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• W1991077173 abstract "Although the outgrowth of micrometastases into macrometastases is the rate-limiting step in metastatic progression and the main determinant of cancer fatality, the molecular mechanisms involved have been little studied. Here, we compared the gene expression profiles of melanoma lymph node micro- and macrometastases and unexpectedly found no common up-regulation of any single growth factor/cytokine, except for the cytokine-like SPP1. Importantly, metastatic outgrowth was found to be consistently associated with activation of the transforming growth factor-β signaling pathway (confirmed by phospho-SMAD2 staining) and concerted up-regulation of POSTN, FN1, COL-I, and VCAN genes—all inducible by transforming growth factor-β. The encoded extracellular matrix proteins were found to together form intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases from various organs. Functional analyses suggested that these newly synthesized protein networks regulate adhesion, migration, and growth of tumor cells, fibroblasts, and endothelial cells. POSTN acted as an anti-adhesive molecule counteracting the adhesive functions of FN1 and COL-I. Further, cellular FN and POSTN were specifically overexpressed in the newly forming/formed tumor blood vessels. Transforming growth factor-β receptors and the metastasis-related matrix proteins, POSTN and FN1, in particular, may thus provide attractive targets for development of new therapies against disseminated melanoma, breast cancer, and possibly other tumors, by affecting key processes of metastasis: tumor/stromal cell migration, growth, and angiogenesis. Although the outgrowth of micrometastases into macrometastases is the rate-limiting step in metastatic progression and the main determinant of cancer fatality, the molecular mechanisms involved have been little studied. Here, we compared the gene expression profiles of melanoma lymph node micro- and macrometastases and unexpectedly found no common up-regulation of any single growth factor/cytokine, except for the cytokine-like SPP1. Importantly, metastatic outgrowth was found to be consistently associated with activation of the transforming growth factor-β signaling pathway (confirmed by phospho-SMAD2 staining) and concerted up-regulation of POSTN, FN1, COL-I, and VCAN genes—all inducible by transforming growth factor-β. The encoded extracellular matrix proteins were found to together form intricate fibrillar networks around tumor cell nests in melanoma and breast cancer metastases from various organs. Functional analyses suggested that these newly synthesized protein networks regulate adhesion, migration, and growth of tumor cells, fibroblasts, and endothelial cells. POSTN acted as an anti-adhesive molecule counteracting the adhesive functions of FN1 and COL-I. Further, cellular FN and POSTN were specifically overexpressed in the newly forming/formed tumor blood vessels. Transforming growth factor-β receptors and the metastasis-related matrix proteins, POSTN and FN1, in particular, may thus provide attractive targets for development of new therapies against disseminated melanoma, breast cancer, and possibly other tumors, by affecting key processes of metastasis: tumor/stromal cell migration, growth, and angiogenesis. Metastasis is a complex process comprising multiple steps, including dissemination of cells from a primary tumor into blood or lymph vessels, survival of the cells in these vessels, arrest and extravasation into a new organ, initiation and maintenance of growth, and vascularization of the metastatic tumor (reviewed in 1Chambers AF Groom AC MacDonald IC Dissemination and growth of cancer cells in metastatic sites.Nat Rev Cancer. 