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Syndecan-1 in Breast Cancer Stroma Fibroblasts Regulates Extracellular Matrix Fiber Organization and Carcinoma Cell Motility

2011; Elsevier BV; Volume: 178; Issue: 1 Linguagem: Inglês

10.1016/j.ajpath.2010.11.039

1525-2191

Ning Yang, Rachel Mosher, Songwon Seo, David J. Beebe, Andreas Friedl,

Skin and Cellular Biology Research

Stromal fibroblasts of breast carcinomas frequently express the cell surface proteoglycan syndecan-1 (Sdc1). In human breast carcinoma samples, stromal Sdc1 expression correlates with an organized, parallel, extracellular matrix (ECM) fiber architecture. To examine a possible link between stromal Sdc1 and the fiber architecture, we generated bioactive cell-free three-dimensional ECMs from cultures of Sdc1-positive and Sdc1-negative murine and human mammary fibroblasts (termed ECM-Sdc1 and ECM-mock, respectively). Indeed, ECM-Sdc1 showed a parallel fiber architecture that contrasted markedly with the random fiber arrangement of ECM-mock. When breast carcinoma cells were seeded into the fibroblast-free ECMs, ECM-Sdc1, but not ECM-mock, promoted their attachment, invasion, and directional movement. We further evaluated the contribution of the structural/compositional modifications in ECM-Sdc1 on carcinoma cell behavior. By microcontact printing of culture surfaces, we forced the Sdc1-negative fibroblasts to produce ECM with parallel fiber organization, mimicking the architecture observed in ECM-Sdc1. We found that the fiber topography governs carcinoma cell migration directionality. Conversely, an elevated fibronectin level in ECM-Sdc1 was responsible for the enhanced attachment of the breast carcinoma cells. These observations suggest that Sdc1 expression in breast carcinoma stromal fibroblasts promotes the assembly of an architecturally abnormal ECM that is permissive to breast carcinoma directional migration and invasion. Stromal fibroblasts of breast carcinomas frequently express the cell surface proteoglycan syndecan-1 (Sdc1). In human breast carcinoma samples, stromal Sdc1 expression correlates with an organized, parallel, extracellular matrix (ECM) fiber architecture. To examine a possible link between stromal Sdc1 and the fiber architecture, we generated bioactive cell-free three-dimensional ECMs from cultures of Sdc1-positive and Sdc1-negative murine and human mammary fibroblasts (termed ECM-Sdc1 and ECM-mock, respectively). Indeed, ECM-Sdc1 showed a parallel fiber architecture that contrasted markedly with the random fiber arrangement of ECM-mock. When breast carcinoma cells were seeded into the fibroblast-free ECMs, ECM-Sdc1, but not ECM-mock, promoted their attachment, invasion, and directional movement. We further evaluated the contribution of the structural/compositional modifications in ECM-Sdc1 on carcinoma cell behavior. By microcontact printing of culture surfaces, we forced the Sdc1-negative fibroblasts to produce ECM with parallel fiber organization, mimicking the architecture observed in ECM-Sdc1. We found that the fiber topography governs carcinoma cell migration directionality. Conversely, an elevated fibronectin level in ECM-Sdc1 was responsible for the enhanced attachment of the breast carcinoma cells. These observations suggest that Sdc1 expression in breast carcinoma stromal fibroblasts promotes the assembly of an architecturally abnormal ECM that is permissive to breast carcinoma directional migration and invasion. Epithelial-stromal interactions play crucial roles in directing mammary gland development and in maintaining normal tissue homeostasis. Conversely, during tumorigenesis, the stroma accelerates carcinoma growth and progression. The predominant cell type within the stromal compartment is the fibroblast, which synthesizes, organizes, and maintains a three-dimensional (3D) network of glycoproteins and proteoglycans known as the extracellular matrix (ECM). Normal stromal fibroblasts and their ECM are believed to exert an inhibitory constraint on tumor growth and progression.1Bauer G. Elimination of transformed cells by normal cells: a novel concept for the control of carcinogenesis.Histol Histopathol. 