral membrane protein 2B <0.01 2.21EFNB3 ephrin-B3 <0.01 2.49The othersATP1B1 ATPase, Na+/K+ transporting, h1 polypeptide <0.001 3.25SRI sorcin <0.001 3.44H1F2 H1 histone family, member 2 <0.001 2.15SUT1 sulfate transporter 1 <0.05 2.15Unknown

Homo sapiens cDNA FLJ11838 fis, clone HEMBA1006624 <0.001 2.21EST <0.001 2.51EST <0.001 2.04

ISYNA1 myo -inositol 1-phosphate synthase A1 <0.001 2.25Homo sapiens cDNA FLJ13549 fis, clone PLACE1007097 <0.001 2.01EST <0.001 2.11EST <0.001 2.09

KIAA0709 endocytic receptor (macrophage mannose receptor family) < 0.001 2.30LOC51243 hypothetical protein <0.001 3.08FJX1 putative secreted ligand homologous to fjx1 <0.001 2.29

EST <0.001 2.42KIAA0442 KIAA0442 protein <0.001 2.42FLJ10769 hypothetical protein FLJ10769 <0.001 2.01

EST <0.001 2.17EST <0.001 2.12

ATP5H ATP synthase, H+ transporting, mitochondrial F1F0, subunit d <0.001 2.02LOC55902 acetyl-CoA synthetase <0.001 2.20

EST <0.01 2.07EST <0.01 2.84

LOC51064 glutathione S-transferase subunit 13 homologue <0.01 3.52Homo sapiens mRNA; cDNA DKFZp586K2123 <0.01 2.29

NBEA neurobeachin <0.01 2.05EST <0.01 2.28EST <0.01 2.03

FLJ10846 hypothetical protein FLJ10846 <0.01 2.17EST <0.01 2.04

BRD7 bromodomain-containing 7 <0.01 2.07D2S448 Melanoma associated gene <0.05 2.23

EST <0.05 2.12EST <0.05 2.69Homo sapiens cDNA FLJ20144 fis, clone COL07809 <0.05 2.20EST <0.05 2.02

c. BoneCell adhesionCELSR1 cadherin, EGF LAG seven-pass G-type receptor 1 <0.001 3.31PLXNC1 plexin C1 <0.001 2.71NEO1 neogenin (chicken) homologue 1 <0.001 2.06PTPRM protein tyrosine phosphatase, receptor type, M <0.001 2.67Cytoskeleton/cell motilityKIAA0855 golgin-67 <0.001 3.48MESDC1 Mesoderm development candidate 1 <0.01 2.58LIMK2 LIM domain kinase 2 <0.01 2.38ECM remodelingKERA keratocan <0.001 2.05COL3A1 collagen, type III, a1 <0.01 3.29Cell-cell signaling (cytokine/chemokine)FST follistatin <0.001 3.97MST1 macrophage stimulating 1 (hepatocyte growth factor-like) < 0.001 2.30FAP fibroblast activation protein, a <0.001 2.01FGFR1 fibroblast growth factor receptor 1 <0.001 2.07