2002; 2: 563-572Crossref PubMed Scopus (3062) Google Scholar, 2Steeg PS Tumor metastasis: mechanistic insights and clinical challenges.Nat Med. 2006; 12: 895-904Crossref PubMed Scopus (1663) Google Scholar, 3Gupta GP Massague J Cancer metastasis: building a framework.Cell. 2006; 127: 679-695Abstract Full Text Full Text PDF PubMed Scopus (3291) Google Scholar). Even before dissemination, the tumor cells may secrete growth factors and cytokines that induce systemic changes and prime the distant site for metastasis (reviewed in 4Psaila B Kaplan RN Port ER Lyden D Priming the ‘soil’ for breast cancer metastasis: the pre-metastatic niche.Breast Dis. 2006; 26: 65-74PubMed Google Scholar, 5Hirakawa S From tumor lymphangiogenesis to lymphvascular niche.Cancer Sci. 2009; 100: 983-989Crossref PubMed Scopus (76) Google Scholar). The early steps in metastasis occur efficiently, in contrast to later steps, where only a small subset of cancer cells at a secondary site initiate growth and form pre-angiogenic micrometastases, and, of these, only a tiny proportion continue to become vascularized and progressively growing macrometastases (reviewed in 4Psaila B Kaplan RN Port ER Lyden D Priming the ‘soil’ for breast cancer metastasis: the pre-metastatic niche.Breast Dis. 2006; 26: 65-74PubMed Google Scholar, 6Fidler IJ The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited.Nat Rev Cancer. 2003; 3: 453-458Crossref PubMed Scopus (3546) Google Scholar). The ability to grow at a distant site is largely dictated by molecular interactions of the cancer cells with the new microenvironment, which may have an even greater impact on cell behavior at a distant site than at the primary tumor location. Since the growth and spread of metastases is the main (90%) cause of death from solid cancers, better therapies are urgently needed. Strategically, the rate-limiting step of metastasis, the colonization of secondary sites, might be a more potent therapeutic target than the earlier efficient steps of metastasis, requiring inhibition of fewer cells. Moreover, at the time of primary-tumor diagnosis the earlier steps may already have occurred. The problem is, however, that although the mechanisms of the earlier steps of metastasis have been fairly extensively studied (though giving rise to differing mechanistic models, reviewed in 7Ma L Weinberg RA Micromanagers of malignancy: role of microRNAs in regulating metastasis.Trends Genet. 2008; 24: 448-456Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 8Pantel K Brakenhoff RH Dissecting the metastatic cascade.Nat Rev Cancer. 2004; 4: 448-456Crossref PubMed Scopus (1061) Google Scholar), the molecules and mechanisms behind the growth of micrometastases to overt macrometastases, especially in actual human cancers, are still little studied and poorly understood. A good model to study the progression of metastasis is human cutaneous melanoma. It is one of the most aggressive malignancies, and it frequently has already disseminated cells to other organs by the time that the primary tumor diagnosis is made; it has no effective treatment after metastasis. The initial route of metastasis in melanoma is thought to mainly occur through the lymphatics to the first lymph nodes (the sentinel LNs) draining the primary melanoma site. Micro- and macrometastases in melanoma are presently defined by their detection method, according to the American Joint Committee on Cancer staging system.9Balch CM Buzaid AC Soong SJ Atkins MB Cascinelli N Coit DG Fleming ID Gershenwald JE Houghton Jr, A Kirkwood JM McMasters KM Mihm MF Morton DL Reintgen DS Ross MI Sober A Thompson JA Thompson JF Final version of the American Joint Committee on Cancer staging system for cutaneous melanoma.J Clin Oncol. 2001; 19: 3635-3648Crossref PubMed Scopus (2275) Google Scholar The patients are classified as having clinically occult (microscopic, detected by sentinel or elective lymphadenectomy) or clinically apparent (macroscopic, detected by physical or radiological examination) metastases without taking into consideration metastasis size. This procedure does not, however, necessarily reflect any biological properties of the metastases. In fact, several studies have shown that size of a metastasis (2 to 3 mm) or tumor burden analyzed by quantitative reverse transcription (RT)-PCR (qRT-PCR) in sentinel LNs is an efficient predictor of recurrence and survival.10Carlson GW Murray DR Lyles RH Staley CA Hestley A Cohen C The amount of metastatic melanoma in a sentinel lymph node: does it have prognostic significance?.Ann Surg Oncol. 2003; 10: 575-581Crossref PubMed Scopus (151) Google Scholar, 11Ranieri JM Wagner JD Azuaje R Davidson D Wenck S Fyffe J Coleman 3rd, JJ Prognostic importance of lymph node tumor burden in melanoma patients staged by sentinel node biopsy.Ann Surg Oncol. 