TGF-β Signaling, Activated Stromal Fibroblasts, and Cysteine Cathepsins B and L Drive the Invasive Growth of Human Melanoma Cells
2012; Elsevier BV; Volume: 181; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2012.08.027
ISSN1525-2191
AutoresMiao Yin, Johanna Soikkeli, Tiina Jahkola, Susanna Virolainen, Olli Saksela, Erkki Hölttä,
Tópico(s)Bone and Dental Protein Studies
ResumoAccumulating evidence indicates that interactions between cancer cells and stromal cells are important for the development/progression of many cancers. Herein, we found that the invasive growth of melanoma cells in three-dimensional–Matrigel/collagen-I matrices is dramatically increased on their co-culture with embryonic or adult skin fibroblasts. Studies with fluorescent-labeled cells revealed that the melanoma cells first activate the fibroblasts, which then take the lead in invasion. To identify the physiologically relevant invasion-related proteases involved, we performed genome-wide microarray analyses of invasive human melanomas and benign nevi; we found up-regulation of cysteine cathepsins B and L, matrix metalloproteinase (MMP)-1 and -9, and urokinase- and tissue-type plasminogen activators. The mRNA levels of cathepsins B/L and plasminogen activators, but not MMPs, correlated with metastasis. The invasiveness/growth of the melanoma cells with fibroblasts was inhibited by cell membrane–permeable inhibitors of cathepsins B/L, but not by wide-spectrum inhibitors of MMPs. The IHC analysis of primary melanomas and benign nevi revealed cathepsin B to be predominantly expressed by melanoma cells and cathepsin L to be predominantly expressed by the tumor-associated fibroblasts surrounding the invading melanoma cells. Finally, cathepsin B regulated TGF-β production/signaling, which was required for the activation of fibroblasts and their promotion of the invasive growth of melanoma cells. These data provide a basis for testing inhibitors of TGF-β signaling and cathepsins B/L in the therapy of invasive/metastatic melanomas. Accumulating evidence indicates that interactions between cancer cells and stromal cells are important for the development/progression of many cancers. Herein, we found that the invasive growth of melanoma cells in three-dimensional–Matrigel/collagen-I matrices is dramatically increased on their co-culture with embryonic or adult skin fibroblasts. Studies with fluorescent-labeled cells revealed that the melanoma cells first activate the fibroblasts, which then take the lead in invasion. To identify the physiologically relevant invasion-related proteases involved, we performed genome-wide microarray analyses of invasive human melanomas and benign nevi; we found up-regulation of cysteine cathepsins B and L, matrix metalloproteinase (MMP)-1 and -9, and urokinase- and tissue-type plasminogen activators. The mRNA levels of cathepsins B/L and plasminogen activators, but not MMPs, correlated with metastasis. The invasiveness/growth of the melanoma cells with fibroblasts was inhibited by cell membrane–permeable inhibitors of cathepsins B/L, but not by wide-spectrum inhibitors of MMPs. The IHC analysis of primary melanomas and benign nevi revealed cathepsin B to be predominantly expressed by melanoma cells and cathepsin L to be predominantly expressed by the tumor-associated fibroblasts surrounding the invading melanoma cells. Finally, cathepsin B regulated TGF-β production/signaling, which was required for the activation of fibroblasts and their promotion of the invasive growth of melanoma cells. These data provide a basis for testing inhibitors of TGF-β signaling and cathepsins B/L in the therapy of invasive/metastatic melanomas. Tumors must be viewed as complex tissues and not as composed of cancer cells alone. Indeed, in the local microenvironment, cancer cells extensively interact with various stromal components, consisting of extracellular matrix (ECM) proteins/polysaccharides and several kinds of cells, such as endothelial cells, inflammatory cells, and fibroblasts, which reciprocally regulate each other.1Bissell M.J. Radisky D. 