Signal transductionPRP4 serine/threonine-protein kinase PRP4 homologue <0.001 2.13PPP3CC protein phosphatase 3 (formerly 2B) <0.001 2.12PTPN1 protein tyrosine phosphatase, non-receptor type 1 <0.001 2.12AKT2 v-akt murine thymoma viral oncogene homologue 2 <0.001 2.14CAV1 caveolin 1, caveolae protein, M r 22,000 <0.001 2.79PRKAR1A tissue-specific extinguisher 1 <0.05 2.01Immune responseMetabolismCell cycle/apoptosis/DNA repairTIA1 TIA1 cytotoxic granule-associated RNA-binding protein <0.001 2.25PRKDC protein kinase, DNA-activated, catalytic polypeptide <0.001 2.62RAD51L3 RAD51 (Saccharomyces cerevisiae )-like 3 <0.001 2.38DDB1 damage-specific DNA binding protein 1 (M r 127,000) <0.001 2.16BAK1 BCL2-antagonist/killer 1 <0.01 2.16CDK3 Cyclin-dependent kinase 3 <0.01 2.00TranscriptionSFRS11 splicing factor, arginine/serine-rich 11 <0.001 2.02DKFZP434P0721 similar to mouse Xrn1/Dhm2 protein <0.001 2.23SIRT5 sir2-like 5 <0.001 2.23TCEB1L transcription elongation factor B (SIII), polypeptide 1-like <0.001 2.46EZH1 enhancer of zeste (Drosophila ) homologue 1 <0.001 2.02HSF2 heat shock transcription factor 2 <0.001 2.15EGR4 early growth response 4 <0.001 2.22HNRPU heterogeneous nuclear ribonucleoprotein U <0.01 3.33GLI3 GLI-Kruppel family member GLI3 <0.01 2.27EGR3 early growth response 3 <0.01 2.95LZTR1 leucine-zipper-like transcriptional regulator, 1 <0.01 2.09SMARCC1 SWI/SNF complex M r 155,000 subunit < 0.05 2.36Protein synthesis/processingMTIF2 mitochondrial translational initiation factor 2 <0.001 2.08MTHFD2 cyclohydrolase, NAD(+)-dependent <0.01 2.27RPL37A ribosomal protein L37a <0.01 2.04SEC63L SEC63, endoplasmic reticulum translocon component like <0.01 2.36LOC54516 similar to prokaryotic-type class I peptide chain release factors <0.01 2.13NeurogenesisDPYSL2 dihydropyrimidinase-like 2 <0.001 3.51GPM6B glycoprotein M6B <0.001 2.50SLIT2 slit (Drosophila ) homologue 2 <0.01 6.01PRPS1 phosphoribosyl pyrophosphate synthetase 1 <0.01 2.10The othersERF Ets2 repressor factor < 0.001 2.24DYT1 dystonia 1, torsion (autosomal dominant; torsin A) <0.001 2.13SLC11A2 solute carrier family 11, member 2 <0.001 2.37MDM2 mouse double minute 2, human homologue of; p53-binding protein <0.001 2.03KTN1 kinectin 1 (kinesin receptor) < 0.001 2.45POV1 prostate cancer overexpressed gene 1 <0.001 3.19STK15 serine/threonine kinase 15 <0.001 2.36INPPL1 inositol polyphosphate phosphatase-like 1 <0.001 2.02GSK3B glycogen synthase kinase 3 h <0.001 2.42KDELR3 KDEL endoplasmic reticulum protein retention receptor 3 <0.001 2.39TRF4-2 topoisomerase-related function protein 4-2 <0.001 2.37RAB2 RAB2, member RAS oncogene family <0.01 2.16KIAA0102 KIAA0102 gene product < 0.01 2.24SMT3H1 SMT3 (suppressor of mif two 3, yeast) homologue 1 <0.01 2.32ENTPD5 ectonucleoside triphosphate diphosphohydrolase 5 <0.01 2.02KIAA0939 KIAA0939 protein <0.01 5.81FUS1 lung cancer candidate <0.01 2.18LLGL2 lethal giant larvae (Drosophila ) homologue 2 <0.01 2.11MYB v-myb avian myeloblastosis viral oncogene homologue <0.01 2.03Unknown

EST <0.001 2.14EST <0.001 2.45

SEC31B-1 Secretory pathway component Sec31B-1 <0.001 3.07EST <0.001 2.04

MAC30 hypothetical protein <0.001 2.14Homo sapiens mRNA; cDNA DKFZp434C136 <0.001 2.55EST <0.001 2.42EST <0.001 3.07EST <0.001 2.83Homo sapiens cDNA: FLJ22562 fis, clone HSI01814 <0.001 2.57EST <0.001 5.12EST <0.001 2.53







KIAA0729 KIAA0729 protein <0.001 2.14EST <0.001 2.24EST <0.001 2.41EST <0.001 3.62

FLJ23399 hypothetical protein FLJ23399 <0.001 2.44KIAA0765 putative brain nuclearly targeted protein <0.001 2.42

EST <0.001 2.28EST <0.001 2.81

KIAA0088 KIAA0088 protein <0.001 2.32PRO2463 PRO2463 protein <0.001 2.02KIAA0459 KIAA0459 protein <0.001 2.19

EST <0.001 2.02KIAA1025 KIAA1025 protein <0.001 2.44AF15Q14 AF15q14 protein <0.001 2.59KIAA1238 Homo sapiens mRNA; cDNA DKFZp586I0521 <0.001 2.03KIAA1278 KIAA1278 protein <0.001 2.27FLJ20195 hypothetical protein FLJ20195 <0.001 2.04VMP1 Likely orthologue of rat vacuole membrane protein 1 <0.001 2.11

EST <0.001 2.42DKFZP761H171 hypothetical GTP-binding protein DKFZp761H171 <0.001 2.63

EST <0.001 2.46EST <0.001 2.49EST <0.001 2.75EST <0.001 2.11EST <0.001 2.20EST <0.001 2.01Homo sapiens cDNA FLJ11998 fis, clone HEMBB1001521 <0.001 2.36EST <0.001 2.27