1996; 11: 237-255PubMed Google Scholar, 2Kuperwasser C. Chavarria T. Wu M. Magrane G. Gray J.W. Carey L. Richardson A. Weinberg R.A. Reconstruction of functionally normal and malignant human breast tissues in mice.Proc Natl Acad Sci U S A. 2004; 101: 4966-4971Crossref PubMed Scopus (630) Google Scholar Major alterations occur in the stromal fibroblasts and ECM during neoplastic transformation, giving rise to a permissive and supportive microenvironment for carcinomas. Compared with their quiescent normal counterpart, carcinoma-associated fibroblasts display an activated phenotype, which is characterized by the expression of smooth muscle markers, an enhanced proliferative and migratory potential, and altered gene expression profiles. Carcinoma-associated fibroblasts produce and deposit elevated amounts and abnormal varieties of ECM components.3Barsky S.H. Green W.R. Grotendorst G.R. Liotta L.A. Desmoplastic breast carcinoma as a source of human myofibroblasts.Am J Pathol. 1984; 115: 329-333PubMed Google Scholar, 4Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Migration-stimulating factor: a genetically truncated onco-fetal fibronectin isoform expressed by carcinoma and tumor-associated stromal cells.Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar, 5Tuxhorn J.A. Ayala G.E. Smith M.J. Smith V.C. Dang T.D. Rowley D.R. Reactive stroma in human prostate cancer: induction of myofibroblast phenotype and extracellular matrix remodeling.Clin Cancer Res. 2002; 8: 2912-2923PubMed Google Scholar Recent evidence6Provenzano P.P. Eliceiri K.W. Campbell J.M. Inman D.R. White J.G. Keely P.J. Collagen reorganization at the tumor-stromal interface facilitates local invasion.BMC Med. 2006; 4: 38Crossref PubMed Scopus (1215) Google Scholar, 7Provenzano P.P. Inman D.R. Eliceiri K.W. Knittel J.G. Yan L. Rueden C.T. White J.G. Keely P.J. Collagen density promotes mammary tumor initiation and progression.BMC Med. 2008; 6: 11Crossref PubMed Scopus (963) Google Scholar indicates that not only ECM composition but also ECM architecture are altered in carcinomas and that these changes may promote tumor progression. However, the contribution of these stromal modifications to tumor development and the molecular mechanisms and signaling events underlying these alterations are incompletely understood. Syndecans (Sdcs) constitute a family of transmembrane heparan sulfate proteoglycans with four known members (Sdc1-4). Via their heparan sulfate glycosaminoglycan (HS-GAG) chains, Sdcs interact with a wide variety of proteins, including growth factors and ECM constituents.8Lopes C.C. Dietrich C.P. Nader H.B. Specific structural features of syndecans and heparan sulfate chains are needed for cell signaling.Braz J Med Biol Res. 2006; 39: 157-167Crossref PubMed Google Scholar, 9Tkachenko E. Rhodes J.M. Simons M. Syndecans: new kids on the signaling block.Circ Res. 2005; 96: 488-500Crossref PubMed Scopus (363) Google Scholar, 10Zimmermann P. David G. The syndecans, tuners of transmembrane signaling.FASEB J. 1999; 13: S9-S100PubMed Google Scholar Consequently, they play roles in cell growth, adhesion, migration, and morphogenesis. Sdc2 appears to be required to assemble laminin and fibronectin (FN) into a fibrillar matrix.11Klass C.M. Couchman J.R. Woods A. Control of extracellular matrix assembly by syndecan-2 proteoglycan.J Cell Sci. 2000; 113: 493-506PubMed Google Scholar Syndecan-4 has also been implied to participate in FN matrix assembly. Concomitant engagement of Sdc4 and integrins promotes Rho GTPase and focal adhesion kinase (FAK) activities, which are crucial for efficient initiation of FN matrix assembly.12Saoncella S. Echtermeyer F. Denhez F. Nowlen J.K. Mosher D.F. Robinson S.D. Hynes R.O. Goetinck P.F. Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers.Proc Natl Acad Sci U S A. 1999; 96: 2805-2810Crossref PubMed Scopus (331) Google Scholar, 13Wilcox-Adelman S.A. Denhez F. Goetinck P.F. Syndecan-4 modulates focal adhesion kinase phosphorylation.J Biol Chem. 2002; 277: 32970-32977Crossref PubMed Scopus (119) Google Scholar, 14Ilic D. Kovacic B. Johkura K. Schlaepfer D.D. Tomasevic N. Han Q. Kim J.B. Howerton K. Baumbusch C. Ogiwara N. Streblow D.N. Nelson J.A. Dazin P. Shino Y. Sasaki K. Damsky C.H. FAK promotes organization of fibronectin matrix and fibrillar adhesions.J Cell Sci. 2004; 117: 177-187Crossref PubMed Scopus (96) Google Scholar, 15Wierzbicka-Patynowski I. Schwarzbauer J.E. Regulatory role for SRC and phosphatidylinositol 3-kinase in initiation of fibronectin matrix assembly.J Biol Chem. 2002; 277: 19703-19708Crossref PubMed Scopus (52) Google Scholar Sdc1 is expressed primarily by epithelial and plasma cells of healthy adult tissue.16Sanderson R.D. Hinkes M.T. Bernfield M. Syndecan-1, a cell-surface proteoglycan, changes in size and abundance when keratinocytes stratify.J Invest Dermatol. 1992; 99: 390-396Abstract Full Text PDF PubMed Google Scholar Recently, we and others17Maeda T. Alexander C.M. Friedl A. Induction of syndecan-1 expression in stromal fibroblasts promotes proliferation of human breast cancer cells.Cancer Res. 2004; 64: 612-621Crossref PubMed Scopus (115) Google Scholar, 18Stanley M.J. Stanley M.W. Sanderson R.D. Zera R. Syndecan-1 expression is induced in the stroma of infiltrating breast carcinoma.Am J Clin Pathol. 1999; 112: 377-383Crossref PubMed Scopus (121) Google Scholar observed the induction of Sdc1 in stromal fibroblasts of invasive breast carcinomas. Syndecan-1, aberrantly expressed by stromal fibroblasts in breast carcinomas, participates in a reciprocal carcinoma growth–promoting feedback loop that requires proteolytic shedding of its ectodomain.17Maeda T. Alexander C.M. Friedl A. Induction of syndecan-1 expression in stromal fibroblasts promotes proliferation of human breast cancer cells.Cancer Res. 2004; 64: 612-621Crossref PubMed Scopus (115) Google Scholar, 19Su G. Blaine S.A. Qiao D. Friedl A. Shedding of syndecan-1 by stromal fibroblasts stimulates human breast cancer cell proliferation via FGF2 activation.J Biol Chem. 2007; 282: 14906-14915Crossref PubMed Scopus (102) Google Scholar, 20Maeda T. Desouky J. Friedl A. Syndecan-1 expression by stromal fibroblasts promotes breast carcinoma growth in vivo and stimulates tumor angiogenesis.Oncogene. 2006; 25: 1408-1412Crossref PubMed Scopus (118) Google Scholar Although the role of Sdc1 in matrix assembly has not been investigated, this molecule has interacted with various ECM components, including FN, fibrillar collagens, laminin, vitronectin, thrombospondin, and tenascin.8Lopes C.C. Dietrich C.P. Nader H.B. Specific structural features of syndecans and heparan sulfate chains are needed for cell signaling.Braz J Med Biol Res. 2006; 39: 157-167Crossref PubMed Google Scholar, 9Tkachenko E. Rhodes J.M. Simons M. Syndecans: new kids on the signaling block.Circ Res. 2005; 96: 488-500Crossref PubMed Scopus (363) Google Scholar, 10Zimmermann P. David G. The syndecans, tuners of transmembrane signaling.FASEB J. 1999; 13: S9-S100PubMed Google Scholar In the present study, we explore the possibility that Sdc1 expression by stromal fibroblasts may be causally involved in altered matrix production of tumor stroma. We find that in mammary stromal fibroblasts, Sdc1 regulates ECM assembly and determines ECM fiber architecture. We further show that cell-free 3D ECMs produced by Sdc1-expressing fibroblasts facilitate the directional migration of mammary carcinoma cells and link this activity to the parallel fiber architecture. Paraffin sections from a tissue microarray containing duplicate tumor samples from 207 patients with breast carcinoma were immunoperoxidase labeled with an antibody to Sdc1, as previously described.17Maeda T. Alexander C.M. Friedl A. Induction of syndecan-1 expression in stromal fibroblasts promotes proliferation of human breast cancer cells.Cancer Res. 2004; 64: 612-621Crossref PubMed Scopus (115) Google Scholar The immunolabeled slides were examined by bright field microscopy and scored manually, using a method developed by Harvey and coworkers.21Harvey J.M. Clark G.M. Osborne C.K. Allred D.C. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer.J Clin Oncol. 1999; 17: 1474-1481Crossref PubMed Google Scholar Sections from the same tumor blocks and those from MMTV-Wnt1–induced mammary tumors (provided by C. Alexander, Ph.D.), were stained with picro-sirius red (sirius red F3B [C.I. 35782; 0.1% w/v] in saturated picric acid aqueous solution) for 60 minutes, followed by two washes in glacial acetic acid (0.5% v/v). Images acquired during polarization microscopy were overlaid with a predesigned template that defined nine evenly distributed measurement points. The fiber closest to each of these points was identified, and the intersection angle of the nearest crossing fiber was measured using an angle measurement tool (ImageJ; http://rsbweb.nih.gov/ij/). Two observers measured the angles in the tissue microarray slides independently in a blinded fashion (intraclass correlation coefficient, 0.82). The human breast carcinoma cell lines MDA-MB-231 were from A.C. Rapraeger, T47D cells were from M. Gould, Ph.D., and NIH-3T3 fibroblasts were from J.S. Malter, MD (University of Wisconsin–Madison). Immortalized human mammary fibroblasts (HMFs) (originally called RMF-EG and HMF herein) were generously provided by C. Kuperwasser, Ph.D.2Kuperwasser C. Chavarria T. Wu M. Magrane G. Gray J.W. Carey L. Richardson A. Weinberg R.A. Reconstruction of functionally normal and malignant human breast tissues in mice.Proc Natl Acad Sci U S A. 2004; 101: 4966-4971Crossref PubMed Scopus (630) Google Scholar Cells were maintained in Dulbecco's modified Eagle's medium supplemented with either 10% fetal bovine serum (T47D cells) or 10% fetal calf serum (all other cells), 2-mmol/L l-glutamine, and 100-U/ml penicillin and streptomycin. The NIH-3T3 cells stably transfected with mouse Sdc1 were previously described.17Maeda T. Alexander C.M. Friedl A. Induction of syndecan-1 expression in stromal fibroblasts promotes proliferation of human breast cancer cells.Cancer Res. 2004; 64: 612-621Crossref PubMed Scopus (115) Google Scholar The HMF cells were stably transfected with a pcDNA3.1 vector containing the cDNA of mouse Sdc1 (a gift from A.C. Rapraeger, Ph.D) or empty vector using a commercially available system (Amaxa Nucleofection System; Lonza, Walkersville, MD) according to the manufacturer's instructions. After transfection, cells were selected with 500-μg/ml G418 and cells expressing Sdc1 at high levels were enriched by fluorescence-activated cell sorting. The HMF mock and Sdc1 cells were maintained in medium containing 250-μg/ml G418. Fibroblast-derived 3D ECM was prepared according to the protocol developed