Identification of Genes Expressed in Malignant Cells That Promote Invasion

[CANCER RESEARCH 63, 8939–8947, December 15, 2003] Identification of Genes Expressed in Malignant Cells That Promote Invasion

Jennifer Walter-Yohrling, Xiaohong Cao, Michele Callahan, William Weber, Sharon Morgenbesser, Stephen L. Madden, Clarence Wang, and Beverly A. Teicher Genzyme Corporation, Framingham, Massachusetts

ABSTRACT study tumor-stromal interactions. Janvier et al. (17) suspended a mixture of fibroblasts, endothelial cells, and human PC3 prostate To systematically identify genes related to invasion a three-dimensional carcinoma cells in collagen or fibrin and demonstrated the importance multicellular matrix invasion assay was used to classify human tumor cell of cellular interactions between endothelial cells and fibroblasts in lines as stromal invasion positive or stromal invasion negative. Cells from two of the primary cell types of the stromal compartment [endothelial cells tube formation. Shekhar et al. (18) showed that breast carcinoma- (HMVEC) and myofibroblasts (HDF)] were assayed for invasion into derived fibroblasts, breast carcinoma epithelial cells, and human um- tumor cell clusters (breast carcinoma, ovarian carcinoma, prostate carci- bilical vascular endothelial cell cultured on a layer of Matrigel devel- noma, lung carcinoma, and melanoma). Four tumor cell lines (MDA- oped a compartmentalized spheroid with a core of fibroblasts and MB231, SKOV-3, A375, and MEL624) scored invasion positive, and four endothelial cells surrounded by epithelial cells. These heterogeneous tumor cell lines (LNCaP, DU145, PC3, and A549) scored invasion nega- spheroids had increased proliferation and invasion, degradation of tive. Serial analysis of gene expression (SAGE) libraries generated from extracellular matrix, and expression of matrix metalloproteinase 9. the tumor cell lines were analyzed by GeneSpring Hierarchical clustering, These cell culture models support the observation that stromal cells ␹2 t test, and test. Clusters emerged that reflected the behavior in the cell play an important role in tumor angiogenesis. culture assay. Of the 47 most highly differentially expressed genes, 30 We have described previously a multicellular stromal invasion were selected for confirmation by real-time PCR, and 9 had good correlation with normalized serial analysis of gene expression tag counts. The assay using three cell types involved in tumor angiogenesis (endothe- strongest correlations were for bone marrow stromal antigen 2, stathmin- lial cells, myofibroblasts, and tumor cells; Ref. 19). SKOV3 ovarian like 3, tumor necrosis factor receptor superfamily member 5, and hepa- tumor cells suspended in a collagen I matrix surrounded by Matrigel tocyte growth factor tyrosine kinase substrate. In situ hybridization of were cultured with fluorescently labeled mature endothelial cells or metastatic and nonmetastatic ovarian cancer demonstrated selective ex- myofibroblasts or with a 1:1 mixed population of mature endothelial pression of bone marrow stromal antigen 2 and tumor necrosis factor cells and myofibroblasts. The mature endothelial cells adhered to the receptor superfamily member 5 in the metastatic disease. This combina- outside of the tumor cell cluster, whereas myofibroblasts invaded the tion approach appears to be a powerful tool for identifying genes that may cluster and localized in the tumor cell cluster. The mixed population be useful as diagnostic markers and/or as therapeutic targets for invasive of mature endothelial cells and myofibroblasts colocalized in the solid tumors. center of the SKOV-3 tumor cluster. Thus, it appeared that the myofibroblasts enabled invasion by the mature endothelial cells. INTRODUCTION Serial analysis of gene expression (SAGE) has been extensively used for expression analyses of various types of cancers for identify- Cancer has been described as a progression of genetic mutations in ing novel diagnostic and prognostic markers and potential therapeutic an aberrant tissue mass, and many oncogenes and tumor suppressor targets, as well as for identifying pathways up-regulated during ma- genes have been