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The Niche Component Periostin Is Produced by Cancer-Associated Fibroblasts, Supporting Growth of Gastric Cancer through ERK Activation

2014; Elsevier BV; Volume: 184; Issue: 3 Linguagem: Inglês

10.1016/j.ajpath.2013.11.012

ISSN

1525-2191

Autores

Yoshinao Kikuchi, Akiko Kunita, Caname Iwata, Daisuke Komura, Takashi Nishiyama, Kazuhiro Shimazu, Kimiko Takeshita, Junji Shibahara, Isao Kii, Yasuyuki Morishita, Masakazu Yashiro, Kosei Hirakawa, Kohei Miyazono, Akira Kudō, Masashi Fukayama, Takeshi Kashima,

Tópico(s)

Signaling Pathways in Disease

Resumo

Overexpression of periostin (POSTN), an extracellular matrix protein, has been observed in several cancers. We investigated the importance of POSTN in gastric cancer. Genome-wide gene expression analysis using publicly available microarray data sets revealed significantly high POSTN expression in cancer tissues from stage II–IV gastric cancer, compared with background normal tissues. The POSTN/vimentin mRNA expression ratio was highly associated with gene groups that regulate the cell cycle and cell proliferation. IHC showed that periglandular POSTN deposition, comprising linear deposition abutting the glandular epithelial cells in normal mucosa, disappeared during intestinal gastric cancer progression. Stromal POSTN deposition was also detected at the invasive front of intestinal-type and diffuse-type cancers. In situ hybridization confirmed POSTN mRNA in cancer-associated fibroblasts, but not in tumor cells themselves. POSTN enhanced the in vitro growth of OCUM-2MLN and OCUM-12 diffuse-type gastric cancer cell lines, accompanied by the activation of ERK. Furthermore, coinoculation of gastric cancer cells with POSTN-expressing NIH3T3 mouse fibroblast cells facilitated tumor formation. The OCUM-2MLN orthotopic inoculation model demonstrated that tumors of the gastric wall in Postn−/− mice were significantly smaller than those in wild-type mice. Ki-67 and p-ERK positive rates were both lower in Postn−/− mice. These findings suggest that POSTN produced by cancer-associated fibroblasts constitutes a growth-supportive microenvironment for gastric cancer. Overexpression of periostin (POSTN), an extracellular matrix protein, has been observed in several cancers. We investigated the importance of POSTN in gastric cancer. Genome-wide gene expression analysis using publicly available microarray data sets revealed significantly high POSTN expression in cancer tissues from stage II–IV gastric cancer, compared with background normal tissues. The POSTN/vimentin mRNA expression ratio was highly associated with gene groups that regulate the cell cycle and cell proliferation. IHC showed that periglandular POSTN deposition, comprising linear deposition abutting the glandular epithelial cells in normal mucosa, disappeared during intestinal gastric cancer progression. Stromal POSTN deposition was also detected at the invasive front of intestinal-type and diffuse-type cancers. In situ hybridization confirmed POSTN mRNA in cancer-associated fibroblasts, but not in tumor cells themselves. POSTN enhanced the in vitro growth of OCUM-2MLN and OCUM-12 diffuse-type gastric cancer cell lines, accompanied by the activation of ERK. Furthermore, coinoculation of gastric cancer cells with POSTN-expressing NIH3T3 mouse fibroblast cells facilitated tumor formation. The OCUM-2MLN orthotopic inoculation model demonstrated that tumors of the gastric wall in Postn−/− mice were significantly smaller than those in wild-type mice. Ki-67 and p-ERK positive rates were both lower in Postn−/− mice. These findings suggest that POSTN produced by cancer-associated fibroblasts constitutes a growth-supportive microenvironment for gastric cancer. The local microenvironment, or niche, plays important roles both in normal tissue maintenance and in cancer development and progression. Extracellular matrix (ECM), a major niche component, is believed to change quantitatively or qualitatively in normal, primary cancer, and metastatic niches. Malanchi et al,1Malanchi I. Santamaria-Martínez A. Susanto E. Peng H. Lehr H.A. Delaloye J.F. Huelsken J. Interactions between cancer stem cells and their niche govern metastatic colonization.Nature. 