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[Cancer Research 65, 4134-4146, May 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Gene Expression Profiling Reveals Cross-talk between Melanoma and Fibroblasts: Implications for Host-Tumor Interactions in Metastasis

Paul G. Gallagher1, Yongde Bao1, Alyson Prorock1, Paola Zigrino2, Roswitha Nischt2, Vincenzo Politi3, Cornelia Mauch2, Bojan Dragulev1 and Jay William Fox1

1 Department of Microbiology, University of Virginia, Charlottesville, Virginia; 2 Department of Dermatology, University of Cologne, Cologne, Germany; and 3 Polifarma SpA, Rome, Italy

Requests for reprints: Jay William Fox, Department of Microbiology, University of Virginia, P.O. Box 800734, Charlottesville, VA 22908-0734. Phone: 434-924-0050; Fax: 434-924-2514; E-mail: jwf8x{at}virginia.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Host-tumor interaction is considered critical in carcinogenesis, tumor invasion, and metastasis. To explore the reciprocal effects of host-tumor interaction, we developed a system to assess the gene expression patterns of A2058 human melanoma cells cocultured in fibrillar collagen with HS-68 primary human fibroblasts. The gene expression pattern of the cocultured A2058 cells was only modestly affected, whereas the HS-68 fibroblast gene expression pattern was significantly altered. Interleukin-11 and inhibitor of DNA-binding domain-1 gene expression in the cocultured A2058 cells was down-regulated, indicative of a proinflammatory response and resistance to apoptosis, respectively. The overall pattern of up-regulated genes indicated triggering of the proinflammatory process. In addition, the melanoma growth and migration stimulatory chemokines CXCL1 and CXCL2 were significantly up-regulated in the cocultured fibroblasts. These results were corroborated by additional coculture experiments with the melanoma cell lines WM-164, BLM, and SK-Mel-28 and immunohistochemistry on invasive human melanoma sections. Taken together, these results indicate that tumor cells cause a proinflammatory and melanoma growth-promoting response in stromal fibroblasts. The role of inflammation in carcinogenesis, tumor promotion, invasion, and metastasis is viewed as being increasingly important and the results of these studies underscore this as well as identify certain key proteins that are expressed as a result of the complex interactive processes in the host-tumor microenvironment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Increasing attention is being focused on the interaction of tumor cells with the host in terms of exploring how that interaction is conducive to the induction, selection, and expansion of the tumor ultimately leading to the malignant progression of the tumor (1–5). For example, studies on human breast carcinomas have shown that vascular stroma formation occurs before invasion by tumor cells. In situ hybridization of breast carcinomas at various stages detected expression of vascular permeability factor and vascular endothelial growth factor, whereas the stromal cells were shown to express thrombospondin-1, collagen type I, fibronectin, versican, and decorin, suggesting that the tumor induces vascularization in regions where they invade and host stroma promotes this vascularization (6).

The microenvironment in which host-tumor interaction occurs has also been implicated in malignancy. The extracellular matrix (ECM) of the microenvironment is well known to regulate a variety of cellular phenomena. Perturbation of the matrix by either proteolysis or alteration of its architecture due to changes in molecular composition and/or stoichiometry resulting from host-tumor interaction can disrupt the homeostasis of the microenvironment (7–9). For example, Thomasset et al. (10) have shown that low-level expression of the transgene matrix metalloproteinase (MMP) stromelysin-1 in mouse mammary epithelia causes an up-regulation of endogenous stromelysin-1 in fibroblasts. These changes resulted in the development of preneoplastic and neoplastic lesions in mice that were associated with the up-regulation of other MMPs, ECM components, and mammary gland vascularization.

The concept that inflammation plays a central role in tumor progression has received increasing attention, particularly in the context of host-tumor interaction (1, 11). For example, macrophage infiltrates of malignant melanoma have been associated with tumor stage and angiogenesis. Production of transforming growth factor-ß, tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin (IL)-1{alpha}, arachidonate metabolites, and extracellular proteinases by the macrophages elicited the expression of IL-8 and vascular endothelial growth factor-A in melanocytes, thereby promoting inflammation and angiogenesis (12). Several studies have shown that host-tumor interaction results in the production of proinflammatory cytokines and chemokines, thereby promoting the recruitment of host leukocytes in the microenvironment of the tumor (13). However, in these investigations and most others, there is always some question as to the cell source of the chemokines and cytokines involved in the development of the proinflammatory pathway within the tumor microenvironment.

Unlike melanocytes, melanoma cells constitutively produce a large number of growth factors and cytokines and their respective receptors that enable the cells to progress to a more aggressive phenotype. Autocrine growth factors (e.g., basic fibroblast growth factor, IL-8, and hepatocyte growth factor) stimulate proliferation and migration of melanoma cells themselves, whereas paracrine factors (e.g., platelet-derived growth factor, transforming growth factor-ß, basic fibroblast growth factor, vascular endothelial growth factor, and monocyte chemoattractant protein-1) are believed to modulate the microenvironment to the benefit of melanoma growth, invasion, and metastasis (14). On the other hand, fibroblasts are also a rich source of growth factors but only after activation. When stimulated by melanoma cell–released factors, they can produce growth factors (e.g., insulin-like growth factor-I, hepatocyte growth factor, and ET-3) that in turn contribute to the orchestra of virtual modulation of cellular activities (15).