identified (1). The notion that tumor progression after lignant transformation and progression (20–23). Herein SAGE was the genetic events that initiate malignancy critically involve interac- used in combination with the tumor cell cluster stromal invasion assay tions between the malignant cells and the normal cells in the tumor with eight human tumor cell lines to systematically investigate the environment has come to the fore more recently (2, 3). It is now genes expressed by tumor cells that promoted interactions between accepted that stromal cells as well as cells from distant locations tumor cells and stromal cells. Combining SAGE with the in vitro actively contribute to tumor growth, invasion, and metastasis (4–6). tumor cluster model to identify genes expressed by tumor cells that Epithelial cells use a wide variety of factors to sense and to respond enabled stromal cell invasion provided a statistically robust gene to their environment. These include cell adhesion molecules, such as expression analysis based on the eight human tumor cell lines. Several intergrins (7), cadherins and the associated catenins (8), and growth genes identified through the analysis were validated using quantita- factors, such as fibroblast growth factor (9), and transforming growth tive, real-time PCR and in situ hybridization. factors ␣ and ␤ (10). Stroma often comprise a major portion of solid tumors such as breast, colon, and prostate carcinoma (11–13). The most prominent MATERIALS AND METHODS stromal cell type, the myofibroblast, secretes large amounts of extra- Materials. Human adult dermal microvascular endothelial cells (HM- cellular matrix proteins responsible for the host desmoplastic response VECs) and EGM2-MV medium were purchased from Clonetics (Walkersville, (14). Myofibroblasts express many growth factors involved in tumor MD). MDA-MB-231, [American Type Culture Collection (ATTC) no. HTB- angiogenesis including vascular endothelial growth factor, basic fi- 26, human breast carcinoma cell line], A-375 (ATCC no. CRL-1619, human broblast growth factor, and transforming growth factor ␤, thus stim- melanoma cell line), LNCaP (ATCC no. CRL-10995, human prostate carci- ulating endothelial cell migration, invasion, proliferation, and vessel noma), A-549 (ATCC no. CCL-185, human lung carcinoma), PC-3 (ATCC no. stability (15, 16). Several in vitro models have been developed to CCL-1435, human prostate carcinoma cell line), DU-145 (ATCC no. HTB-81, human prostate carcinoma cell line), and SKOV-3 cells (ATCC no. HTB-77, human ovarian carcinoma cell line) were purchased from ATTC (Manassas, Received 8/8/03; revised 10/1/03; accepted 10/7/03. The costs of publication of this article were defrayed in part by the payment of page VA). The melanoma cell line, Mel624 was a gift from Dr. Steven Rosenberg charges. This article must therefore be hereby marked advertisement in accordance with (National Cancer Institute, Bethesda, MD). Human adult dermal fibroblasts 18 U.S.C. Section 1734 solely to indicate this fact. (HDFs) were a gift from Dr. James Gailit (State University of New York, Notes: Drs. Walter-Yohrling and Cao contributed equally to this work. Stony Brook, NY). DMEM, fetal bovine serum, and F12/Ham’s medium were Requests for reprints: Beverly A. Teicher, Genzyme Corporation, 1 Mountain Road, Framingham, MA 01701-9322. Phone: (508) 271-2843; Fax: (508) 620-1203; Email: purchased from Life Technologies, Inc. (Gaithersburg, MD). Falcon tissue [email protected]. culture flasks, 24-well plates, and Matrigel were purchased from Becton 8939

Table 1 Sample and tag summary for SAGEa libraries included in the analysis SAGE libraries for various tumor cell lines were constructed as described previously. There were 67,735 total unique tags. Stromal invasion positive Stromal invasion negative

Cell line Tissue of origin Total SAGE tag counts Cell line Tissue of origin Total SAGE tag counts MDA-231 Breast 31221 DU145 Prostate 36977 SKOV3 Ovary 50349 PC-3 Prostate 43223 A375 Melanoma 18245 A549 #1 Lung 11396 MEL624 Melanoma 52307 A549 #2 Lung 26279 LNCaP #1 Prostate 23029 LNCaP #2 Prostate 41625 a SAGE, serial analysis of gene expression.