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Cutaneous melanoma.Lancet. 2005; 365: 687-701Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar During the past few years, however, we have learned much about the molecular and genetic alterations associated with melanoma development and progression,10Davies H. Bignell G.R. Cox C. Stephens P. Edkins S. Clegg S. et al.Mutations of the BRAF gene in human cancer.Nature. 2002; 417: 949-954Crossref PubMed Scopus (8310) Google Scholar, 11Berger M.F. Levin J.Z. Vijayendran K. Sivachenko A. Adiconis X. Maguire J. Johnson L.A. Robinson J. Verhaak R.G. Sougnez C. Onofrio R.C. Ziaugra L. Cibulskis K. Laine E. Barretina J. Winckler W. Fisher D.E. Getz G. Meyerson M. Jaffe D.B. Gabriel S.B. Lander E.S. Dummer R. Gnirke A. Nusbaum C. Garraway L.A. Integrative analysis of the melanoma transcriptome.Genome Res. 2010; 20: 413-427Crossref PubMed Scopus (219) Google Scholar, 12Scott K.L. Nogueira C. Heffernan T.P. van Doorn R. Dhakal S. Hanna J.A. Min C. Jaskelioff M. Xiao Y. Wu C.J. 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With the current awareness of the importance of the tumor microenvironment in tumor progression, it is reasonable to expect that studies elucidating the interplay between the melanoma cells and stromal components could reveal new effective targets for therapy. The acquisition of invasive potential by tumor cells is a key prerequisite step for metastasis. In invasion/metastasis, proteolytic degradation/processing of the basement membrane/ECM and signaling components is needed. This is a complex process, thought to involve several proteases from five different catalytic classes (ie, serine, threonine, aspartic, cysteine, and metalloproteases). In previous studies, most attention has been paid to matrix metalloproteases (MMPs) and the urokinase-type plasminogen activator (uPA)14de Vries T.J. Quax P.H. Denijn M. Verrijp K.N. Verheijen J.H. Verspaget H.W. Weidle U.H. Ruiter D.J. van Muijen G.N. Plasminogen activators, their inhibitors, and urokinase receptor emerge in late stages of melanocytic tumor progression.Am J Pathol. 1994; 144: 70-81PubMed Google Scholar, 15Shi Z. Stack M.S. Urinary-type plasminogen activator (uPA) and its receptor (uPAR) in squamous cell carcinoma of the oral cavity.Biochem J. 2007; 407: 153-159Crossref PubMed Scopus (46) Google Scholar, 16Kessenbrock K. Plaks V. Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment.Cell. 2010; 141: 52-67Abstract Full Text Full Text PDF PubMed Scopus (3589) Google Scholar, 17Mason S.D. Joyce J.A. Proteolytic networks in cancer.Trends Cell Biol. 2011; 21: 228-237Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar and less attention has been paid to cathepsins17Mason S.D. Joyce J.A. Proteolytic networks in cancer.Trends Cell Biol. 2011; 21: 228-237Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 18Turk V. Kos J. Turk B. Cysteine cathepsins (proteases): on the main stage of cancer.Cancer Cell. 2004; 5: 409-410Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 19Mohamed M.M. Sloane B.F. Cysteine cathepsins: multifunctional enzymes in cancer.Nat Rev Cancer. 