by Cukierman.22Cukierman E. Cell migration analyses within fibroblast-derived 3-D matrices.Methods Mol Biol. 2005; 294: 79-93PubMed Google Scholar, 23Cukierman E. Preparation of Extracellular Matrices Produced by Cultured Fibroblasts.in: Bonifacino J.S. Dasso M. Lippincott-Schwartz J. Harford J.B. Yamada K.M. John K. Wiley & Sons, New York2002: 10.19.11-10.19.14Google Scholar Briefly, NIH-3T3 and HMF cells were cultured in a highly confluent state for 7 days in the presence of 50-μg/ml ascorbic acid (Fisher Scientific Inc., Pittsburgh, PA). The matrix cultures were then treated with alkaline detergent solution (25-mmol/L Tris-HCl, pH 7.4; 150-mmol/L sodium chloride; 0.5% Triton X-100; and 20-mmol/L ammonia hydroxide) to remove the fibroblasts. Cellular remnants were washed away with PBS, leaving an intact 3D cell-free ECM attached to the culture surface. Unextracted 3D cultures were fixed/permeabilized in 4% paraformaldehyde and 0.5% Triton X-100 in PBS for 5 minutes at room temperature and then fixed with 4% paraformaldehyde for an additional 15 minutes. Cells were blocked with 5% fetal bovine serum in PBS for 1 hour at room temperature and then incubated with mouse anti-FN (1 μg/ml; BD Biosciences, San Jose, CA) and rat anti-mouse Sdc1 antibody (5 μg/ml; a gift from A.C. Rapraeger, Ph.D) at 4°C overnight. The cell-free 3D ECMs were directly blocked with 5% fetal bovine serum and incubated with primary antibodies overnight. After washing with PBS, goat anti-mouse IgG (Alexa Fluor488-conjugated) and goat anti-rat IgG (Alexa Fluor647-conjugated) (both 5 μg/ml; Invitrogen, Carlsbad, CA) were added to 3D culture or cell-free ECM for 1 hour at room temperature. The preparations were analyzed with a laser-scanning confocal microscope (MRC 1024; Bio-Rad Laboratories, Inc., Hercules, CA). Whole-cell lysates of NIH-3T3 and HMF cells were prepared using a radioimmunoprecipitation assay (RIPA) buffer (Boston BioProducts, Worcester, MA). The ECM proteins of cell-free ECM-mock and ECM-Sdc1 were solubilized using TUT buffer (8-mol/L urea, 10-mmol/L Tris, 1-mmol/L sodium sulfate, and 0.1% Triton X-100, pH 8.0). Equal amounts of protein lysates were either blotted directly onto the nitrocellulose membranes or fractionated on 4% to 12% precast gel (Criterion XT; Bio-Rad Laboratories, Inc.) before transfer to polyvinylidene difluoride membranes. The membranes were probed overnight with rat anti-Sdc1 (1-μg/ml) or mouse anti-FN (0.25-μg/ml) antibodies. Horseradish peroxidase–conjugated anti-rat (20-ng/ml) or anti-mouse (100-ng/ml) IgG (Sigma, St. Louis, MO) was used as a secondary antibody. FN or Sdc1 was then visualized using a maximum sensitivity substrate SuperSignal West Femto (Pierce, Thermo Fisher Scientific, Rockford, IL). Confocal images of the cell-free ECMs were overlaid with a predesigned template that defined nine evenly distributed measurement points. Fiber-to-fiber angles were measured as previously described for the human tumor samples. A minimum of 10 images were analyzed for each condition. The MDA-MB-231 (2 × 104 cells) and T47D (5 × 104 cells) were plated on cell-free NIH-3T3 and HMF mock and Sdc1 ECMs and cultured for up to 6 days. Carcinoma cells were collected every day by trypsinization and counted with a hemocytometer. Semiconfluent cultures of MDA-MB-231 and T47D cells were stained with a nuclear dye (Hoechst 33342; 2.5 μg/ml; Invitrogen, Carlsbad, CA). Labeled cells were added to the glass-bottom dishes (MatTek Corporation, Ashland, MA) precoated with NIH-3T3 or HMF-derived ECM-mock, ECM-Sdc1, or soluble FN and incubated at 37°C for 10 minutes. The unattached cells were removed by washing with PBS, and attached cells were fixed with ice-cold 100% methanol. Images of