KIAA0986 KIAA0986 protein <0.001 2.93EST <0.001 3.02EST <0.001 2.04EST <0.001 2.12EST <0.01 2.31Homo sapiens cDNA: FLJ22530 fis, clone HRC12866 <0.01 2.26EST <0.01 2.22

FLJ23293 hypothetical protein FLJ23293 <0.01 2.16EST <0.01 2.28EST <0.01 2.31EST <0.01 2.63EST <0.01 2.02EST <0.01 2.85Homo sapiens cDNA FLJ13213 fis, clone NT2RP4001126 <0.01 2.18

KIAA0105 Wilms’ tumour 1-associating protein <0.01 2.43EST <0.01 2.23EST <0.01 2.32EST <0.01 2.51

LOC51153 FT005 protein <0.01 3.14EST <0.01 2.36Homo sapiens clone FLB3024 PRO0756 mRNA, complete cds <0.01 2.17EST <0.01 2.22EST <0.01 2.76

S164 S164 protein <0.01 2.38Human clone 23589 mRNA sequence <0.01 2.15

LANO LAP (leucine-rich repeats and PDZ) and no PDZ protein <0.01 2.16Homo sapiens cDNA: FLJ23538 fis, clone LNG08010 <0.01 2.13EST <0.05 2.07EST <0.05 2.11

KIAA0161 KIAA0161 gene product < 0.05 2.44EST <0.05 2.60EST <0.05 2.10EST <0.05 2.07EST <0.05 2.22

DKFZP564K247 DKFZP564K247 protein <0.05 2.26SBB103 hypothetical SBBI03 protein <0.05 2.27

EST <0.05 2.22Homo sapiens cDNA: FLJ21693 fis, clone COL09609 <0.05 2.78EST <0.05 2.64

d. LiverCell adhesionIGFBP7 insulin-like growth factor binding protein 7 <0.001 2.29CDH2 cadherin 2, type 1, N-cadherin (neuronal) < 0.01 3.01







Cytoskeleton/cell motilityCBX1 chromobox homologue 1 (Drosophila HP1 h) < 0.001 2.51HECH heterochromatin-like protein 1 <0.001 2.61MYPT1 myosin phosphatase, target subunit 1 <0.001 3.00SDCBP syndecan binding protein (syntenin) <0.001 2.26CD2AP CD2-associated protein <0.01 2.21ECM remodelingCTSL2 cathepsin L2 <0.01 2.06P4HA1 prolyl 4-hydroxylase, a-1 subunit < 0.01 3.11ADAM17 A disintegrin and metalloproteinase domain 17 <0.01 2.04Cell-cell signaling (cytokine/chemokine)LIF leukemia inhibitory factor < 0.001 3.49

IFN-g antagonist cytokine <0.001 2.23PBEF pre-B-cell colony-enhancing factor < 0.01 2.52Signal transductionGNAS1 GNAS complex locus <0.001 2.61TIEG TGFB inducible early growth response <0.001 2.11RHEB2 Ras homologue enriched in brain 2 <0.001 2.49YWHAQ 14-3-3 protein H <0.01 2.22PTPN12 protein tyrosine phosphatase, non-receptor type 12 <0.01 2.17SSH3BP1 spectrin SH3 domain binding protein 1 <0.05 2.10LOC56990 non-kinase Cdc42 effector protein SPEC2 <0.05 2.11MTM1 myotubular myopathy 1 <0.05 2.14Immune responseMetabolismPGK1 phosphoglycerate kinase 1 <0.001 2.57PDK1 pyruvate dehydrogenase kinase, isoenzyme 1 <0.001 6.21LDHB lactate dehydrogenase B <0.001 3.37ATQ1 antiquitin 1 <0.001 2.22AGL amylo, 6-glucosidase, 4-a-glucanotransferase <0.001 2.18PHKB phosphorylase kinase, h <0.001 2.71ACLY ATP citrate lyase <0.05 2.40Cell cycle/apoptosis/DNA repairCCNG1 cyclin G1 <0.001 2.17CAP-C chromosome-associated polypeptide C <0.001 2.53BNIP3L BCL2/adenovirus E1B 19kD-interacting protein 3-like <0.01 2.15REV3L REV3 (yeast homologue)-like <0.01 2.45TranscriptionTOP3 DNA topoisomerase III < 0.001 2.00HNRPA2B1 heterogeneous nuclear ribonucleoprotein A2/B1 <0.001 2.73DDX15 DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 15 <0.001 3.26HNRPA1 heterogeneous nuclear ribonucleoprotein A1 <0.001 2.45RBMX RNA binding motif protein, X chromosome <0.001 2.21HTATSF1 HIV TAT specific factor 1 <0.001 2.31TAF172 TBP-associated factor 172 <0.001 2.18TAF2B TBP-associated factor 2 <0.001 2.64LRRFIP1 leucine rich repeat (in FLII) interacting protein 1 <0.001 2.24BTF3 basic transcription factor 3 <0.001 2.03MYC v-myc avian myelocytomatosis viral oncogene homologue <0.001 2.50BHLHB2 basic helix-loop-helix domain containing, class B, 2 <0.001 5.45DBY DEAD/H box polypeptide, Y chromosome <0.001 2.33MXI1 MAX-interacting protein 1 <0.01 2.86ZNF9 zinc finger protein 9 <0.01 2.62BAZ1A bromodomain adjacent to zinc finger domain, 1A <0.05 2.13Protein synthesis/processingRABGGTB Rab geranylgeranyltransferase, h subunit < 0.001 2.76RPL9 ribosomal protein L9 <0.001 2.27EIF3S6 eukaryotic translation initiation factor 3, subunit 6 (M r 48,000) <0.001 2.11RPL6 ribosomal protein L6 <0.001 2.10RPL7 ribosomal protein L7 <0.001 2.40RPS3A ribosomal protein S3A <0.001 2.73RPS4X ribosomal protein S4, X-linked <0.001 2.16EEF1A1 eukaryotic translation elongation factor 1 a1 <0.001 2.57LOC51280 golgi membrane protein GP73 <0.01 2.16NeurogenesisGPM6B glycoprotein M6B <0.001 3.87STMN2 Stathmin-like 2 <0.001 3.78GPI glucose phosphate isomerase <0.001 2.44The othersCSPG6 chondroitin sulfate proteoglycan 6 (bamacan) <0.001 2.97ST13 suppression of tumorigenicity 13(Hsp70-interacting protein) < 0.001 2.29BET1 Golgi vesicular membrane trafficking protein p18 <0.001 2.15