2006; 6: 764-775Crossref PubMed Scopus (1011) Google Scholar, 20Frohlich E. Proteases in cutaneous malignant melanoma: relevance as biomarker and therapeutic target.Cell Mol Life Sci. 2010; 67: 3947-3960Crossref PubMed Scopus (23) Google Scholar; most studies have been performed in two-dimensional–cell culture or animal models and examined only cancer cell–specific proteases. In this study, we aimed at identifying the invasion-related proteases in melanomas relevant for the development and progression of the human disease. First, we used three-dimensional (3D)–Matrigel/collagen-I matrices, mimicking the tumor microenvironment, to study the interplay between human melanoma cells (harboring BRAF or NRAS mutations) and skin fibroblasts in the invasive growth. We further performed genome-wide gene expression analyses of primary melanomas and benign nevi to identify the physiologically relevant proteases involved in invasion/metastasis, and used specific protease inhibitors to assess their potential functional significance. In addition, we analyzed by immunohistochemistry (IHC) the cellular expression patterns of the key proteases identified, cathepsins B and L, in the tumor tissues. Finally, we studied the potential role of cathepsin B in the processing of transforming growth factor (TGF)-β and the significance of the TGF-β signaling in the invasion process. Purified cathepsin L from human liver (specific activity, 6.036 U/mg protein), purified cathepsin B from human liver (specific activity, 32.7 U/mg protein), the wide-spectrum MMP inhibitors GM6001 (Ilomastat and its negative control), BB-2516 (Marimastat), cathepsin L inhibitors I and II, and cathepsin B inhibitors CA-074 and CA-O74Me (a cell membrane–permeable analogue of CA-074) were obtained from Merck/Calbiochem (Darmstadt, Germany). The MMP inhibitor BB-3103 (inhibitory concentration of 50%, 2 nmol/L for MMP-1, 10 nmol/L for MMP-2, 30 nmol/L for MMP-3, 20 nmol/L for MMP-7, 7 nmol/L for MMP-9, and 4 nmol/L for MMP-13) was obtained from British Biotech Pharmaceuticals Ltd (Oxford, UK), the TGF-β receptor I (ALK5) inhibitor SB-505124 was obtained from Sigma-Aldrich (St Louis, MO), the neutralizing antibodies to TGF-β (monoclonal anti–TGF-β1, anti–TGF-β2, and anti–TGF-β3 antibody) was obtained from R&D Systems (Minneapolis, MN), uPA inhibitor was obtained from American Diagnostica Inc. (Stamford, CT), and recombinant human TGF-β1 was obtained from Humanzyme (Chicago, IL). Fibroblast medium-serum free was obtained from ScienCell Research Laboratories (Carlsbad, CA), growth factor–reduced Matrigel and rat tail collagen type I, high concentration, were obtained from Becton Dickinson Biosciences (Bedford, MA), and Celltracker Green CMFDA and Celltracker Red CMTPX were obtained from Invitrogen–Molecular Probes, Inc. (Eugene, OR). The nonmetastatic (or poorly metastatic) human vertical growth phase (VGP) melanoma cell lines WM115 (BRAFV600D mutation; obtained from ATCC-LGC Standards, Borås, Sweden) and WM793 (BRAFV600E; provided by Dr. Meenhard Herlyn, Wistar Institute, Philadelphia, PA),21Hsu M. Elder D.E. Herlyn M. The Wistar (WM) melanoma cell lines.in: Palsson J.Ma.B. Human Cell Culture. Kluwer Academic Publishers, London1999: 259-274Google Scholar the metastatic melanoma cell lines SK-MEL-28 (BRAFV600E; obtained from ATCC-LGC Standards), SK-MEL-103 (NRASQ61R), and SK-MEL-147 (NRASQ61R) (both originating from Dr. Alan Houghton, Memorial Sloan-Kettering Cancer Center, New York, NY)22Gorden A. Osman I. Gai W. He D. Huang W. Davidson A. Houghton A.N. Busam K. Polsky D. Analysis of BRAF and N-RAS mutations in metastatic melanoma tissues.Cancer Res. 2003; 63: 3955-3957PubMed Google Scholar, 23Fernandez Y. Verhaegen M. Miller T.P. Rush J.L. Steiner P. Opipari Jr, A.W. Lowe S.W. Soengas M.S. Differential regulation of noxa in normal melanocytes and melanoma cells by proteasome inhibition: therapeutic implications.Cancer Res. 2005; 65: 6294-6304Crossref PubMed Scopus (191) Google Scholar; provided by Dr. Maria Soengas, Spanish National Cancer Research Center, Madrid, Spain), and WM239 (BRAFV600D; obtained from Dr. M. Herlyn),21Hsu M. Elder D.E. Herlyn M. The Wistar (WM) melanoma cell lines.in: Palsson J.Ma.B. Human Cell Culture. Kluwer Academic Publishers, London1999: 259-274Google Scholar the embryonic skin fibroblasts (HES), and adult skin fibroblasts (HAS)24Soikkeli J. Podlasz P. Yin M. Nummela P. Jahkola T. Virolainen S. Krogerus L. Heikkila P. von Smitten K. Saksela O. Holtta E. Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.Am J Pathol. 2010; 177: 387-403Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA) and antibiotics (penicillin and streptomycin) at 37°C in a 5% CO2 atmosphere. Cells in exponential growth were washed twice with serum-free RPMI 1640 medium, incubated in prewarmed medium containing Celltracker Green CMFDA or Celltracker Red CMTPX (5 or 10 μmol/L, respectively; Invitrogen–Molecular Probes, Inc.) at 37°C in a 5% CO2 atmosphere for 45 minutes. After that, the medium was replenished with normal growth medium, and the cells were grown overnight before use in the experiments. The invasive/aggressive behavior of the cells was studied using 3D-Matrigel invasion/outgrowth assays, essentially as previously described.25Ravanko 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 Briefly, 1 to 2 × 104 melanoma cells and fibroblasts (HES or HAS) alone and 104 melanoma cells mixed with 104 HES or HAS cells in the co-culture assays were seeded onto 24-well plates precoated with 300 μL of Matrigel (diluted 1:3 in RPMI 1640 medium), and allowed to adhere for 1 hour at 37°C. Then, a second 250-μL layer of Matrigel (1:3) was cast above the cells. Finally, 500 μL of RPMI 1640 medium containing 10% fetal bovine serum was added on top of the Matrigel matrix. More important, we and other researchers have previously found that this thick, two-layer Matrigel assay (measuring the invasive, migratory, and proliferative ability of the cells) correlates better with the tumor cell invasiveness/aggressiveness in vivo than the Matrigel-coated filter/Boyden chamber assays.26Zajchowski D.A. Bartholdi M.F. Gong Y. Webster L. Liu H.L. Munishkin A. Beauheim C. Harvey S. Ethier S.P. Johnson P.H. Identification of gene expression profiles that predict the aggressive behavior of breast cancer cells.Cancer Res. 2001; 61: 5168-5178PubMed Google Scholar, 27Paasinen-Sohns A. Kaariainen E. Yin M. Jarvinen K. Nummela P. Holtta E. Chaotic neovascularization induced by aggressive fibrosarcoma cells overexpressing S-adenosylmethionine decarboxylase.Int J Biochem Cell Biol. 2011; 43: 441-454Crossref PubMed Scopus (10) Google Scholar Corresponding invasion assays were also performed with cells embedded in a mixture of Matrigel (2 mg/mL) and collagen-I (2 or 4 mg/mL),28Seton-Rogers S.E. Lu Y. Hines L.M. Koundinya M. LaBaer J. Muthuswamy S.K. Brugge J.S. Cooperation of the ErbB2 receptor and transforming growth factor beta in induction of migration and invasion in mammary epithelial cells.Proc Natl Acad Sci U S A. 2004; 101: 1257-1262Crossref PubMed Scopus (191) Google Scholar, 29Wang S.E. Shin I. Wu F.Y. Friedman D.B. Arteaga C.L. HER2/Neu (ErbB2) signaling to Rac1-Pak1 is temporally and spatially modulated by transforming growth factor beta.Cancer Res. 2006; 66: 9591-9600Crossref PubMed Scopus (93) Google Scholar because collagen-I is the major matrix protein in dermis/connective tissues, acting as a barrier to cellular invasion. In testing the effects of various inhibitors, the experiments were performed with or without MMP inhibitors GM6001 (or its negative control), BB-2516, or BB-3103 (0.5, 5, 10, and 15 μmol/L), cathepsin B inhibitors CA-074 or CA-074Me (1, 2, and 5 μmol/L), cathepsin L inhibitors I and II (0.5, 5, and 15 μmol/L), TGF-β receptor I (ALK5) inhibitor SB-505124 (1, 2.5, 5, and 10 μmol/L), or the neutralizing antibodies to TGF-β (2.5, 5, and 10 μg/mL), added into the Matrigel and the growth medium. The growth medium was replenished every third day. The growth pattern and morphological characteristics of the cells were documented daily by phase-contrast microscopy and photography using an Olympus CK2 microscope, equipped with phase-contrast optics and a digital camera, or an Olympus IX71 microscope and an Olympus DP70 camera (Tokyo, Japan). Whole cell extracts and secreted proteins in conditioned media were analyzed by using Western blot analysis, as previously described.27Paasinen-Sohns A. Kaariainen E. Yin M. Jarvinen K. Nummela P. Holtta E. Chaotic neovascularization induced by aggressive fibrosarcoma cells overexpressing S-adenosylmethionine decarboxylase.Int J Biochem Cell Biol. 2011; 43: 441-454Crossref PubMed Scopus (10) Google Scholar, 30Kielosto M. Nummela P. Katainen R. Leaner V. Birrer M.J. Holtta E. Reversible regulation of the transformed phenotype of ornithine decarboxylase- and ras-overexpressing cells by dominant-negative mutants of c-Jun.Cancer Res. 2004; 64: 3772-3779Crossref PubMed Scopus (22) Google Scholar Monoclonal antibodies to TGF-β (R&D Systems), polyclonal antibodies to human hepatocyte growth factor (HGF; R&D Systems), monoclonal antibodies to cathepsin B (CB 59-4B11; Alexis/Enzo Life Sciences, Lausanne, Switzerland), and monoclonal antibodies to α-smooth muscle actin (α-SMA; Dako, Glostrup, Denmark) were used to detect the respective proteins. Polyclonal antibodies to α-tubulin (Rockland Immunochemicals, Gilbertsville, PA) and monoclonal antibodies to actin (EMD Millipore–Calbiochem, Darmstadt, Germany) were used as a loading control. Fresh benign nevi (n = 21) from healthy volunteers and primary melanoma specimens [n = 27: Breslow's thickness, ≤2.0 mm (median, 1.5 mm; range, 0.6 to 2.0 mm), n = 11; and >2.0 mm (median, 6.4 mm; range, 2.4 to 27 mm), n = 16] (for microarray analyses) were obtained by surgical excision at Helsinki University Central Hospital, Helsinki, Finland. Adjacent sections were subjected to pathological and IHC examinations by an experienced dermatopathologist (S.V.). The protocols for taking the specimens were approved by the Ethics Committees of the Helsinki University Central Hospital. Furthermore, informed consent was obtained from all patients. Formalin-fixed, paraffin-embedded nevi (n = 13) and primary melanomas (n = 60) (for IHC analyses) were from the archives of the Department of Pathology, University of Helsinki, Helsinki. Microarray analyses with the Affymetrix Human Genome U133 Set and U133 Plus 2.0 arrays were performed according to the protocols of the manufacturer (Affymetrix Inc., Santa Clara, CA), as previously described.24Soikkeli J. Podlasz P. Yin M. Nummela P. Jahkola T. Virolainen S. Krogerus L. Heikkila P. von Smitten K. Saksela O. Holtta E. Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.Am J Pathol. 2010; 177: 387-403Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 31Soikkeli 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 The data were processed by RMAExpress version 1.0.4 (http://rmaexpress.bmbolstad.com), and the probe sets were ordered both by significance analysis of microarrays (SAM)32Tusher V.G. Tibshirani R. Chu G. Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci U S A. 2001; 98: 5116-5121Crossref PubMed Scopus (9771) Google Scholar and fold-change ranking, combined with P value calculations,33Shi L. Reid L.H. Jones W.D. Shippy R. Warrington J.A. et al.MAQC ConsortiumThe MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements.Nat Biotechnol. 