2011; 481: 85-89Crossref PubMed Scopus (983) Google Scholar using the MMTV-PyMT mouse breast cancer model, recently demonstrated that periostin (POSTN; alias osteoblast-specific factor 2) plays a primary role in spontaneous metastasis to the lung as ECM of the metastatic niche. POSTN, which is expressed by fibroblasts in normal tissue and in the stroma of the primary tumor, is necessary for cancer stem cell maintenance. Our research group originally cloned and characterized POSTN, a 90-kDa secreted matricellular protein and a member of the fasciclin (Fas) family.2Takeshita S. Kikuno R. Tezuka K. Amann E. Osteoblast-specific factor 2: cloning of a putative bone adhesion protein with homology with the insect protein fasciclin I.Biochem J. 1993; 294: 271-278Crossref PubMed Scopus (543) Google Scholar, 3Horiuchi K. Amizuka N. Takeshita S. Takamatsu H. Katsuura M. Ozawa H. Toyama Y. Bonewald L.F. Kudo A. Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor beta.J Bone Miner Res. 1999; 14: 1239-1249Crossref PubMed Scopus (801) Google Scholar POSTN comprises an amino-terminal EMI domain, a tandem repeat of four Fas I domains, and a carboxyl-terminal domain including a heparin-binding site at its C-terminal end.4Kudo A. Periostin in fibrillogenesis for tissue regeneration: periostin actions inside and outside the cell.Cell Mol Life Sci. 2011; 68: 3201-3207Crossref PubMed Scopus (225) Google Scholar POSTN has been regarded as regulating fibrillogenesis in tissue subjected to mechanical stress, such as the endocardium, fascia, periosteum, articular surface, and periodontal ligament.4Kudo A. Periostin in fibrillogenesis for tissue regeneration: periostin actions inside and outside the cell.Cell Mol Life Sci. 2011; 68: 3201-3207Crossref PubMed Scopus (225) Google Scholar POSTN has also been recognized as playing a key role in tissue repair. Our research group has previously shown that POSTN is expressed by myofibroblasts and that it regulates tissue repair processes in myocardial infarction5Shimazaki M. Kudo A. Impaired capsule formation of tumors in periostin-null mice.Biochem Biophys Res Commun. 2008; 367: 736-742Crossref PubMed Scopus (28) Google Scholar and wound healing.6Nishiyama T. Kii I. Kashima T.G. Kikuchi Y. Ohazama A. Shimazaki M. Fukayama M. Kudo A. Delayed re-epithelialization in periostin-deficient mice during cutaneous wound healing.PLoS One. 2011; 6: e18410Crossref PubMed Scopus (105) Google Scholar POSTN is regarded as an interventional molecular target for myocardial infarction therapy.7Segers V.F. Lee R.T. Protein therapeutics for cardiac regeneration after myocardial infarction.J Cardiovasc Transl Res. 2010; 3: 469-477Crossref PubMed Scopus (94) Google Scholar In neoplastic lesions, overexpression of POSTN has been identified in various cancers, including those of the lung (non–small-cell subtypes),8Sasaki H. Dai M. Auclair D. Fukai I. Kiriyama M. Yamakawa Y. Fujii Y. Chen L.B. Serum level of the periostin, a homologue of an insect cell adhesion molecule, as a prognostic marker in nonsmall cell lung carcinomas [Erratum appeared in Cancer 2002, 95:2580].Cancer. 2001; 92: 843-848Crossref PubMed Scopus (96) Google Scholar ovary,9Gillan L. Matei D. Fishman D.A. Gerbin C.S. Karlan B.Y. Chang D.D. Periostin secreted by epithelial ovarian carcinoma is a ligand for alpha(V)beta(3) and alpha(V)beta(5) integrins and promotes cell motility.Cancer Res. 2002; 62: 5358-5364PubMed Google Scholar breast,10Grigoriadis A. Mackay A. Reis-Filho J.S. Steele D. Iseli C. Stevenson B.J. Jongeneel C.V. Valgeirsson H. Fenwick K. Iravani M. Leao M. Simpson A.J. Strausberg R.L. Jat P.S. Ashworth A. Neville A.M. O'Hare M.J. Establishment of the epithelial-specific transcriptome of normal and malignant human breast cells based on MPSS and array expression data.Breast Cancer Res. 2006; 8: R56Crossref PubMed Scopus (111) Google Scholar, 11Shao R. Bao S. Bai X. Blanchette C. Anderson R.M. Dang T. Gishizky M.L. Marks J.R. Wang X.F. Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through up-regulation of vascular endothelial growth factor receptor 2 expression.Mol Cell Biol. 2004; 24: 3992-4003Crossref PubMed Scopus (281) Google Scholar colon,12Bao S. Ouyang G. Bai X. Huang Z. Ma C. Liu M. Shao R. Anderson R.M. Rich J.N. Wang X.F. Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway.Cancer Cell. 2004; 5: 329-339Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 13Kikuchi Y. Kashima T.G. Nishiyama T. Shimazu K. Morishita Y. Shimazaki M. Kii I. Horie H. Nagai H. Kudo A. Fukayama M. Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.J Histochem Cytochem. 2008; 56: 753-764Crossref PubMed Scopus (107) Google Scholar pancreas,14Baril P. Gangeswaran R. Mahon P.C. Caulee K. Kocher H.M. Harada T. Zhu M. Kalthoff H. Crnogorac-Jurcevic T. Lemoine N.R. Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the beta4 integrin and the PI3k pathway.Oncogene. 2007; 26: 2082-2094Crossref PubMed Scopus (241) Google Scholar, 15Erkan M. Kleeff J. Gorbachevski A. Reiser C. Mitkus T. Esposito I. Giese T. Buchler M.W. Giese N.A. Friess H. Periostin creates a tumor-supportive microenvironment in the pancreas by sustaining fibrogenic stellate cell activity.Gastroenterology. 2007; 132: 1447-1464Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 16Fukushima N. Kikuchi Y. Nishiyama T. Kudo A. Fukayama M. Periostin deposition in the stroma of invasive and intraductal neoplasms of the pancreas.Mod Pathol. 2008; 21: 1044-1053Crossref PubMed Scopus (78) Google Scholar, 17Kanno A. Satoh K. Masamune A. Hirota M. Kimura K. Umino J. Hamada S. Satoh A. Egawa S. Motoi F. Unno M. Shimosegawa T. Periostin, secreted from stromal cells, has biphasic effect on cell migration and correlates with the epithelial to mesenchymal transition of human pancreatic cancer cells.Int J Cancer. 2008; 122: 2707-2718Crossref PubMed Scopus (120) Google Scholar bile duct,18Utispan K. Thuwajit P. Abiko Y. Charngkaew K. Paupairoj A. Chau-in S. Thuwajit C. Gene expression profiling of cholangiocarcinoma-derived fibroblast reveals alterations related to tumor progression and indicates periostin as a poor prognostic marker.Mol Cancer. 2010; 9: 13Crossref PubMed Scopus (103) Google Scholar and head and neck.19Kudo Y. Ogawa I. Kitajima S. Kitagawa M. Kawai H. Gaffney P.M. Miyauchi M. Takata T. Periostin promotes invasion and anchorage-independent growth in the metastatic process of head and neck cancer.Cancer Res. 2006; 66: 6928-6935Crossref PubMed Scopus (182) Google Scholar However, expression and function of POSTN in gastric cancer, the second most common cause of cancer death worldwide,20Ferlay J. Shin H.R. Bray F. Forman D. Mathers C. Parkin D.M. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.Int J Cancer. 2010; 127: 2893-2917Crossref PubMed Scopus (13608) Google Scholar have not been studied sufficiently,21Li J.S. Sun G.W. Wei X.Y. Tang W.H. Expression of periostin and its clinicopathological relevance in gastric cancer.World J Gastroenterol. 2007; 13: 5261-5266Crossref PubMed Scopus (35) Google Scholar, 22Liu Y. Liu B.A. Enhanced proliferation, invasion, and epithelial–mesenchymal transition of nicotine-promoted gastric cancer by periostin.World J Gastroenterol. 2011; 17: 2674-2680Crossref PubMed Scopus (42) Google Scholar especially with respect to the two distinctive histological groups, the intestinal and diffuse types.23Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification.Acta Pathol Microbiol Scand. 1965; 64: 31-49Crossref PubMed Scopus (5048) Google Scholar The morphology, molecular and genetic background, and clinical features of the two subtypes differ completely.24Hu B. El Hajj N. Sittler S. Lammert N. Barnes R. Meloni-Ehrig A. Gastric cancer: Classification, histology and application of molecular pathology.J Gastrointest Oncol. 