the adhesion of cancer cells to the vessel wall in specific

organs (10). Consequently, the adhesive interaction between

cancer cells and endothelium is likely to be associated with the

organ selectivity of metastasis. Our data suggest that lectin,

a family of h-galactoside-binding proteins implicated in

modulating cell-cell and cell-matrix interactions, may play an

important role in organ preference because one member of this

molecular family, LGALS1 , is highly expressed in pulmonary

metastases and another, LGALS9 , in renal metastases. In

addition, LGALS3 has shown an association with pulmonary

metastasis of osteosarcoma (19), and its binding protein

LGALS3BP, an indicator of the metastatic propensity of lung

cancer (20), was also highly expressed in pulmonary metastases

in our murine model.


SLC2A1 solute carrier family 2, member 1 <0.001 3.11SLC16A1 solute carrier family 16, member 1 <0.001 2.35RANBP2 RAN binding protein 2 <0.001 2.33ATP5A1 mitochondrial ATP synthetase, oligomycin-resistant < 0.001 2.01P115 vesicle docking protein p115 <0.01 2.54SEC23B Sec23 (Saccharomyces cerevisiae ) homologue B <0.01 2.05SCP2 sterol carrier protein 2 <0.01 2.56UBA2 SUMO activating enzyme subunit 2 <0.05 2.50SLC2A3 solute carrier family 2, member 3 <0.05 2.96SSFA2 sperm specific antigen 2 <0.05 2.17UnknownARFGAP1 ADP-ribosylation factor GTPase activating protein 1 <0.001 2.54LOC51187 60S ribosomal protein L30 isolog <0.001 2.26KIAA1223 KIAA1223 protein <0.001 2.00LOC51606 CGI1 protein <0.001 2.05CL25022 hypothetical protein <0.001 3.10

Homo sapiens CDA08 mRNA, complete cds <0.001 2.39Homo sapiens cDNA FLJ13018 fis, clone NT2RP3000685 <0.001 2.77

KIAA0725 KIAA0725 protein <0.001 2.04EST <0.001 2.04

KIAA0863 KIAA0863 protein <0.001 2.34EST <0.001 2.34Homo sapiens cDNA: FLJ22844 fis, clone KAIA5181 <0.001 2.09

E2IG5 hypothetical protein, estradiol-induced <0.001 2.20EST <0.001 2.14

PGK1 phosphoglycerate kinase 1 <0.001 2.77sequence 1 from US patent < 0.001 2.29

LIV LIV protein, estrogen regulated <0.001 2.55Homo sapiens cDNA FLJ14041 fis, clone HEMBA1005780 <0.001 2.21EST <0.001 2.02EST <0.001 2.16