2006; 24: 1151-1161Crossref PubMed Scopus (1727) Google Scholar essentially as previously described.24Soikkeli J. Podlasz P. Yin M. Nummela P. Jahkola T. Virolainen S. Krogerus L. Heikkila P. von Smitten K. Saksela O. Holtta E. Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.Am J Pathol. 2010; 177: 387-403Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Cathepsin B and L gene expression levels in melanoma cells and fibroblasts were detected by RT-PCR, which was performed essentially as previously described.31Soikkeli 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, 34Nummela P. Lammi J. Soikkeli J. Saksela O. Laakkonen P. Hölttä E. Transforming growth factor beta-induced (TGFBI) is an anti-adhesive protein regulating the invasive growth of melanoma cells.Am J Pathol. 2012; 180: 1663-1674Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Briefly, 1 μg of total RNA extracted from the cells with the RNeasy kit (Qiagen, Crawley, UK) was reverse transcribed to cDNA. The primers (forward and reverse) designed for cathepsin B were as follows: 5′-AAGCTTCGATGCACGGGAACAATG-3′ and 5′-TCCAGCCACCACTTCTGATTCGAT-3′, respectively; cathepsin L, 5′-GTCAGTGTGGTTCTTGTTGG-3′ and 5′-AAGGACTCATGACCTGCATC-3′, respectively; and β-actin, 5′-GCTCGTCGTCGACAACGGCTC-3′ and 5′- CAAACATGATCTGGGTCATCTTCTC-3′, respectively. The PCRs, optimized to be in the linear range, consisted of denaturation (94°C for 2 minutes), followed by 20 to 25 cycles of denaturation at 94°C for 60 seconds, annealing at 56°C for 45 seconds, extension at 72°C for 90 seconds, and a final elongation of 7 minutes at 72°C. The products were resolved in 2% agarose gels, stained with GelStar nucleic acid gel stain (Cambrex Bio Science Rockland, Rockland, ME), and visualized under UV light. The gel runs were documented with Alpha Imager HP and Alpha EaseFC Software, version 5.01 (Alpha Innotech Corporation, San Leandro, CA). Immunostainings were performed essentially as previously described.35Kaariainen E. Nummela P. Soikkeli J. Yin M. Lukk M. Jahkola T. Virolainen S. Ora A. Ukkonen E. Saksela O. Holtta E. Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion.J Pathol. 2006; 210: 181-191Crossref PubMed Scopus (58) Google Scholar We first tested the performance of several commercial antibodies to cathepsin B (data not shown), because of the known unsatisfactory function of many previously used ones (eg, polyclonal sheep antibodies) in IHC.36Sinha A.A. Wilson M.J. Gleason D.F. Reddy P.K. Sameni M. Sloane B.F. Immunohistochemical localization of cathepsin B in neoplastic human prostate.Prostate. 1995; 26: 171-178Crossref PubMed Scopus (77) Google Scholar Antibody staining conditions were then optimized. The slides were exposed to heat-induced epitope retrieval and incubated with the antibodies to cathepsin B (5 g/mL; CB 59-4B11) and cathepsin L (7 g/mL, ab7431; Abcam, Cambridge, UK). A two-tailed Welch's t-test was used for the significance analyses. P < 0.05 was considered statistically significant. The Kaplan-Meier survival curves were analyzed with PASW Statistics 18 software (SPSS Inc., Chicago, IL), using the log-rank (Mantel-Cox) test. We and other researchers have shown that fibroblasts contribute to melanoma progression/invasion in vitro37Wandel E. Raschke A. Hildebrandt G. Eberle J. Dummer R. Anderegg U. Saalbach A. Fibroblasts enhance the invasive capacity of melanoma cells in vitro.Arch Dermatol Res. 2002; 293: 601-608Crossref PubMed Scopus (22) Google Scholar, 38Eves P. Katerinaki E. Simpson C. Layton C. Dawson R. Evans G. Mac Neil S. Melanoma invasion in reconstructed human skin is influenced by skin cells: investigation of the role of proteolytic enzymes.Clin Exp Metastasis. 2003; 20: 685-700Crossref PubMed Scopus (46) Google Scholar, 39Li L. Dragulev B. Zigrino P. Mauch C. Fox J.W. The invasive potential of human melanoma cell lines correlates with their ability to alter fibroblast gene expression in vitro and the stromal microenvironment in vivo.Int J Cancer. 