2002; 9: 975-981Crossref PubMed Google Scholar, 12Soikkeli J Lukk M Nummela P Virolainen S Jahkola T Katainen R Harju L Ukkonen E Saksela O Holtta E Systematic search for the best gene expression markers for melanoma micrometastasis detection.J Pathol. 2007; 213: 180-189Crossref PubMed Scopus (33) Google Scholar These measures may provide a division into pre-angiogenic micrometastases and more persistently growing macrometastases. In this study, our goal was to identify genes required for the outgrowth of melanoma metastases by comparing gene expression profiles of melanoma micro- and macrometastases from LNs to understand the mechanisms involved and to identify rational targets for therapy. Expression of the genes identified was then analyzed in the metastases by immunohistochemistry (IHC) and confocal microscopy. Our results revealed the metastatic outgrowth to be associated with formation of an intricate network by four specific extracellular matrix (ECM) proteins: collagen-I (COL-I), fibronectin 1 (FN1), periostin (POSTN), and versican (VCAN), which appear to regulate tumor/stromal cell adhesion, migration, growth, and angiogenesis/tubulogenesis. We further studied breast cancer LN metastases, as well as melanoma and breast cancer metastases from other organs, to assess the generality of these findings. We divided the melanoma LN metastases into micrometastases (n = 42, maximum diameter ≤2.5 mm) and macrometastases (n = 65, maximum diameter >2.5 mm). We based this division on our gene expression clustering analyses, on a systematic study of metastasis diameters (0.3 to 10 mm) identifying 3 mm as the most significant cut-point predictive of survival,11Ranieri JM Wagner JD Azuaje R Davidson D Wenck S Fyffe J Coleman 3rd, JJ Prognostic importance of lymph node tumor burden in melanoma patients staged by sentinel node biopsy.Ann Surg Oncol. 2002; 9: 975-981Crossref PubMed Google Scholar and on studies using a 2-mm cut-point10Carlson GW Murray DR Lyles RH Staley CA Hestley A Cohen C The amount of metastatic melanoma in a sentinel lymph node: does it have prognostic significance?.Ann Surg Oncol. 2003; 10: 575-581Crossref PubMed Scopus (151) Google Scholar—a diameter arbitrarily chosen for clinical breast cancer staging. The results and conclusions were, however, unaffected if we used a 2- or 3-mm cut-point. The breast cancer LN metastases were analyzed both with a 2.0- and 2.5-mm cut-point for macrometastases. The sixth edition of the TNM (tumor-nodes-metastasis) classification defines metastases between 0.2 mm and 2 mm in largest diameter as micrometastases and ≤0.2-mm metastases as isolated tumor cells,13Sobin LH Wittekind C TNM Classification of Malignant Tumours. 6th Edition. John Wiley & Sons, New Jersey2002: 1-239Google Scholar but these measures are not evidence based and not used in melanoma diagnostics. As micrometastatic deposits are usually not spherical, measurements of the largest diameter alone may not be sufficient for the tumor size estimation. The melanoma metastases from LNs (n = 107), liver (n = 2), and lung (n = 12), and breast cancer metastases from LNs (n = 45), liver (n = 3), and lung (n = 2), as well as noncancerous control LNs (n = 39; see12Soikkeli J Lukk M Nummela P Virolainen S Jahkola T Katainen R Harju L Ukkonen E Saksela O Holtta E Systematic search for the best gene expression markers for melanoma micrometastasis detection.J Pathol. 2007; 213: 180-189Crossref PubMed Scopus (33) Google Scholar) were obtained by surgical excision at Helsinki University Central Hospital. Tissue specimens were either fixed in formalin or immediately frozen in liquid nitrogen for the various analysis methods. Protocols for taking the tissue specimens were approved by the Ethics Committees of Helsinki University Central Hospital. Further, all patients gave their informed consent before the operations. Total RNA was extracted from cells and frozen tissues with the RNeasy kit (Qiagen, Crawley, UK) and pigment, if present, was removed by Bio-Gel P-60 gel filtering, as described.12Soikkeli J Lukk M Nummela P Virolainen S Jahkola T Katainen R Harju L Ukkonen E Saksela O Holtta E Systematic search for the best gene expression markers for melanoma micrometastasis detection.J Pathol. 