LOC56270 hypothetical protein 628 <0.001 2.22LOC51012 CGI07 protein <0.001 3.03RANBP6 RAN binding protein 6 <0.001 2.47KIAA1615 KIAA1615 protein <0.001 2.03

EST <0.001 2.39EST <0.01 2.45

KIAA0430 Human Chromosome 16 BAC clone CIT987SK-A-362G6 <0.01 2.08EST <0.01 2.22

CH1 membrane protein CH1 <0.01 2.75EST <0.01 2.15EST <0.01 2.35

KIAA0637 KIAA0637 gene product < 0.01 2.28KIAA1014 KIAA1014 protein <0.01 2.56LCP host cell factor homologue <0.01 2.46FLJ10582 hypothetical protein FLJ10582 <0.01 2.82FHL1 four and a half LIM domains 1 <0.01 2.34

Homo sapiens cDNA: FLJ23093 fis, clone LNG07264 <0.01 2.34DKFZP434N093 DKFZP434N093 protein <0.01 2.13LOC51030 CGI48 protein <0.01 2.24

Homo sapiens mRNA; cDNA DKFZp586G2222 <0.01 2.41FLJ10461 hypothetical protein FLJ10461 <0.01 2.80KIAA0601 KIAA0601 protein <0.05 2.44FLJ10595 hypothetical protein FLJ10595 <0.05 2.93DKFZP564I052 DKFZP564I052 protein <0.05 2.07SNX2 sorting nexin 2 <0.05 2.58FLJ10326 hypothetical protein FLJ10326 <0.05 2.29

Homo sapiens cDNA: FLJ21962 fis, clone HEP05564 <0.05 2.94SH3BGRL SH3 domain binding glutamic acid-rich protein like <0.05 2.11

EST <0.05 2.22MPDU1 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’’).





A number of genes associated with the cytoskeleton or

with cell motility were differentially expressed among the

four organ groups. Especially in pulmonary metastatic foci,

actin isoforms and related genes such as RHOC , ARPC4 ,

PFN1 , and PTK9L were expressed more strongly than in

metastatic lesions of the other three organs. RHOC is a

member of the Ras-related GTP-binding protein family and

regulates reorganization of the actin cytoskeleton; its

enhanced expression has been associated with pulmonary

metastasis of melanoma cells (21). ARPC4 and PFN1 are

implicated in directional movement of cells by promoting

actin polymerization and controlling formation of filopodia

and lamellipodia (22). On the other hand, PTK9L is

associated with actin depolymerization. Therefore, up-regulation

of these genes might reflect active cellular movement of

cancer cells. Because cellular movement is essential for

migration and invasion of cancer cells, genes involved in the

cellular cytoskeleton and motility may well contribute to

metastasis. In addition, a number of genes having various

functions such as remodeling of the ECM, or participating in

immune responses or signal transduction, were differentially

expressed in each metastatic site.

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 ,

ACTA2 , TMSB10 , MYH7 , FLNA , and ARPC4 , the expres-

sions of which were elevated in micrometastasis, coordin-

ately form lamellipodia and new adhesion sites at the

leading edge of the invading cells, and move the cell

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),

was also up-regulated in the smaller lesions. On the other

hand, none of the genes belonging to the categories

documented above were highly expressed in the larger

lesions. Enhanced expression of these genes in the smaller

lesions might reflect active cellular movement and invasion

of cancer cells in micrometastasis. Because the differential

expression of 105 genes between the two groups might

reflect differences in the biological features of these tumors,

further investigations of nearly half of the genes of

unknown functions listed here should provide important

insights into the progression from micrometastasis to

macrometastasis.

In summary, we identified dozens of molecules that might

be associated with organ-preferential metastasis or with tumor

progression from micrometastasis to macrometastasis. Our

results support the notion that metastasis is a complicated,

multistep phenomenon and that each step requires several key

molecules. Further analysis using clinical materials might help

to clarify the mechanism of organ-specific metastasis and to

further define the genes of importance. This should eventually

lead to molecular target-based chemotherapy and prevention

of metastasis.

FIGURE 2. Cluster analysis of 25 metastatic lesions. Horizontal row,single gene; vertical column, metastatic lesion. Red indicates a high levelof expression relative to the mean; blue indicates a low level of expressionrelative to the mean. Highly expressed genes in lung metastases (a ), inkidney metastases (b), in bone metastases (c ), and in liver metastases (d).The lists of the genes specifically expressed in each of the fourmetastasized organs (a – d) are shown in Table 2, a –d, respectively.