Dickinson (Franklin Lakes, NJ). The fluorescent label, PKH26, was purchased group comparison, and ␹2 test for the comparison of the averages of each from Sigma Chemical Company (St. Louis, MO). Collagen I (Vitrogen) was group. Confidence interval levels (90% for the ␹2 test and 95% for the t test) supplied by Cohesion Technologies (Palo Alto, CA). PCR primers were were used as significance filters. Hierarchical clustering was performed on purchased from Integrated DNA Technologies (Coralville, IA). Trizol filtered libraries using GeneSpring software release 5.0.2 build number 954 was purchased from Sigma, and the RNA Extraction kit was obtained from (Silicon Genetics, Redwood City, CA). Pearson correlation for similarity Qiagen (Valencia, CA). The High Capacity cDNA Archive kit, Taqman rRNA measurement and the minimum distance was set to 0.001. SAGE tags were Control Reagents, Taqman Universal PCR Master Mix, and Sybr Green mapped to UniGene (Build #157) clusters using modified tag to gene mapping. PCR Master Mix were purchased from Applied Biosystems (Foster City, This mapping was generated to maximize both the specificity and coverage of CA). Tissue sections from a human ovarian metastatic tumor (Coopera- the UniGene clusters that mapped to a single tag. UniGene cluster numbers tive Human Tissue Network #25412) and a nonmetastatic tumor (Cooperative were additionally used as links to obtain functional and cellular localization Human Tissue Network #25439) were obtained from the Cooperative Human annotation for individual genes. Tissue Network at the National Cancer Institute (Bethesda, MD). Quantitative PCR. Each of the eight human tumor cell lines were grown Cell Culture. All of the cells were grown in a humidified incubator at 37°C to confluence in T75 flasks suspended and lysed using TRIzol. Cellular RNA and 5% CO2. HMVECs (up to passage 9) were maintained in EGM2-MV. The was isolated by phenol:chloroform extraction followed by column isolation eight human tumor cell lines and human dermal fibroblasts were grown in using the Qiagen RNA Extraction kit. cDNA was generated using the High DMEM-supplemented 10% fetal bovine serum and 10 units/ml penicillin/10 Capacity cDNA Archive kit. Real-time PCR for was performed with Sybr ␮g/ml streptomycin (Invitrogen Life Technologies, Inc., Grand Island, NY). Green PCR Master Mix using PCR primers on an ABI Prism 7900 Sequence Human dermal fibroblast cultures at high passage number were confirmed to Detection System (Applied Biosystems). Relative mRNA expression was be substantially comprised of myofibroblasts as indicated by ␣-smooth muscle determined by dividing the threshold of each sample by the threshold of 18S, actin expression analyzed by fluorescence activated flow cytometry (data not solving for 2x and adjusting the relative value to the whole number. shown; Ref. 11). In Situ Hybridization. In situ hybridization was carried out using a mod- In Vitro Model. The in vitro tumor cell cluster model of stromal invasion ification of reported procedures (21). cDNA fragments for the BST2 and has been described (19). Briefly, a thick layer of Matrigel (300 ␮l) was added TNFRS5 mRNAs were generated by PCR amplification of fragments ranging to each well of a 24-well plate and allowed to polymerize. A plug of Matrigel from 200 bases to 650 bases, using primers with T7 promoters incorporated of ϳ1 mm in diameter was removed using a glass pipette under light vacuum. into the antisense primers. Digoxigenin riboprobes were generated by in vitro The resulting space was filled with tumor cells (1 ϫ 106) suspended in a 2.4 transcription in the presence of digoxigenin, according to manufacturer’s mg/ml collagen I solution (5 ␮l) prepared according to the manufacturer’s instructions (Roche, Indianapolis, IN). Human ovarian tumor sections were suggestions. The collagen was allowed to polymerize for 30 min. Myofibro- deparaffinized in xylene, washed in 100% ethanol, and then hydrated in 85%, blasts (HDF) were fluorescently labeled with PKH26 (red) according to the 75%, and 50% ethanol in distilled water. After incubation in diethylpyrocar- manufacturer’s suggested protocol. Briefly, myofibroblasts suspended in bonate-treated