2007; 213: 180-189Crossref PubMed Scopus (33) Google Scholar Quality of the purified RNA was assessed by gel electrophoresis or by Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Melanoma LN micrometastases (n = 9) and macrometastases (n = 13; 11 detected by palpation and 2 by sentinel LN biopsy; in this group, all patients but one—who had undergone multiple surgical operations—subsequently died within 2 years) were analyzed with the Human Genome U133 Set (Affymetrix, Santa Clara, CA), as described.12Soikkeli J Lukk M Nummela P Virolainen S Jahkola T Katainen R Harju L Ukkonen E Saksela O Holtta E Systematic search for the best gene expression markers for melanoma micrometastasis detection.J Pathol. 2007; 213: 180-189Crossref PubMed Scopus (33) Google Scholar None of the patients received any chemo- or radiotherapy before sample collection. Breast cancer LN metastases (n = 11, 6 ductal and 5 lobular) and normal LNs (n = 7) were analyzed with the Human Genome U133 Plus 2.0 Array (Affymetrix). In addition, the normal LNs (n = 11) analyzed with the HG-U133 Set12Soikkeli J Lukk M Nummela P Virolainen S Jahkola T Katainen R Harju L Ukkonen E Saksela O Holtta E Systematic search for the best gene expression markers for melanoma micrometastasis detection.J Pathol. 2007; 213: 180-189Crossref PubMed Scopus (33) Google Scholar served for control purposes. The HG-U133A and -B chips were analyzed separately. The individual probe signals were summarized by the RMA algorithm (RMAExpress version 0.3, http://rmaexpress.bmbolstad.com/). To reveal all of the gene expression changes in melanoma cells and stromal cells, the expression data from the micro- and macrometastatic lymph nodes were first quantile–normalized together.14Bolstad BM Irizarry RA Astrand M Speed TP A comparison of normalization methods for high density oligonucleotide array data based on variance and bias.Bioinformatics. 2003; 19: 185-193Crossref PubMed Scopus (6420) Google Scholar Gene probe sets with a mean difference <100 and a fold-change <1.5 between micro- and macrometastases were filtered off (to remove probe sets with low signals and with small expression level changes, which are not measured reliably). The remaining probe sets were ordered by significance analysis of microarrays (SAM) 3.015Tusher VG Tibshirani R Chu G Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci USA. 2001; 98: 5116-5121Crossref PubMed Scopus (9771) Google Scholar (http://www-stat.stanford.edu/∼tibs/SAM/), and their class specificity was evaluated by 5000 random permutations. Delta was chosen so that the false discovery rate was <1%. The list of significant genes was then filtered for a fourfold difference. In addition, the data were normalized to melanocyte marker genes (SOX10, MLANA, TYR) to find genes with potentially specific up-regulation in melanoma cells. To confirm the reproducibility of the list of differentially expressed genes, SAM analysis was also performed using data preprocessed with MAS 5.0 algorithm (Affymetrix). Further, filtering of RMA-summarized data before SAM analysis was performed in various ways: by removing only probe sets with absent calls in all samples, probe sets with expression signal values <20 in all samples, or probe sets with a mean difference <100 between the groups. In addition to SAM analysis, we produced a list of differentially expressed genes using fold change-ranking combined with t-statistics (Volcano plot).16Shi L Jones WD Jensen RV Harris SC Perkins RG Goodsaid FM Guo L Croner LJ Boysen C Fang H Qian F Amur S Bao W Barbacioru CC Bertholet V Cao XM Chu TM Collins PJ Fan XH Frueh FW Fuscoe JC Guo X Han J Herman D Hong H Kawasaki ES Li QZ Luo Y Ma Y Mei N Peterson RL Puri RK Shippy R Su Z Sun YA Sun H Thorn B Turpaz Y Wang C Wang SJ Warrington JA Willey JC Wu J Xie Q Zhang L Zhang L Zhong S Wolfinger RD Tong W The balance of reproducibility, sensitivity, and specificity of lists of differentially expressed genes in microarray studies.BMC Bioinformatics. 