Table 3. Genes Predominantly Expressed in Micrometastasis and Macrometastasis


a. MicrometastasisCell adhesionGP110 Adhesion regulating molecule 1 <0.001 2.00SDC1 syndecan 1 <0.01 2.30STEAP six transmembrane epithelial antigen of the prostate <0.01 2.03CEACAM4 carcinoembryonic antigen-related cell adhesion molecule 4 <0.05 2.25PCDHGC3 protocadherin g subfamily C, 3 <0.05 2.01Cytoskeleton/cell motilityHSPB3 heat shock M r 27,000 protein 3 <0.001 2.04ARPC4 actin related protein 2/3 complex, subunit 4 (M r 20,000) <0.001 2.03ACTA2 actin, a2, smooth muscle, aorta <0.01 3.08ACTB actin, h <0.01 2.42MYH7 myosin, heavy polypeptide 7, cardiac muscle, h <0.01 2.34TMSB10 thymosin, h10 <0.05 2.04ECM remodelingMMP1 matrix metalloproteinase 1 (interstitial collagenase) < 0.05 2.55HTF9C Hpa II tiny fragments locus 9C (collagen type iii) < 0.05 2.43Cell-cell signaling (cytokine/chemokine)FGF19 fibroblast growth factor 19 <0.001 2.03SCYB13 small inducible cytokine B subfamily, member 13 <0.05 2.05EGFR epidermal growth factor receptor <0.05 5.79Signal transductionNR1I3 nuclear receptor subfamily 1, group I, member 3 <0.001 2.14FSTL1 follistatin-like 1 <0.001 2.03SHC1 SHC (Src homology 2 domain-containing) transforming protein 1 <0.01 2.04IFITM1 interferon induced transmembrane protein 1 (9 – 27) <0.05 2.76Immune responseMD-2 MD-2 protein <0.001 2.00IFITM2 interferon induced transmembrane protein 2 (1-8D) <0.01 2.56IGKC immunoglobulin n constant < 0.05 2.27

DC class II histocompatibility antigen a-chain <0.05 3.45MetabolismMAN1B1 mannosidase, a, class 1B, member 1 <0.001 2.23FBP2 fructose, 6-bisphosphatase 2 <0.001 2.29NUCB1 nucleobindin 1 <0.01 2.39PMM2 phosphomannomutase 2 <0.05 2.09Cell cycle/apoptosis/DNA repairTranscriptionEEF1E1 eukaryotic translation elongation factor 1 q1 <0.001 2.00NFX1 nuclear transcription factor, X-box binding 1 <0.05 2.07Protein synthesis/processingHUGT1 UDP-glucose:glycoprotein glucosyltransferase 1 <0.05 2.14PPP1CA protein phosphatase 1, catalytic subunit, a isoform <0.05 2.45NeurogenesisTHY1 Thy cell surface antigen <0.001 2.84The othersRAB32 RAB32, member RAS oncogene family <0.001 2.00GBF1 golgi-specific brefeldin A resistance factor 1 <0.001 2.66ATP1B1 ATPase, Na+/K+ transporting, h1 polypeptide <0.01 4.31FRZB frizzled-related protein <0.05 6.47KIAA1011 synaptic nuclei expressed gene 2 <0.05 2.00COPE coatomer protein complex, subunit q <0.05 2.04Unknown

EST <0.001 2.19Homo sapiens mRNA for FLJ00116 protein, partial cds <0.001 2.39EST <0.001 2.14EST <0.001 2.19EST <0.001 2.14EST <0.001 2.26EST <0.001 2.02Homo sapiens cDNA FLJ13945 fis, clone Y79AA1000969 <0.01 2.22

FLJ10846 hypothetical protein FLJ10846 <0.01 2.50LOC51690 U6 snRNA-associated Sm-like protein LSm7 <0.01 2.10

EST <0.01 2.07EST00098 hypothetical protein EST00098 <0.01 2.06RGC32 RGC32 protein <0.01 2.61

Homo sapiens cDNA FLJ12595 fis, clone NT2RM4001344 <0.01 2.36Homo sapiens mRNA; cDNA DKFZp586F1323 <0.01 2.47EST <0.05 2.17

LOC55895 M r 22,000 peroxisomal membrane protein-like <0.05 2.30Homo sapiens cDNA: FLJ21880 fis, clone HEP02743 <0.05 2.67EST <0.05 2.16





Materials and MethodsCell Line

Human SCLC cell line SBC-5 was kindly provided by Dr. K.

Hiraki (Okayama University, Okayama, Japan). The SBC-5 cells

were maintained in Eagle’s MEM supplemented with 10% fetal

bovine serum (Life Technologies, Inc., Grand Island, NY),

gentamycin (Schering-Plough, Osaka, Japan), and 4mMHEPES.