2012; 3: 251-261PubMed Google Scholar In the present study, we investigated the expression and function of POSTN in gastric cancer. First, we evaluated POSTN expression in normal and neoplastic stomach using both bioinformatics and an extensive tissue sample examination to clarify the cell types that predominantly produce POSTN. Next, we examined the function of POSTN using POSTN-producing fibroblasts and an orthotopic inoculation model of diffuse-type gastric cancer in Postn−/− mice. Our results demonstrated that POSTN is overexpressed by cancer-associated fibroblasts (CAFs) and suggested that POSTN constitutes the primary tumor niche by supporting cancer cell proliferation through ERK signaling pathway in gastric cancer. We compared POSTN mRNA levels using the following public domain microarray data sets from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo): GSE15460, 25 gastric cancer cell lines and 161 cases of gastric cancer (8 cases in T1, 44 cases in T2, 108 cases in T3, and 1 case in T4); GSE7307, 12 cases of normal stomach; and GSE19826, 12 cases of gastric cancer and matched normal tissues in which the clinical TNM stage was available, 3 cases for each stage. Raw data were retrieved and processed using the MAS5 algorithm with target intensity of 500 (Affymetrix, Santa Clara, CA).25Hubbell E. Liu W.M. Mei R. Robust estimators for expression analysis.Bioinformatics. 2002; 18: 1585-1592Crossref PubMed Scopus (494) Google Scholar Gene Set Enrichment Analysis (GSEA) software26Subramanian A. Tamayo P. Mootha V.K. Mukherjee S. Ebert B.L. Gillette M.A. Paulovich A. Pomeroy S.L. Golub T.R. Lander E.S. Mesirov J.P. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles.Proc Natl Acad Sci USA. 2005; 102: 15545-15550Crossref PubMed Scopus (26525) Google Scholar implemented in a Java GSEA desktop application version 2.0 (http://www.broadinstitute.org/gsea/index.jsp) was used to determine whether POSTN has a function correlated with the cell cycle and cell proliferation. Gene sets for use with GSEA were taken from REACTOME (http://www.reactome.org)27Vastrik I. D'Eustachio P. Schmidt E. Gopinath G. Croft D. de Bono B. Gillespie M. Jassal B. Lewis S. Matthews L. Wu G. Birney E. Stein L. Reactome: a knowledge base of biologic pathways and processes.Genome Biol. 2007; 8: R39Crossref PubMed Scopus (481) Google Scholar and Gene Ontology (GO) (http://www.geneontology.org).28Ashburner M. Ball C.A. Blake J.A. Botstein D. Butler H. Cherry J.M. Davis A.P. Dolinski K. Dwight S.S. Eppig J.T. Harris M.A. Hill D.P. Issel-Tarver L. Kasarskis A. Lewis S. Matese J.C. Richardson J.E. Ringwald M. Rubin G.M. Sherlock G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium.Nat Genet. 2000; 25: 25-29Crossref PubMed Scopus (26989) Google Scholar Pearson's correlation coefficient of POSTN per stromal cell [as the ratio of POSTN to vimentin (POSTN/VIM)] and each gene expression level across all samples >100 were used to rank genes. Expression levels of probe sets 210809_s_at and 201426_s_at were used as POSTN and VIM mRNA expression, respectively. Human tissue materials (32 non-neoplastic gastric mucosae, 87 intestinal-type gastric cancers, and 50 diffuse-type gastric cancers) were obtained during surgery and retrieved from the archives of the University of Tokyo Hospital during 2005 to 2006. This study was approved by the Ethics Committee of the Graduate School of Medicine of the University of Tokyo (no. 2381). Human diffuse-type gastric cancer cell lines OCUM-2MLN and OCUM-1229Fujihara T. Sawada T. Hirakawa K. Chung Y.S. Yashiro M. Inoue T. Sowa M. Establishment of lymph node metastatic model for human gastric cancer in nude mice and analysis of factors associated with metastasis.Clin Exp Metastasis. 1998; 16: 389-398Crossref PubMed Scopus (53) Google Scholar and the mouse fibroblast cell line NIH3T3 expressing POSTN (NIH3T3POSTN) and its EGFP-expressing control (NIH3T3EGFP) were established as described previously.13Kikuchi Y. Kashima T.G. Nishiyama T. Shimazu K. Morishita Y. Shimazaki M. Kii I. Horie H. Nagai H. Kudo A. Fukayama M. Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.J Histochem Cytochem. 2008; 56: 753-764Crossref PubMed Scopus (107) Google Scholar OCUM-2MLN, OCUM-12, and the NIH3T3 transfectants were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (CELLECT FBS, lot no. 5766H; MP Biomedicals, Santa Ana, CA) and 100 U/mL penicillin–100 μg/mL streptomycin (Sigma-Aldrich) at 37°C in a humid atmosphere saturated with 5% CO2. C57BL/6 Postn−/− mice (previously established in our laboratory30Kii I. Amizuka N. Minqi L. Kitajima S. Saga Y. Kudo A. Periostin is an extracellular matrix protein required for eruption of incisors in mice.Biochem Biophys Res Commun. 