2009; 125: 1796-1804Crossref PubMed Scopus (43) Google Scholar and in vivo.24Soikkeli J. Podlasz P. Yin M. Nummela P. Jahkola T. Virolainen S. Krogerus L. Heikkila P. von Smitten K. Saksela O. Holtta E. Metastatic outgrowth encompasses COL-I, FN1, and POSTN up-regulation and assembly to fibrillar networks regulating cell adhesion, migration, and growth.Am J Pathol. 2010; 177: 387-403Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 35Kaariainen E. Nummela P. Soikkeli J. Yin M. Lukk M. Jahkola T. Virolainen S. Ora A. Ukkonen E. Saksela O. Holtta E. Switch to an invasive growth phase in melanoma is associated with tenascin-C, fibronectin, and procollagen-I forming specific channel structures for invasion.J Pathol. 2006; 210: 181-191Crossref PubMed Scopus (58) Google Scholar To assess the importance of stromal fibroblasts for the invasive growth of melanoma cells, we first analyzed the growth patterns of VGP WM793 melanoma cells cultured alone or mixed in a 1:1 ratio with embryonic (HES) or adult (HAS) skin fibroblasts in 3D-Matrigel and Matrigel/collagen-I matrices. The early VGP-derived WM793 cells exhibited no or only poor invasion during the first 24 hours (Figure 1, A and D; see also Supplemental Figure S1A at http://ajp.amjpathol.org), but showed some invasive growth during prolonged culture (see Supplemental Figure S1B at http://ajp.amjpathol.org). The HES or HAS fibroblasts, in turn, were not able to grow or invade on their own in 3D-Matrigel, but remained as isolated, round to oval cells, and died during the prolonged culture (Figure 1, B and E; data not shown). Interestingly, when HES or HAS cells were admixed and co-cultured with WM793 cells, the fibroblasts became spindle shaped/activated, and assembled in direct contact with the melanoma cells. This interaction appeared to dramatically increase the invasive growth potential of both the fibroblasts and WM793 cells, resulting in a rapidly expanding network of invading cells (Figure 1, C and F). A clear, but less intense, promotion of the invasive growth was also observed when WM793 cells were seeded in the middle of Matrigel and fibroblasts were seeded on top of Matrigel, so that the melanoma cells and fibroblasts could not contact each other directly when plated (Figure 1, G–L). After 24 hours of culture, small conglomerates of fibroblasts and melanoma cells in direct contact were nevertheless observed (Figure 1, I and L). Furthermore, we found HES and HAS cells seeded on top of Matrigel to become activated on addition of conditioned growth media (CM) from WM793 cells (see Supplemental Figure S2 at http://ajp.amjpathol.org). These data suggested that melanoma cells secrete some factor(s) that can activate even the quiescent adult skin fibroblasts at a distance and induce their migration to a physical contact with the melanoma cells. Reciprocally, we also found that the secreted factors/CM from HAS and particularly from HES increased the invasive activity of WM793 cells (see Supplemental Figure S3A at http://ajp.amjpathol.org). Notably, the CM from HES appeared to induce in WM793 cells an epithelial-mesenchymal transition–like conversion in morphological characteristics (see Supplemental Figure S3A at http://ajp.amjpathol.org). The higher potency of the CM from HES than HAS was understandable, because the embryonic cells represented activated fibroblasts, as evidenced by their expression of increased amounts of HGF and α-SMA (ACTA2), markers of myofibroblasts (see Supplemental Figure S3B at http://ajp.amjpathol.org). To better define the invasion pat
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