ReagentAnti-mouse interleukin 2 receptor h-chain monoclonal

antibody, TM-h1 (IgG2b), was supplied by Drs. M. Miyasaka

and T. Tanaka (Osaka University, Osaka, Japan) (26). None of

this material contained endotoxins, as judged by the Limulus

amoebocyte assay (Seikagaku Kogyo, Tokyo, Japan; minimum

detection level 0.1 ng/ml).


HSPC157 HSPC157 protein <0.05 3.24EST <0.05 2.06EST <0.05 2.25EST <0.05 2.40EST <0.05 2.09EST <0.05 2.03EST <0.05 2.57EST <0.05 3.55Homo sapiens cDNA FLJ12425 fis, clone MAMMA1003104 <0.05 2.32EST <0.05 2.41

b. MacrometastasisCell adhesionCytoskeleton/cell motilityECM remodelingCell-cell signaling (cytokine/chemokine)PDGFRA platelet-derived growth factor receptor, a polypeptide <0.05 2.08TGFBR2 transforming growth factor, h receptor II (M r 70,000 –80,000) <0.05 2.72Signal transductionRHEB2 Ras homologue enriched in brain 2 <0.001 2.01RalGPS1A Ral guanine nucleotide exchange factor RalGPS1A <0.01 2.42AKAP9 A kinase (PRKA) anchor protein (yotiao) 9 <0.05 2.20RAB2L RAB2, member RAS oncogene family-like <0.05 2.02GNAS1 G protein, a stimulating activity polypeptide 1 <0.05 2.22CD47 CD47 antigen (Rh-related antigen, integrin-associated signal transducer) < 0.05 2.31Immune responseMetabolismLDHA lactate dehydrogenase A <0.01 2.51Cell cycle/apoptosis/DNA repairBNIP3L BCL2/adenovirus E1B M r 19,000-interacting protein 3-like <0.01 3.15TranscriptionBTF3 basic transcription factor 3 <0.01 2.22ZNF277 zinc finger protein 277 <0.05 2.34SMARCE1 SWI/SNF related, matrix associated, actin-dependent regulator of chromatin, subfamily e, member 1 <0.05 2.13Protein synthesis/processingSMT3H1 SMT3 (suppressor of mif two 3, yeast) homologue 1 <0.001 2.19HUGT1 UDP-glucose:glycoprotein glucosyltransferase 1 <0.05 2.41NeurogenesisSYNGR3 synaptogyrin 3 <0.001 2.01The othersNARF nuclear prelamin A recognition factor < 0.01 2.08GMPS guanine monophosphate synthetase <0.05 2.22DNMT2 DNA (cytosine-5-)-methyltransferase 2 <0.05 2.83Unknown

ESTs <0.001 2.07ESTs, highly similar to I38945 melanoma ubiquitous mutated protein <0.001 2.18Homo sapiens mRNA; cDNA DKFZp761K2024 <0.001 2.60ESTs <0.001 2.09

PTD004 hypothetical protein <0.01 2.48KIAA1265 KIAA1265 protein <0.01 2.33KIAA0071 KIAA0071 protein <0.01 2.18CHAC Chorea acanthocytosis < 0.05 2.00

pseudo-keratin K16 type I <0.05 2.13KIAA1250 likely homologue of rat kinase D-interacting substance of M r 220,000 <0.05 3.04FLJ10120 hypothetical protein FLJ10120 <0.05 2.11

Homo sapiens cDNA FLJ10533 fis, clone NT2RP2001056 <0.05 2.11SENP2 sentrin-specific protease <0.05 2.07MUL Mulibrey nanism <0.05 2.21

ESTs <0.05 2.12ESTs <0.05 2.34

FLJ13433 hypothetical protein FLJ13433 <0.05 2.08Homo 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’’)





AnimalsMale SEB-17/Icr-scid mice, 6–8 weeks old, were obtained

from Charles River Laboratories (Yokohama, Japan) and

maintained under specific pathogen-free conditions throughout

the experiment. Experiments were performed following the

ethical guidelines of our university.

Experimental Metastasis of SBC-5 in Mice LackingNK Cells

To facilitate the metastasis of human SBC-5, NK cells were

depleted in SCID mice by i.p. injection of TM-h1Ab (1 mg/1 ml

in PBS/mouse) 2 days before inoculation of tumor cells (12). The

tumor cells were first harvested and washed with Ca2+- and

Mg2+-free PBS; cell viability was determined by the trypan blue

exclusion test, and only cell suspensions showing >90% viability

were used. We injected 0.3 ml of tumor-cell suspension (1–5 �106 cells) into the lateral tail vein of non-anesthetized mice. The

mice were sacrificed 35 days after inoculation, and four organs

(lung, liver, kidney, and bone tissues) containing macroscopic

lesions were excised, embedded in TissueTek OCT medium

(Sakura, Tokyo, Japan), and snap frozen at �80jC.