2008; 9 Suppl 9: S10Crossref PubMed Scopus (189) Google Scholar Similar lists of top-ranked genes were obtained with all methods. Additionally, the genes (with a mean difference ≥100 and a fold-change ≥1.5 between groups) were clustered with Pearson correlation measurement and average linkage as implemented in GeneSpring GX 7.3 (Agilent Technologies) to explore the coordinate expression of selected genes in melanoma and breast cancer metastases. We also subjected the SAM-ordered probes (mean difference ≥ 100 and fold-change ≥ 1.5) to gene set enrichment analysis.17Subramanian A Tamayo P Mootha VK Mukherjee S Ebert BL Gillette MA Paulovich A Pomeroy SL Golub TR Lander ES Mesirov JP 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 (26834) Google Scholar The Molecular Signature Database (MSigDB, v2.5, Broad Institute, MA, http://www.broad.mit.edu/gsea/msigdb/index.jsp) gene set collections C2 to C5 were analyzed (C2 comprised of 1892 curated gene sets). The data were also analyzed by GenMAPP 2.1 and Ingenuity Pathway analysis tools (Ingenuity Systems, Redwood City, CA). One μg of total RNA was reverse-transcribed into cDNA, and PCR was performed essentially as described.12Soikkeli J Lukk M Nummela P Virolainen S Jahkola T Katainen R Harju L Ukkonen E Saksela O Holtta E Systematic search for the best gene expression markers for melanoma micrometastasis detection.J Pathol. 2007; 213: 180-189Crossref PubMed Scopus (33) Google Scholar POSTN primers were designed to cover the C-terminal region of POSTN mRNA (NM_006475), which is known to be differentially spliced. The forward primer was 5′-GTTTGTTCGTGGTAGCACCT-3′ and reverse 5′-TGTTGGCTTGCAACTTCCTCAC-3′, amplifying from the full-length POSTN mRNA a fragment of 500 bp. PCR was performed in a DYAD DNA engine (MJ Research, Waltham, MA) under the following conditions: 94°C for 15 s, 57°C for 30 s, and 72°C for 1 minute 10 seconds for 32 cycles. β-actin (ACTB) served as the control. PCR products were separated by electrophoresis on a 2% agarose gel, visualized by SYBR Green I staining, and documented by a video camera system. For sequencing, PCR fragments (separated on high resolution MetaPhor agarose, Cambrex/Lonza, Basel, Switzerland) were purified with Ultrafree-DA centrifugal filter units (Millipore, Billerica, MA) and with the Illustra GFX PCR DNA and gel band purification kit (GE Health care, Waukesha, WI). DNA sequencing of the PCR products was performed with the BigDye Terminator v3.1 kit and 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). Real-time qRT-PCR analysis for POSTN mRNA was performed with the ABI PRISM 7700 Sequence Detection System instrument and software (Applied Biosystems) using a Taqman Gene Expression Assay Hs00170815_m1 for POSTN and a Taqman pre-developed assay for β-actin. POSTN and ACTB cDNA levels were measured in triplicate. Standard curves were generated for both genes, and the relative POSTN expression values were normalized to that of ACTB. Paraffin-embedded sections (5 μm) were deparaffinized, rehydrated through a graded ethanol series, and washed in distilled water. Antigen retrieval was performed by trypsin treatment (0.5% in PBS) at 37°C for 30 minutes, except for POSTN and phospho-SMAD2 (pSMAD2) staining, where the samples were heated in a microwave oven at 850 W for 3 minutes and at 170 W for 7 minutes in 10 mmol/L citric acid, pH 6.0. For both paraffin-embedded and frozen sections (5 μm), the endogenous peroxidase activity was blocked with 1% H2O2 in methanol for 30 minutes. After blocking in 1 to 10% goat serum in PBS or in CAS-block reagent (Zymed Laboratories, San Francisco, CA), the sections were incubated with the primary antibodies (see supplemental Table S1 at http://ajp.amjpathol.org/) diluted in the blocking buffer at 4°C overnight. Immunodetection was performed with the StreptABComplex/HRP Duet kit (Dako, Glostrup, Denmark) according to the manufacturer’s protocol. The chromogen was either 3,3′-diaminobenzidine or 3-amino-9-ethylcarbatzole. Slides were counterstained with Mayer’s hematoxylin and mounted with Mountex (Histolab Products, Göteborg, Sweden) or Aquamount (BDH Laboratories, Poole, UK). Images were obtained with a Nikon Eclipse E800 or 80i microscope, Nikon DXM1200 digital camera, or Digital Sight DS-5M camera, and the ACT-1 or NIS-Elements F 2.20 software (Nikon, Tokyo, Japan). Frozen tissue samples were embedded in Tissue-Tek optimal cutting temperature compound (Miles Inc., Elkhart, IN) and cut into 40- to 80-μm thicknesses. Sections were mounted onto SuperFrost Plus slides (Menzel-Glaser, Braunschweig, Germany) and air-dried at room temperature for 1 hour. The slides were washed with PBS pH 7.4, pre-incubated with a blocking mixture containing 0.25% Triton