the nuclei were acquired using an inverted microscope. The number of attached cells was determined using ImageJ software (ImageJ), and the average value obtained on the FN control was normalized as 1 arbitrary unit. The NIH-3T3 or HMF cells were cultured in the insert of invasion chambers (BD Biocoat Matrigel Invasion Chambers; BD Biosciences) for 7 days and then extracted to leave cell-free ECMs on the upper side of the insert. MDA-MB-231 cells were loaded onto these fibroblast-derived ECMs and cultured at 37°C for 24 hours in Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum. The lower chambers were filled with DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal calf serum as a source of chemoattractants. Noninvading MDA-MB-231 cells remaining on the upper side of the insert were removed, whereas the invading cells attached to the lower side of the insert were fixed with 100% methanol and then stained with Hoechst dye 33342. Images of the nuclei were acquired using an inverted microscope (Olympus) and analyzed using ImageJ software. Live MDA-MB-231 cells were labeled using a cytoplasmic membrane staining kit (CellBrite; Biotium Inc., Hayward, CA) according to the manufacturer's instructions. Labeled cells were added to glass-bottom tissue culture plates precoated with NIH-3T3 and HMF ECMs. After overnight incubation, the plates were placed in the environmentally controlled chamber of a confocal bioimager (BD Pathway; BD Biosciences). Cell movements were monitored in real time for 5 to 6 hours, and images were captured every 30 minutes with a cooled 12-bit charge-coupled device camera (Autovision; BD Biosciences). The resulting images were stacked using ImageJ software (ImageJ). The migratory directionality of each individual cell was determined by tracing the path of the manually detected cell center using the Fragment Line tool in ImageJ. The results represent the average of two to three independent experiments analyzing 50 to 100 cells each. Elastomeric polydimethylsiloxane stamps with line or square patterns were fabricated in the Beebe laboratory. After optimization, lines with a width of 150 μm and spacing of 150 μm were used. The size of squares used as controls was 450 × 450 μm, and the spacing between adjacent squares was 150 μm. These stamps were used to print FN patterns directly onto the glass-bottom tissue culture dishes.24Bernard A. Delamarche E. Schmid H. Michel B. Bosshard H.R. Biebuyck H. Printing patterns of proteins.Langmuir. 1998; 14: 2225-2229Crossref Scopus (417) Google Scholar Briefly, the stamps were cleaned by sonication, immersed in FN solution (50 μg/ml in PBS) for 2 hours to allow for protein adsorption, and then air dried in a tissue culture hood. The FN-coated stamps were placed onto the glass bottom of the culture dishes, pressed down gently, and kept in this position for approximately 15 minutes to ensure the transfer of FN onto the culture dishes. After the removal of the stamps, the dishes were blocked with 5% heat-inactivated fetal bovine serum for 1 hour. The HMF mock and Sdc1 cells were then added to the patterned dishes and incubated for 20 minutes at 37°C. The HMF cells preferentially attached to the FN patterns, leaving the spacing regions unoccupied. After washing away the unattached cells, the remaining cells were cultured for 7 days to produce 3D ECMs (see previous data). Either the Student's t-test or one-way analysis of variance was used, depending on the number of groups to be compared. When analysis of variance yielded significance compared with multiple groups, pairwise comparisons using the Tukey's test were performed. Fiber angle data from the mouse tissues and from ECMs produced in vitro were compared with the