Laser-Capture MicrodissectionWe prepared 8-Am-thick frozen sections, which were fixed

in 70% ethanol for 30 s, stained with H&E, and dehydrated first

with 99.5% ethanol for 5 min and then with xylene for 1 min.

The stained tissues were observed microscopically; 25

metastatic lesions (10 lung, 5 liver, 5 kidney, and 5 bone

tumors) were selected for laser-capture microdissection with a

PixCell II LCM system, according to the manufacturer’s

protocols (Arcturus Engineering, Mountain View, CA).

RNA Extraction and T7-Based RNA AmplificationTotal RNA was extracted from each captured cancer tissue

using the RNeasy mini kit (Qiagen, Valencia, CA) and RNase-

free DNase according to the manufacturer’s protocols. Total

RNAs extracted from each of the 25 metastatic lesions were

subjected to T7-based RNA amplification, as described

previously (27). Two rounds of amplification yielded 40–200

Ag of aRNA (over 100,000-fold) from each sample. Aliquots

(2.5 Ag) of a RNA from individual lesions (test probes) and

from mixture of aRNAs from all 25 lesions (a control probe)

were labeled respectively with Cy5-dCTP or Cy3-dCTP.

cDNA MicroarraysOur ‘‘genome-wide’’ cDNA microarray system contains

23,040 cDNAs selected from the UniGene database of the

National Center for Biotechnology Information (28). Fabrica-

tion of the microarray, hybridization, washing, and detection of

signal intensities were described previously (27). To normalize

the amount of mRNA between tumors and controls, the Cy5/

Cy3 ratio for each gene’s expression was adjusted so that the

averaged Cy5/Cy3 ratio of 52 housekeeping genes was equal to

1. We assigned a cut-off value to each microarray slide, using a

variance analysis. Genes, the Cy3 or Cy5 signal intensities of

which were lower than the cut-off values, were excluded from

further investigation. We also excluded data from genes where

the signal/noise ratio was <3.

Cross-Hybridization of Mouse Messenger RNATo assess the influence of contamination of normal mouse

mRNA, we microdissected normal mouse cells in individual

organs and hybridized on the human cDNA microarrays by the

same method as described above.

Cluster Analysis of Gene-Expression ProfilesTo identify genes that were expressed differently among the

four types of metastatic tissue, we applied random-permutation

tests to estimate the ability of each gene to distinguish between

two groups (each organ-specific metastasis versus the mixture

of metastases in the other three organs). The comparative

combinations were as follows: (a) 10 lung metastases versus 15

metastases in three other organs; (b) 5 liver metastases versus

20 others; (c) 5 kidney metastases versus 20 others; and (d) 5

bone metastases versus 20 others. Mean (l) and standard (r)deviations were calculated from the log-transformed relative

expression ratios of each gene in both groups. A discrimination

score (DS) for each gene was defined as follows:

DS ¼ ðl1 � l2Þ=ðr1 þ r2Þ

The samples were randomly permutated 10,000 times for

each pair of groups. Because the DS data set of each gene

showed a normal distribution, we calculated a P value for the

user-defined grouping (29).

A hierarchical clustering analysis was applied to the 25

metastatic loci and the 435 genes extracted by the random

permutation tests, using Web-available software (‘‘Cluster’’ and

‘‘TreeView’’) written by M. Eisen. (http://genome-www5.

stanford.edu/MicroArray/SMD/restech.html).

Identification of Genes Differentially Expressed BetweenMicrometastasis and Macrometastasis

To compare gene-expression profiles between small and

large metastatic foci, we also applied a random permutation test

to 9 of the 10 lesions in lung: 5 that were <1 mm (average size:

0.60 mm, SD: 0.22); and 4 that were >2 mm (average: 2.40 mm,

SD: 0.32) (29). To determine the size of the tumors, we first

sliced each lesion from the top to the bottom (8 Am thick) and

measured the maximum axis of the largest tumor section. The

Union Internationale Centre le Cancer advocates the use of the

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

micrometastasis as a lesion smaller than 1 mm in diameter.

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2003;1:485-499. Mol Cancer Res Soji Kakiuchi, Yataro Daigo, Tatsuhiko Tsunoda, et al. Promotion of Science (no. 00L01402) to Y.N.Future'' Program Grant of The Japan Society for the

''Research for the11Human Small Cell Lung Cancer in MiceGenome-Wide Analysis of Organ-Preferential Metastasis of

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