water, sections were permeabilized by treatment with pepsin in serum-free medium were incubated in the presence of 2.5 ␮M PKH67 diluted 0.2 N hydrochloric acid, washed briefly in PBS, then fixed in 4% paraform- for 5 min. The labeling was terminated with 1 ml of fetal bovine serum for 1 aldehyde. The sections were acetylated in acetic anhydride/0.1 M triethanola- min followed by three washes in serum-containing medium. After the washes, mine (pH. 8.0), equilibrated for 10 min in 5ϫ SSC, and prehybridized for 1–2 the labeled myofibroblasts were suspended in EGM2-MV and counted. HM- hat55°C in mRNA hybridization buffer (DAKO, Carpinteria, CA) and then VECs were infected with enhanced green fluorescent protein-expressing ade- hybridized with digoxigenin riboprobes (100–200 ng/ml) in mRNA hybrid- novirus at 200 multiplicity of infection for 18 h. A total of 30,000 cells (15,000 ization buffer (DAKO) overnight at 55°C. After removing unbound riboprobes HMVECs and 15,000 myofibroblasts) were added to each well in EGM2-MV by washing, sections were incubated with RNase (Ambion, Austin, TX) to (1 ml). After 48 h, the fluorescently labeled cells were visualized using a remove any nonspecifically bound riboprobe and treated with peroxidase block fluorescein (enhanced green fluorescent protein/HMVEC) or rhodamine (DAKO) to eliminate any endogenous peroxidase, then blocked with a 1% (PKH26/myofibroblasts) filter. In some experiments, 4Ј,6-diamidino-2- blocking reagent (DIG nucleic acid detection kit; Roche), containing rabbit phenylindole-labeled human MDA-MB231 breast carcinoma cells and human immunoglobulin fraction (DAKO) in Tris-buffered saline). Rabbit antidigoxi- PC-3 prostate carcinoma cells were used. Fluorescent and bright field images genin-horseradish peroxidase (DAKO) was used to detect the riboprobes and were captured with a ϫ4 objective on a Sony DXC-390 digital camera using to catalyze the deposition of biotinylated tyramide (Gen-Point; DAKO) ac- Scion Image version 1.62c. Experiments were repeated three times. cording to the manufacturer’s instructions. Final detection was accomplished Construction and Analysis of SAGE Libraries. SAGE libraries for the through rabbit antibiotin-conjugated to alkaline phosphatase (DAKO). Alka- eight tumor cell lines were constructed as described (24). The SAGE libraries line phosphatase was visualized with Fast Red (DAKO) for 10–60 min at corresponding to each tumor cell line were retrieved from either the Genzyme room temperature and then counterstained in hematoxylin. The nuclei are proprietary database or the public Gene Expression Omnibus database.1 Table blued with ammonium hydroxide for 30 s, and then mounted with crystal- 1 lists the sample and tag information for the libraries. Tag counts were mount (BioMeda, Foster City, CA). normalized to 50,000 total library counts for each library. Initially 2 tag counts were removed from at least 2 of the 11 libraries as a filter for erroneous tags. RESULTS Libraries were separated into the “Stromal Invasion” group and the “Non- Stromal Invasion” group based on the behavior of the cell lines in the in vitro To determine whether tumor cell lines could be grouped by their assay. Two statistical tests were applied to the SAGE tag counts, t test for the ability to enable stromal invasion, eight human tumor cell lines were analyzed in the tumor cell cluster model for stromal invasion (19). 1 Internet address: http://www.ncbi.nlm.nih.gov/geo/. Tumor cell clusters (1 ϫ 106 cells) suspended in collagen and sur- 8940

Fig. 1. Comparison of the eight human tumor cell lines. A, 1:1 mixture of human adult dermal microvascular endothelial cells (HMVECs; green) and myofibroblasts (human adult dermal fibroblasts; red) was cocultured with human tumor cell clusters for 48 h. Bright field and fluorescence images were collected. In some wells, HMVECs and myofibroblasts formed a shell around the tumor cell clusters (stromal invasion negative). In other wells, HMVECs and myofibroblasts invaded the tumor cell clusters (stromal invasion positive). rounded by Matrigel were cultured in the presence of 1:1 mixtures of tion of the collagen induced by the myofibroblasts and in part from fluorescently labeled