X-100 and 10% normal horse serum in PBS (1 hour, room temperature), and incubated overnight at room temperature with the primary antibodies (see supplemental Table S1 at http://ajp.amjpathol.org/). The polyclonal and monoclonal antibodies to FN1 showed similar staining patterns. After incubation with primary antibodies, the slides were washed with PBS and incubated with Alexa-conjugated (488, 568, 647) secondary antibodies (Invitrogen, Carlsbad, CA) diluted 1:500 in PBS containing Triton X-100 and 10% normal horse serum for 1 hour at room temperature. The slides were then washed with PBS and mounted with glycerol and PBS (1:1). Specimens were examined with a Leica DM RXA epifluorescence microscope and Leica TCS MP SP confocal microscopy system. The emission wavelengths were set up for Alexa 488 at 495 to 550 nm, for Alexa 568 at 600 to 650 nm, and for Alexa 647 at 660 to 750 nm. The fluorophores were excited by use of an argon-krypton laser beam at 488 nm (Lasos, Jena, Germany) or by an 85 YCA-series diode-pumped solid-state yellow laser beam at 561 nm and a helium-neon laser at 633 nm (Omnichrome, Melles Griot, Carlsbad, CA). Acquisition of data and the three-dimensional reconstruction imaging were performed with Leica TCS NT/SP Scanware software. Binding of the 90-kDa full-length recombinant human POSTN (R&D Systems, Minneapolis, MN), the 75-kDa fragment of human POSTN (BioVendor, Heidelberg, Germany), human plasma FN (pFN, Chemicon International, Temecula, CA), human cellular FN (cFN, United States Biological, Swampscott, MA), COL-I (Sigma-Aldrich, St. Louis, MO), human tenascin C (TNC, Chemicon International), and human secreted phosphoprotein 1/osteopontin (SPP1, R&D Systems) to each other was analyzed with the Biacore 2000 surface plasmon resonance-based biosensor (Biacore, Uppsala, Sweden). The purity of all proteins was >90%, mostly >95%. CM5 sensor chip surfaces were coupled with pFN/cFN, COL-I, and full-length POSTN to final resonance unit values of 3900, 3700, and 1800, respectively (in the experiment shown), using the standard amine coupling kit (Biacore) according to the manufacturer’s protocol. Flow cell 1 was activated and blocked without any protein for controlling any nonspecific binding. Binding experiments were performed in PBS at a flow rate of 10 μl/minute. All proteins served as the ligand coupled to the sensor surface and as the soluble analyte injected over the sensor surfaces, at 50 to 200 nmol/L concentrations for 2 minutes (followed by monitoring of dissociation) with five separate chips. The responses obtained from a blank control surface were subtracted from the data from the protein-coated surfaces, and the sensorgrams were analyzed with the BIAevalution 3.1 Software (Biacore). The chip surface was regenerated after each injection by adding 100 mmol/L HCl for 30 s, followed by a re-equilibration with PBS. Primary human melanocytes and melanoma cells were isolated and cultured as described.18Alanko T Rosenberg M Saksela O FGF expression allows nevus cells to survive in three-dimensional collagen gel under conditions that induce apoptosis in normal human melanocytes.J Invest Dermatol. 1999; 113: 111-116Crossref PubMed Scopus (52) Google Scholar Melanoma cell lines WM793 and WM239 were kindly provided by Dr. M. Herlyn (Wistar Institute, Philadelphia, PA), and primary human adult and embryonic skin fibroblasts by Dr. A-M. Ranki and Dr. A. Vaheri (University of Helsinki, Finland), respectively. Breast cancer cell line MB-MDA-231 was from ATCC (Teddington, UK). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. Primary human microvascular endothelial cells (HMVECs, adult dermis, Invitrogen) were cultured in growth factor–supplemented Medium 131 (Invitrogen). WM793, WM239, HMVECs, and primary human embryonic and adult fibroblasts were transduced with short hairpin RNA (shRNA) lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA) targeting FN1 (sc-29315-V), POSTN (sc-61324-V), or with negative (scrambled) control shRNA particles (sc-108080) according to the manufacturer’s instructions. Transductions were performed twice in duplicates in 12- or 24-well plates, and puromycin-resistant pools of cells were used in the assays (to avoid clonal variation). Flat-bottomed 96-well plates were incubated with 10 μg/ml bovine