2006; 342: 766-772Crossref PubMed Scopus (111) Google Scholar) were intercrossed with C57BL/6 Rag2−/− mice (Taconic Farms, Germantown, NY) to obtain POSTN and RAG-2 double-knockout mice. The method using Rag2−/− mice had been adopted in a study to survey the function of every type of host-derived matrix metalloproteinase in the cancer microenvironment.31Acuff H.B. Carter K.J. Fingleton B. Gorden D.L. Matrisian L.M. Matrix metalloproteinase-9 from bone marrow-derived cells contributes to survival but not growth of tumor cells in the lung microenvironment.Cancer Res. 2006; 66: 259-266Crossref PubMed Scopus (143) Google Scholar Genotyping procedures for Postn and Rag2 were performed as described previously30Kii I. Amizuka N. Minqi L. Kitajima S. Saga Y. Kudo A. Periostin is an extracellular matrix protein required for eruption of incisors in mice.Biochem Biophys Res Commun. 2006; 342: 766-772Crossref PubMed Scopus (111) Google Scholar and according to the supplier's protocol. Xenograft studies were performed using female BALB/c nude mice, obtained from Charles River Laboratories International (Kanagawa, Japan; Wilmington, MA). Mice were handled under specific-pathogen-free conditions in accordance with the policies of the Animal Ethics Committee of the University of Tokyo (no. 1822T-020). Tissue samples were fixed in 4% neutral formalin and were embedded in paraffin routinely. The primary antibodies used for the present study included a previously established rabbit polyclonal anti-POSTN,5Shimazaki M. Kudo A. Impaired capsule formation of tumors in periostin-null mice.Biochem Biophys Res Commun. 2008; 367: 736-742Crossref PubMed Scopus (28) Google Scholar anti–αsmooth muscle actin (anti–α-SMA) (1:50; 1A4; Dako–Agilent Technologies, Glostrup, Denmark), anti–human Ki-67 (1:200; MIB-1; Dako–Agilent Technologies), and anti–p-p44/42 MAPK (1:200; no. 9101; Cell Signaling Technology, Danvers, MA). IHC and immunofluorescence microscopy were performed (sections 5 μm thick) as described previously.13Kikuchi Y. Kashima T.G. Nishiyama T. Shimazu K. Morishita Y. Shimazaki M. Kii I. Horie H. Nagai H. Kudo A. Fukayama M. Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.J Histochem Cytochem. 2008; 56: 753-764Crossref PubMed Scopus (107) Google Scholar For semiquantifying POSTN expression, we scored four groups according to prevalence: score 0, 80%. We regarded scores 2 and 3 as significant and positive. Antisense and sense complementary (c)RNA probes were prepared by in vitro transcription of EcoRI–XbaI fragment of human and mouse POSTN cDNA using a DIG labeling mix (Roche Applied Science, Indianapolis, IN) as described previously.32Kashima T.G. Nishiyama T. Shimazu K. Shimazaki M. Kii I. Grigoriadis A.E. Fukayama M. Kudo A. Periostin, a novel marker of intramembranous ossification, is expressed in fibrous dysplasia and in c-Fos-overexpressing bone lesions.Hum Pathol. 2009; 40: 226-237Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Nonradioactive mRNA in situ hybridization was performed manually on formalin-fixed, paraffin-embedded sections (5 μm thick) as described previously.13Kikuchi Y. Kashima T.G. Nishiyama T. Shimazu K. Morishita Y. Shimazaki M. Kii I. Horie H. Nagai H. Kudo A. Fukayama M. Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.J Histochem Cytochem. 2008; 56: 753-764Crossref PubMed Scopus (107) Google Scholar OCUM-2MLN and either of the NIH3T3 transfectants (NIH3T3POSTN or NIH3T3EGFP), each at a final concentration of 1 × 106/mL, were suspended in 1 mL of the mixture of type I collagen gel. Three-dimensional coculturing was then performed for 2 weeks, as described previously.13Kikuchi Y. Kashima T.G. Nishiyama T. Shimazu K. Morishita Y. Shimazaki M. Kii I. Horie H. Nagai H. Kudo A. Fukayama M. Periostin is expressed in pericryptal fibroblasts and cancer-associated fibroblasts in the colon.J Histochem Cytochem. 