endothelial cells (HMVECs) and myofibroblasts migration of the tumor cells from the cluster (Figs. 1 and 2; Ref. 25). (HDFs; Fig. 1). After 48 h, the wells were scored for fluorescence In a second experiment, human MD-MBA-231 breast carcinoma cells localization within the tumor cell cluster. Among the eight human and human PC-3 prostate carcinoma cells were labeled with a tracer tumor cell lines, the breast carcinoma cell line MDA-MB-231, the concentration of 4Ј,6-diamidino-2-phenylindole and were subjected to ovarian carcinoma cell line SKOV-3, and the melanoma cell lines the same assay described above. After 48 h, fluorescent images A375 and Mel624 had the greatest concentration of fluorescence showed the MDA-MB231 cells moving into the Matrigel, whereas the within the tumor cell cluster. However, in the wells containing tumor PC-3 cells remained in the collagen plug (Fig. 2). cell clusters of the non-small cell lung carcinoma cell line A549, and SAGE data were used to obtain a comprehensive, unbiased com- the prostate carcinoma cell lines LNCaP, PC-3, and DU145 most of parison of gene expression between the human tumor cell lines that the fluorescence remained outside of the tumor cell cluster. Tumor underwent efficient invasion by the myofibroblasts and endothelial cell clusters that were efficiently invaded by the myofibroblasts and cells with those cell lines that did not. In each SAGE library, sequence endothelial cells decreased in size in part possibly from the contrac- tags of 10–11 bases from the last CATG site at the 3Ј end of each

Fig. 2. Comparison of invasive and noninvasive human tumor cells. Clusters of 4Ј,6-diamidino-2- phenylindole-labeled human MDA-MB231 breast carcinoma cells and human PC-3 prostate carcinoma cells were cocultured with human adult dermal microvascular endothelial cells (HMVEC), 1:1 HMVECs and myofibroblasts (human adult dermal fibroblasts; MyoF), or myofibroblasts for 48 h. Fluorescence images were collected showing MDA-MB231 cells moving into the Matrigel, whereas PC-3 cells remained within the collagen plug.

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Fig. 3. Hierarchical clustering of serial analysis of gene expression libraries. Tags with Ͼ2 counts in only 2 of the 10 libraries were filtered out to remove erroneous tags. Tumor cell lines that enabled stromal invasion (red) and tumor cell lines that did not enable stromal invasion (black) did not form distinctive subclusters. Thus, genes that enable the invasive behavior of the stromal cells did not dictate the general gene expression profiles of tumor cells. transcript and their respective counts were acquired. The relative was applied for group comparisons. The t test was not able to abundance of a specific tag in a SAGE library was considered to be eliminate tags with very low counts in one group and zeros in the proportional to the expression level of its corresponding mRNA (24). other group. Therefore the ␹2 test, a frequency-based test, was used as There were total 64,478 unique SAGE tags generated from 10 SAGE an additional filter. When significance values of P Յ 0.05 for t test libraries obtained from monolayer cultures of the eight human tumor and 0.1 for c2 test were applied, 99 SAGE tags emerged with signif- cell lines. After normalization of each of the libraries to 50,000 SAGE icant expression differentials between the two groups of tumor cell tag counts, 7,791 tags had counts of Ն2 in at least 2 of the 10 libraries. lines. These SAGE tags corresponded to 75 known genes, 25 previ- To compare the expression profiles of the eight human tumor cell ously un-named genes, and 3 unmatched tags. When the 10 libraries lines, an initial hierarchical clustering of all of the SAGE libraries was were subjected to GeneSpring analysis using the 99 differentially carried out using GeneSpring software (Fig. 3). There was no clear expressed SAGE tags, two distinct groups corresponding to the effi- grouping in the clustering tree among those tumor cell lines that were ciency of invasion by the stromal cells into the tumor clusters formed efficiently invaded by the myofibroblasts and endothelial cells and (Fig. 4A). those that were not. Thus, the expressed genes