nonparametric Mann-Whitney U test. Spearman correlation analysis was used to examine the relationship between angle measurements in human tumor tissue microarray and tumor parameters on a continuous scale. Statistical significance was defined as a two-tailed P < 0.05. We and others17Maeda T. Alexander C.M. Friedl A. Induction of syndecan-1 expression in stromal fibroblasts promotes proliferation of human breast cancer cells.Cancer Res. 2004; 64: 612-621Crossref PubMed Scopus (115) Google Scholar, 18Stanley M.J. Stanley M.W. Sanderson R.D. Zera R. Syndecan-1 expression is induced in the stroma of infiltrating breast carcinoma.Am J Clin Pathol. 1999; 112: 377-383Crossref PubMed Scopus (121) Google Scholar have previously reported that Sdc1 is aberrantly expressed by stromal fibroblasts in human breast carcinomas. During microscopic examination of breast carcinoma tissues, it appears that Sdc1 expression in fibroblasts is frequently associated with an organized desmoplastic stroma. To confirm this subjective impression, we examined a previously characterized tissue microarray that contains duplicate tumor tissue samples from 207 patients with breast carcinoma.25Baba F. Swartz K. van Buren R. Eickhoff J. Zhang Y. Wolberg W. Friedl A. Syndecan-1 and syndecan-4 are overexpressed in an estrogen receptor-negative, highly proliferative breast carcinoma subtype.Breast Cancer Res Treat. 2006; 98: 91-98Crossref PubMed Scopus (94) Google Scholar, 26Bauer M. Eickhoff J.C. Gould M.N. Mundhenke C. Maass N. Friedl A. Neutrophil gelatinase-associated lipocalin (NGAL) is a predictor of poor prognosis in human primary breast cancer.Breast Cancer Res Treat. 2008; 108: 389-397Crossref PubMed Scopus (178) Google Scholar Stromal Sdc1 was detected by immunohistochemistry and quantified by manual scoring21Harvey J.M. Clark G.M. Osborne C.K. Allred D.C. Estrogen receptor status by immunohistochemistry is superior to the ligand-binding assay for predicting response to adjuvant endocrine therapy in breast cancer.J Clin Oncol. 1999; 17: 1474-1481Crossref PubMed Google Scholar, 25Baba F. Swartz K. van Buren R. Eickhoff J. Zhang Y. Wolberg W. Friedl A. Syndecan-1 and syndecan-4 are overexpressed in an estrogen receptor-negative, highly proliferative breast carcinoma subtype.Breast Cancer Res Treat. 2006; 98: 91-98Crossref PubMed Scopus (94) Google Scholar (Figure 1, A and B). The ECM fiber architecture was visualized by polarization microscopy of picro-sirius red–stained paraffin sections27Junqueira L.C. Bignolas G. Brentani R.R. Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections.Histochem J. 1979; 11: 447-455Crossref PubMed Scopus (1958) Google Scholar (Figure 1, C and D). Because of tissue loss or absence of identifiable ECM fibers, 21 tumors had to be eliminated from the analysis. The ECM fiber-to-fiber angles (mean ± SD; 45.6 ± 10.1 degrees; n = 186) correlated negatively with stromal Sdc1 expression (r = −0.21; P = 0.004). As expected, when the tumors were dichotomized along the median into Sdc1- positive and Sdc1-negative cases, the fiber angles were more acute in carcinomas with Sdc1-positive stroma (Table 1). Conversely, the ECM fiber-to-fiber angles did not correlate with any other tumor parameter, including size, grade, lymph node status, Ki-67 proliferative index, estrogen or progesterone receptor expression, human epidermal growth factor receptor 2 (Her-2) overexpression, or patient age. To examine more rigorously a potential link between stromal Sdc1 and ECM organization, we compared the ECM fiber architecture in MMTV-Wnt1–induced mouse mammary tumors arising in genetically Sdc1-deficient and wild-type animals. In this animal model, we have previously demonstrated that strong Sdc1 