2008; 56: 753-764Crossref PubMed Scopus (107) Google Scholar Subsequently, 10% phosphate-buffered and formalin-fixed, paraffin-embedded sections were studied with H&E staining. Microphotographs of >50 foci were taken at random locations and cancer cells per high-power field were counted. Proliferation assay was performed using a cell-counting kit (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) as described in the technical manual. The final concentration was supplemented with 0.1% bovine serum albumin and either recombinant human POSTN (250 ng/mL, BioVendor, Heidelberg, Germany) dissolved in PBS or vehicle alone. Phosphorylated MAP kinase protein array analysis was performed using a human p-MAPK array kit (Proteome Profiler antibody array; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Samples of cell lysates were obtained after 24 hours incubation in a no-serum condition, supplemented with 1 μg/mL of recombinant human POSTN (BioVendor) or equal amounts of PBS. p-MAPK levels were detected using chemiluminescence. The signal intensity was quantified using an LAS-3000 imaging system (Fujifilm, Tokyo, Japan) and Multi Gauge software version 2.2 (Fujifilm). For detection of ERK phosphorylation by POSTN, cells were cultured overnight in a no-serum condition after 1 hour of incubation supplemented with 250 ng/mL of recombinant human POSTN (BioVendor) or equal amounts of PBS. Cell lysate samples were prepared in radioimmunoprecipitation assay buffer separated on 8% polyacrylamide gels. They were electroblotted onto a polyvinylidene difluoride membrane. After blocking, the membranes were incubated for 1 hour at room temperature with an anti-ERK (1:1000; sc-94; Santa Cruz Biotechnology) or an anti–p-p44/42 MAPK (1:1000; no. 9101; Cell Signaling Technology). The membranes were washed again and incubated for 1 hour with secondary antibodies. The antigen was then detected using enhanced chemiluminescence Western blot detection reagents (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions. OCUM-2MLN or OCUM-12 (5 × 106 cells) and NIH3T3POSTN or NIH3T3EGFP (1 × 106 cells) were suspended in 50 μL of PBS and were coinjected subcutaneously in the flanks (left flank with NIH3T3POSTN; right flank with NIH3T3EGFP) of nude mice. Tumor volumes were calculated using the following formula: volume V = (W2 × L)/2, where W is the short diameter and L is the long diameter. Histological features of the xenografts were examined with H&E staining. H&E sections were scanned using a Nanozoomer 2.0HT system (Hamamatsu Photonics, Hamamatsu, Japan) and the tumor area was analyzed using NDP.view viewer software version 1.1.27 (Hamamatsu Photonics). A total of 5 × 106 cells suspended in 50 μL of PBS were inoculated subserosally into the gastric walls of mice, as described previously.29Fujihara T. Sawada T. Hirakawa K. Chung Y.S. Yashiro M. Inoue T. Sowa M. Establishment of lymph node metastatic model for human gastric cancer in nude mice and analysis of factors associated with metastasis.Clin Exp Metastasis. 1998; 16: 389-398Crossref PubMed Scopus (53) Google Scholar For the present study, we used Postn−/−Rag2−/− mice (n = 6) and Postn+/+Rag2−/− mice (n = 7), aged 4 to 5 weeks. At 5 weeks after inoculation, the mice were sacrificed for subsequent evaluation procedures. For analysis of publicly available microarray data sets, the data were evaluated using Wilcoxon signed-rank test for statistical comparisons. The trend test was performed with a Jonckheere–Terpstra test. For the distribution of POSTN expression in IHC, a Cochrane–Armitage trend test was applied. These statistical tests were performed using R software version 2.11.0 (http://www.r-project.org). In vitro and in vivo growth data were evaluated with two-way analysis of variance using GraphPad Prism software version 4 (GraphPad Software, San Diego, CA). All other numerical results are expressed as means ± SEM. Data were evaluated using U-tests for statistical comparisons using StatView software version 4 (SAS Institute, Cary, NC). P < 0.05 was considered significant for all statistical tests. To study whether POSTN expression is up-regulated in gastric cancer, we first analyzed POSTN mRNA levels in normal gastric tissue, gastric cancer tissue, and gastric cancer cell lines from publicly available microarray data sets (GSE15460 and GSE7307). POSTN expression was found in both normal and neoplastic tissues, without any marked differences, although all gastric cancer cell lines (excepting only YCC11) expressed negligible POSTN (Figure 1A). In a closer comparison of POSTN mRNA expression levels between cancer and matched normal stomach samples, we analyzed another data set including an annotation for the