that allowed efficient Two of the SAGE libraries included in the statistical analysis had stromal invasion were likely represented by a small percentage of the Ͻ20,000 tags and, thus, may have been artificially elevated or reduced total transcripts in the tumor cells and differences in the expression when the tag counts were normalized to 50,000 (27). To evaluate the levels of these genes from cell line to cell line were not sufficient to effect of library size on the analysis, a statistical comparison was direct the clustering process. Tumor cell lines from the same tissue of carried out on the eight SAGE libraries that exceeded 20,000 tags. origin were not always nearest neighbors and did not form distinct After applying the same statistical criteria described above, 87 SAGE subclusters in the GeneSpring analysis. This type of observation has tags emerged as significantly differentially expressed between tumor been made previously when analyzing endothelial cells from human cell lines that were efficiently invaded by stromal cells and those that tumors along with human umbilical vascular endothelial cell and were not. These SAGE tags corresponded to 64 known genes, 21 HMVEC from cell culture, and when comparing normal cells and previously un-named genes, and 5 unmatched tags. The eight libraries malignant cells from the same tissues of origin. Therefore, the mixing with Ͼ20,000 SAGE tags formed two clusters when subjected to of different tumor cell lines in the clustering diagram was not sur- GeneSpring analysis (Fig. 4B). Forty-seven of the 87 SAGE tags from prising (20, 21). the eight library analysis overlapped with the 99 tags from the 10 The availability of 10 SAGE libraries representing the eight human library analysis. tumor cell lines provides multiple samples in each phenotypic group To validate the differential gene expression profiles identified by allowing statistical methods to be applied to the data (26, 27). A analysis of the SAGE libraries, 30 genes were selected for real-time combination of two statistical tests was used to extract tags with PCR examination in the eight tumor cell lines. The differential gene significant differential expression. t test, which has been widely used expression of 9 of the genes analyzed by real-time PCR showed good when comparing gene expression data from microarray experiments, correlation with normalized SAGE tag counts. The strongest correla- 8942

Fig. 4. Hierarchical clustering of serial analysis of gene expression libraries with statistically selected genes. A, experimental and gene tree from full library analysis. B, experimental and gene tree from analysis of 20kϩ libraries. Tumor cell lines that enabled stromal invasion (red), and tumor cell lines that did not enable stromal invasion (black) formed distinct subclusters. Genes “up-regulated” are shown in red and “Down-regulated” are shown in blue. 8943

fibroblasts, and/or extracellular matrix materials (2, 4, 28–31). Many stromal-derived growth factors are involved in tumor progression, including urokinase plasminogen activator, hepatocyte growth factor, transforming growth factor-␤, and epidermal growth factor (2, 4, 6, 32–34). Several studies have used SAGE to identify genes involved in malignant invasion, and have used SAGE libraries of colorectal and pancreatic cancer to compared gene expression in normal and cancer cells (35–39). Ryu et al. (37) applied the biocomputational tools, Cluster and Treeview, for hierarchical clustering to identify genes associated with invasion in 10 SAGE libraries including 2 samples derived from normal colon mucosa, 2 primary colorectal cancers, 2 colon cancer cell lines, 2 pancreatic cancer samples, and 2 pancreatic cancer cell lines. The result was 90 SAGE tags associated with invasion that mapped to 74 known genes. In situ hybridization was Fig. 5. Real-time PCR measurements and serial analysis of gene expression (SAGE) used to characterize 12 of the genes in tissue sections of human library counts. Relative expressions for reverse transcription-PCR results are shown in pancreatic carcinoma (35). Eight genes were expressed within the columns, and normalized SAGE counts are shown in lines. In general, both reverse