serum albumin (fatty acid-free, Sigma-Aldrich), pFN, cFN, full-length POSTN, POSTN fragment, TNC, COL-I, or combinations of these proteins for 2 hours at 37°C and washed three times with PBS. Cells were suspended in serum-free medium at a density of 2 × 105 cells/ml, and 0.1 ml of the cell suspension was added to each well of the coated plate. After 1 hour of incubation at 37°C, attached and spread cells were photographed and counted. Experiments were repeated three to eight times (depending on the coated protein). Adhesion of cells expressing the control, FN1, or POSTN shRNAs was studied the same way but using uncoated plates. Falcon cell culture inserts (8 μm, BD Biosciences, Franklin Lakes, NJ) were coated on the outer surface with 45 μg/ml cFN or COL-I (40 μl; to enable attachment of the migrated cells in serum-free medium) and on the inner surface with 90 μg/ml cFN or COL-I (40 μl), or with a mixture (40 μl) of 45 μg/ml cFN or COL-I and 45 μg/ml full-length POSTN or POSTN fragment. Cells were suspended in the medium at a density of 1.5 × 105 cells/ml, and 0.2 ml of the cell suspension was added to each insert. The lower chamber was filled with 800 μl of serum- and growth factor-free medium specifically to measure spontaneous cell migration (rather than chemoinvasion). After a 16- to 18-hour incubation (depending on the cell type), the insert filters were fixed in 3.5% paraformaldehyde and washed three times with PBS. The fixed cells were stained with 0.5% crystal violet (in 20% methanol) for 2 hours and washed with distilled water. Cells on the upper face of the filter were scraped off, and migrated cells on the lower face of the filter were photographed and counted. Experiments were repeated three to six times, and the results were analyzed by two-tailed t-test. The effect of POSTN and cFN on melanoma cell invasion was assayed in thick three-dimensional growth factor–reduced Matrigel (BD Biosciences, Franklin Lakes, NJ) essentially as described.19Ravanko K Jarvinen K Helin J Kalkkinen N Holtta E Cysteine cathepsins are central contributors of invasion by cultured adenosylmethionine decarboxylase-transformed rodent fibroblasts.Cancer Res. 2004; 64: 8831-8838Crossref PubMed Scopus (48) Google Scholar The lower and upper Matrigel layers as well as the growth medium were supplemented or not with POSTN, cFN, or both at 0.1, 1, 2.5, or 10 μg/ml. Then, 20,000 WM793 cells in 100 μl of RPMI-1640 were plated on top of the Matrigel and allowed to adhere for 1 hour at 37°C. Excess medium was removed, and 250 μl Matrigel (supplemented or not with POSTN, cFN, or both at 0.1, 1, 2.5, or 10 μg/ml) was layered above the cells. Finally, 500 μl of growth medium without or with POSTN, cFN, or both (0.1, 1, 2.5, or 10 μg/ml) was added on top of the Matrigel matrix. The growth medium was replenished every third day. The growth pattern of the cells in Matrigel was followed daily by microscopy and photography. It has become increasingly evident that metastatic growth is not only dependent on the tumor cells, but also on the activation or suppression of resident stromal cells (fibroblasts, inflammatory and immune cells, and endothelial cells), and/or recruitment of various progenitor cells from the bone marrow. To identify the genes associated with the macroscopic growth of melanoma metastases, we performed gene expression analyses of micro- and of macrometastatic LNs with metastasis diameters of ≤2.5 mm and of >2.5 mm. Specifically, we were interested to discover any common denominator(s) for the metastatic growth. Significance analysis of microarrays resulted in 166 probe sets representing 131 genes significantly overexpressed (Table 1, and supplemental Tables S2 and S3 at http://ajp.amjpathol.org/) and 145 probe sets representing 121 genes underexpressed ≥fo" @default.
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• W1991077173 date "2010-07-01" @default.
• W1991077173 modified "2023-10-17" @default.
• W1991077173 title "Metastatic Outgrowth Encompasses COL-I, FN1, and POSTN Up-Regulation and Assembly to Fibrillar Networks Regulating Cell Adhesion, Migration, and Growth" @default.
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• W1991077173 doi "https://doi.org/10.2353/ajpath.2010.090748" @default.
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