clinical stage (GSE19826). It is particularly interesting that no difference between cancer and normal tissue samples was found for stage I gastric cancer (P = 0.98, Wilcoxon signed-rank test; n = 3), although cancer tissue samples showed significantly higher POSTN expression than each corresponding background normal tissue for stage II, III, and IV gastric cancer (P = 0.049, Wilcoxon signed-rank test; n = 9) (Figure 1B). We further analyzed the correlation between POSTN mRNA level and tumor progression using the GSE15460 data set. The trend test results showed that the POSTN mRNA expression in tumor samples was proportionally greater at the higher T stages (P = 0.0033, Jonckheere–Terpstra test) (Figure 1C). To correlate the stromal amount and VIM expression, we analyzed the data set GSE8218, comprising 136 cases of prostate cancer for which the stromal percentage was available for each tumor sample (Supplemental Figure S1). Raw data were retrieved and processed using the MAS5 algorithm (Affymetrix). Pearson correlation coefficients indicated correlation between VIM mRNA expression and the amount of stroma in prostate cancer (R = 0.525, P = 5.35 × 10−11) (Supplemental Figure S1). No correlation was found between keratin 18 mRNA expression and the stromal amount (data not shown). To standardize the number of stromal cells, we next performed a trend test against POSTN/VIM mRNA expression ratio. The POSTN/VIM mRNA expression ratio in tumors was also associated significantly with the T stage (P = 0.0077, Jonckheere–Terpstra test) (Figure 1C). We extensively investigated POSTN protein expression in both non-neoplastic and neoplastic gastric tissues. Two distinctive immunostaining patterns, indicative of periglandular and stromal POSTN deposition, were noted in the ECM of normal gastric mucosa. Periglandular-type staining showed linear POSTN immunoreactivity abutting the gastric foveolus and surrounding the proper gastric glands (Figure 2, A–C). Stromal staining, however, was indicative of intense POSTN deposition in the ECM of reactive gastric mucosa, in which there is an inflammatory cell infiltrate and an increase in vascularity, in addition to more pronounced periglandular immunoreactive POSTN in the intestinal metaplasia area (Figure 2D). The stromal staining pattern was barely visible in the normal lamina propria mucosae. Immunofluorescent double staining for POSTN and α-SMA showed a focal close approximation of α-SMA+ cells to POSTN immunoreactivity, which encircled normal gastric glands (Figure 2E). No specific staining of epithelial cells or other mesenchymal cell types was noted, with the exception of occasional POSTN deposition in the vascular wall. In the submucosal and muscular layer, POSTN expression was focally detected in perivascular cells of capillaries (data not shown). Next, we examined POSTN expression in gastric adenocarcinomas according to the two histological subtypes, the intestinal type (n = 87) and the diffuse type (n = 50).23Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification.Acta Pathol Microbiol Scand. 1965; 64: 31-49Crossref PubMed Scopus (5048) Google Scholar Periglandular POSTN staining was found in all of the non-neoplastic tissues (n = 14), although it was dramatically lower in carcinoma of the intestinal subtype at Tis, in 19% (10/52) of cases, which did not involve the basement membrane (Figure 3, A–C, and Supplemental Table S1). Furthermore, periglandular POSTN staining was observed in only 8% (1/13) of cases of the invasive intestinal-type adenocarcinomas at T1, which involved the lamina muscularis mucosae and which extended into the submucosa (Figure 3C). Periglandular POSTN expression tended to decrease with progressing stage (P = 7.62 × 10−8). Invasive intestinal-type adenocarcinomas in stages T2 to T4 showed prominent stromal POSTN deposition, rather than periglandular staining (Figure 3D), in contrast to noninvasive intestinal-type cancers. The immunoreactive POSTN was fibrillar and was confined at the invasive front of invasive tumor nests. However, all diffuse-type adenocarcinomas, especially the linitis plastica type, showed prominent stromal-type POSTN staining in

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