transcription-PCR and SAGE counts showed distinctive expression differences between stromal and/or angioendothelial cells, 4 of the genes were expressed the stromal invasion positive and stromal invasion negative cell lines. by the stromal cells immediately adjacent to the invasive neoplastic epithelium, and 4 genes were expressed by the invasive neoplastic epithelium. Iacobuzio-Donahue et al. (35) elucidated genes associated tions were for bone marrow stromal antigen 2 (BST2 or HM1.24), with invasion by infiltrating breast carcinoma. SAGE tags from 11 stathmin-like 3 (STMN3), tumor necrosis factor receptor superfamily libraries including 2 normal mammary samples, 2 ductal carcinoma in member 5 (TNFRSF5), and hepatocyte growth factor tyrosine kinase situ samples, 2 invasive breast cancers, 2 lymph node metastases, and substrate (HGS or HRS; Fig. 5). The frequency of the expression of 3 breast cancer cell lines were analyzed (40). One hundred and three BST2, STMN3, TNFRS5, and HGS in 150 SAGE libraries including 87 SAGE tags associated with invasive breast carcinomas corresponding libraries representing malignant cell lines or tissues and 63 libraries to 68 known genes were identified. Six genes were investigated by in representing normal cells or tissues was determined (Table 2). The situ hybridization and were found in five different regions of the most widely expressed gene was HGS, which was observed in Ն50% tumor corresponding to different cell types. of 147 of the SAGE libraries, the exceptions being fibroblast cells, The current study used human tumor cell lines from several major normal ovary, and renal cell carcinoma. The most highly tumor- tumor types in cell culture with human dermal fibroblasts and HM- selective gene expression was observed for BST2. BST2 was ex- VECs. An earlier study found that HMVECs in coculture with the pressed by 100% of the brain tumors, renal cell carcinomas, hepato- tumor cells do not invade tumor cell clusters from any tumor cell cellular carcinomas, and lymphomas, 75% of the pancreatic lines, whereas myofibroblasts in coculture with the tumor cells do carcinomas, and Ͼ50% of the breast cancers, melanomas, and ovarian invade tumor cell clusters, and 1:1 mixtures of HMVEC and myofi- cancers. BST2 was also found frequently in normal kidney, liver, and broblasts allow both cell types to invade some of the tumor cell in dendritic cells. The tumors that most frequently expressed all four clusters (19). Among the well-established human tumor cell lines used of these genes were brain tumors, hepatocellular carcinoma, lymphomas, and pancreatic cancers. Normal kidney SAGE libraries also highly expressed these genes (STMN3, 100%; TNFRS5, 85%; BST2, Table 2 Frequency of expression of BST2, STMN3, TNFRS5, and HGS in a 50%; and HGS, 50%). The other normal tissues that had relatively SAGE libraries high expression of these representative genes were liver and dendritic Number of cells. The 29 endothelial cell SAGE libraries includes both tumor Library source libraries BST2 STMN3 TNFRS5 HGS endothelial cells and normal endothelial cells, and all four of the genes Malignant cells or tissues Brain tumors 2 2 2 0 2 had some level of expression in the endothelial cell libraries ranging Breast cancer 13 7 4 1 9 from 90% for HGS to 10% for TNFRS5. Colon cancer 15 5 4 0 12 Renal cell carcinoma 7 7 1 2 2 In situ hybridization for BST2 and STMN3 was performed on Hepatocellular carcinoma 1 1 1 0 1 samples of metastatic and nonmetastatic ovarian cancer (Fig. 6). Lung cancer 10 4 6 2 9 Substantially higher levels of both BST2 and STMN3 were observed in Lymphoma 4 4 0 2 3 Melanoma 11 7 3 2 7 the metastatic ovarian cancer tissue than the nonmetastatic ovarian Ovarian cancer 10 6 5 3 8 cancer tissue. Pancreatic cancer 4 3 3 1 2 Prostate cancer 8 1 2 1 4 Normal cells or tissues DISCUSSION Brain 1 0 1 0 1 Breast 4 1 1 1 3 The tumor stromal compartment is comprised of nonmalignant cells Colon 2 0 1 0 1 Kidney 6 3 6 5 3 including immune cells, inflammatory cells, smooth muscle cells, Liver 2 2 2 0 2 pericytes, myofibroblasts, and vascular endothelial cells (2). Mobili- Lung 4 0 0 0 2 Skin 2 0 0 0 2 zation of these normal cells by malignant cells is critical to continuing Ovary 1 0 0 1 0 tumor growth and malignant progression (1, 2, 4, 5). However, the Prostate 3 1 1 0 3 genes expressed by malignant cells that enable invasion by stromal Endothelial cells 29 12 11 3 26 Cardiomyocytes 4 0 0 0 3 cells have yet to be fully elucidated. Many human tumor xenograft Dendritic cells 3 2 0 2 2 models have enhanced take rates and growth in immunodeficient mice Fibroblasts 3 0 0 0 0 when tumor cells were implanted along with stromal cells, activated a SAGE, serial analysis of gene expression. 8944