induction in stromal fibroblasts occurs in the abundant stroma.17Maeda T. Alexander C.M. Friedl A. Induction of syndecan-1 expression in stromal fibroblasts promotes proliferation of human breast cancer cells.Cancer Res. 2004; 64: 612-621Crossref PubMed Scopus (115) Google Scholar Similar to the human breast carcinomas, the fiber-to-fiber angles were significantly (P < 0.011) more acute in tumors arising in Sdc1+/+ animals (Figure 1E and Table 1) than in tumors from Sdc1−/− mice (Figure 1F). These findings in human and mouse tumors suggest a modulatory effect of Sdc1 on tumor stroma ECM organization.Table 1ECM Fiber-to-Fiber Angles in Human Breast Carcinomas⁎These angles were dichotomized along the median into stroma Sdc1 positive (H-score ≥5) and Sdc1 negative (H-score <5) and in MMTV-Wnt1 mouse mammary tumors arising in an Sdc1+/+ and an Sdc1−/− genetic background.StromaHumanMouseSdc1 positive†Data are given as mean ± SD.43.8 ± 10.324.4 ± 7.2Sdc1 negative†Data are given as mean ± SD.47.2 ± 9.935.0 ± 12.8P value0.0240.011 These angles were dichotomized along the median into stroma Sdc1 positive (H-score ≥5) and Sdc1 negative (H-score <5) and in MMTV-Wnt1 mouse mammary tumors arising in an Sdc1+/+ and an Sdc1−/− genetic background.† Data are given as mean ± SD. Open table in a new tab To model ECM production in vitro, we used an experimental system developed by Amatangelo and co-workers.22Cukierman E. Cell migration analyses within fibroblast-derived 3-D matrices.Methods Mol Biol. 2005; 294: 79-93PubMed Google Scholar, 28Amatangelo M.D. Bassi D.E. Klein-Szanto A.J. Cukierman E. Stroma-derived three-dimensional matrices are necessary and sufficient to promote desmoplastic differentiation of normal fibroblasts.Am J Pathol. 2005; 167: 475-488Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 29Cukierman E. Preparation of extracellular matrices produced by cultured fibroblasts.in: Harford J.B. Yamada K.M. Lippincott-Schwartz, Philadelphia2002: 10.19.11-10.19.14Google Scholar In this model, fibroblasts are permitted to grow to an overconfluent state under culture conditions that favor ECM production. The fibroblasts are then removed by detergent extraction, leaving a cell-free 3D ECM scaffold behind, which can then be reseeded with other cell types. We studied ECMs produced by generic mouse NIH-3T3 cells and organotypic immortalized HMFs.2Kuperwasser C. Chavarria T. Wu M. Magrane G. Gray J.W. Carey L. Richardson A. Weinberg R.A. Reconstruction of functionally normal and malignant human breast tissues in mice.Proc Natl Acad Sci U S A. 2004; 101: 4966-4971Crossref PubMed Scopus (630) Google Scholar The induction of Sdc1 in fibroblasts of breast carcinoma stroma was simulated in vitro by stable forced expression of Sdc1. The expression of Sdc1 in transfected cells was confirmed by immunofluorescence staining (Figure 2A) and dot blot analysis (Figure 2C). After detergent extraction of fibroblasts, cell-free ECMs remained attached to the culture vessel. To characterize the ECMs, they were immunofluorescently labeled with antibodies to FN and examined by laser confocal microscopy (Figure 2B). FN immunolabeling proved a robust method to visualize ECM fibers, although labeling with antibodies to collagen I yielded similar results (data not shown). The thickness of the ECMs produced by Sdc1-positive and Sdc1-negative fibroblasts (subsequently referred to as ECM-Sdc1 and ECM-mock, respectively, herein) was uniform throughout the culture vessel and averaged 34 μm for NIH-3T3 ECMs and 14 μm for HMF (z-axis views of the cell-free matrices, Figure 2B). Importantly, Sdc1 was undetectable in the ECMs by immunofluorescence microscopy (Figure 2B) and dot blot analysis (Figure 2C), demonstrating th