Fig. 6. Tumor tissue section staining images of BST2 and TNFRS5 by in situ hybridization. Substantially higher level of signal staining was observed in metastatic tumor sample than the nonmetastatic tumor sample for both genes. in this study, the MDA-MB-231 cells have been recognized as inva- (48). The expression of BST2/HM1.24 gene may be regulated by the sive/metastatic in vivo when grown in nude mice (41–44), whereas the activation of signal transducers and activators of transcription 3 in prostate cancer cell lines such as LNCaP have been recognized as less response to the vascular endothelial growth factor secretion by the invasive/metastatic (45, 46). The SAGE libraries analyzed in this myeloma cells. A monoclonal antibody against HM1.24 can induce study were from tumor cell lines on the assumption that genes ex- antibody-dependent cellular cytotoxicity of multiple myeloma cells in pressed by the tumor cell lines in monolayer will allow the identifi- vitro and in vivo (49, 50). A humanized monoclonal antibody to cation of genes important to malignant invasion. The initial Gene- HM1.24 is currently in clinical trial as an immunotherapy to treat Spring clustering did not group the tumor cell lines by differential patients with multiple myeloma (48, 50–55). BST2/HM1.24 was behavior in the experimental system, in agreement with the notion that selectively expressed by a wide variety of solid tumors in the current only a small percentage of expressed genes is involved in malignant study as well as by 100% of the four lymphoma SAGE libraries invasion (20). Refinement of the clustering analysis allowed the studied. identification of several genes that may be associated with a more Stathmin-like 3/SCLIP belongs to the stathmin/oncoprotein 18 fam- invasive malignant phenotype. ily of microtubule-destabilizing phosphoproteins (56–62). It has 70% Several of the genes identified in this study have been found identity with SCG10 protein, and is involved in signal transduction previously to be involved in aspects of tumor invasion and progres- and regulation of microtubule dynamics (59). Stathmin-like 3/SCLIP sion. BST2, also known as HM1.24 antigen and EMP24, is a type II is expressed mainly in neural structures, and is found at comparable membrane protein found in the endoplasmic reticulum and cell sur- levels in neonatal and adult rat brain, suggesting a potential role not face, and expressed at high levels by multiple myeloma cells (46). The only in the acquisition, but also in the expression of differentiated promoter region of the BST2/HM1.24 gene has a tandem repeat of neuronal functions (61). Although under normal circumstances stath- three cis- elements for a transcription factor, signal transducers and min-like 3/SCLIP is expressed mainly in the nervous system, it has activators of transcription 3, which mediates interleukin 6 response been found in brain tumors and mammary gland tumors. Many pro- gene expression (47). Interleukin 6 is a differentiation factor for B teins have been found to play important roles both in neurite and cells and a paracrine growth factor for multiple myeloma cells. Vas- angiogenesis regulation, such as vascular endothelial growth factor cular endothelial growth factor triggers the increase in the interleukin (63), basic fibroblast growth factor (64, 65), pigment epithelium- 6 secretion level by the marrow stromal cells and, in turn, enhances derived factor (66), and thrombospondin-1 (67). Analysis of SAGE the paracrine interactions between myeloma and marrow stromal cells libraries found expression of stathmin-like 3/SCLIP in 100% of brain 8945

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