Several investigators have shown that host-tumor interactions also play a crucial role in the regulation of connective tissue breakdown in different tumors. Tumor cell–derived factors, such as extracellular MMP inducer, have been shown to be expressed by various tumor cells, including melanoma, and to induce production of MMP-1, MMP-2, and MMP-3 in normal fibroblasts (16). Recently, we could show in human primary melanoma and in lymph node metastases that the activity of MMPs (e.g., MMP-2 and MMP-9) was located primarily in those areas where tumor cells lay adjacent to the ECM or within connective tissue septa among the aggregates of melanoma cells (17). This observation stresses the importance of host-tumor interactions with structural and cellular components of the surrounding ECM.

In this investigation, we report the results of assays designed to assess the reciprocal effect of soluble cofactors resulting from the coculture of human melanoma cell lines A2058, BLM, WM-164, and SK-Mel-28 with human HS-68 fibroblasts on their respective gene expression profiles using oligonucleotide microarrays. Interestingly, the gene expression profile of the fibroblasts was dramatically altered under coculture conditions, whereas the effect on the melanoma A2058 cell gene expression profile was more modest. Overall, the fibroblasts responded to coculture by up-regulation of a variety of genes associated with the proinflammatory pathway and cellular proliferation and this up-regulation was confirmed by quantitative real-time PCR (qRT-PCR). Several transcripts of ECM components were observed to be down-regulated in cocultured fibroblasts suggesting a potential alteration of the extracellular environment. Furthermore, the presence of several of these proteins was detected by immunohistochemistry of invasive human melanoma.

In summary, we observed a profound effect on the gene expression profile of fibroblasts when in coculture with melanoma cells. Analysis of the gene expression profiles indicates the initiation of the proinflammatory pathway and an altered ECM, conditions that have been implicated in tumor progression, invasion, and metastasis. However, the response to coculture with fibroblasts by the melanoma cell line is modest. Therefore, we conclude based on this study that the stroma, due to signals originating from the tumor, is primarily responsible for the generation of a microenvironment that is proinflammatory, proproliferation, proinvasion, and prometastatic.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
HS-68 fibroblast and melanoma cell culture and RNA isolation. Human primary foreskin fibroblasts (HS-68, American Type Culture Collection, Manassas, VA) were grown in DMEM/10% FCS, and the melanoma cell lines A2058, BLM, WM-164, and SK-Mel-28 were grown in DMEM/10% FCS and 1 µmol/L sodium pyruvate. Fibroblasts and melanoma cells were seeded individually during polymerization of the collagen type I lattice and cultured on opposite sides of a 1 µm pore, six-well cell culture insert (Becton Dickinson, Sparks, MD). Collagen lattices containing cells were composed of 1x DMEM, 0.9 mg/mL bovine skin collagen type I (IBFB, Leipzig, Germany), 14.8 mmol/L NaOH, 10% FCS, and 3 x 105 cells/mL. Collagen/cell mixture (540 µL) was added to each side of the insert and allowed to polymerize to form the cell-embedded lattice. The fibroblast/collagen lattice was situated above the filter and melanoma cell/collagen lattice below. After collagen fibers had formed, DMEM/10% FCS (1 mL) was injected below each insert and DMEM/10% FCS (250 µL) was overlaid on top of the fibroblast/collagen lattice. Monoculture control samples were generated by culturing only melanoma or fibroblast cells in collagen lattices on the same side of the filter as in the coculture design. Cells were incubated for 2 or 72 hours at 37°C/5% CO2, and total RNA was isolated from two inserts for each cell type using TRIzol reagent (Invitrogen, Carlsbad, CA). All experiments were done in duplicate.

Microarray hybridization. Biotin-labeled cRNA was generated from total RNA samples according to standard Affymetrix GeneChip protocols (Santa Clara, CA). Briefly, a poly-T oligo primer containing a 5' T7 RNA polymerase promoter was used to generate double-stranded cDNA followed by biotin-labeled cRNA synthesis using BioArray High-Yield RNA Transcript Labeling kit (Enzo, New York, NY). cRNA was fragmented according to Affymetrix standard protocol, and fragmented cRNA (10 µg) was hybridized to Affymetrix Hg-U95A probe arrays for 16 hours. The arrays were washed and stained in an Affymetrix Automated Fluidics Station 400 and scanned with a HP GeneArray scanner.

Gene expression analysis. Scanned gene array images were first examined for visible defects and then checked for the fitness of the gridding. When passed, the image file was analyzed to generate composite data files ("cell files"). From this point on, a coordination of two paths of analysis was carried on using the Affymetrix Microarray Analysis Suite version 5.0 and the Dchip software version 1.1 (18). The detection of a particular gene, called "present", "absent", or "marginal", was made using the nonparametric Wilcoxon ranked score algorithm in Microarray Analysis Suite version 5.0. Those detection calls were later imported into and used by the Dchip program. Scatter plots were also generated using this software to inspect the reproducibility of the replicates as well as the degree of changes of the samples under comparison. Quantification of gene expression was obtained using Dchip, which applied a model-based approach to derive the probe sensitivity index and expression index. The two indices were used in a linear regression to quantify a particular gene. When certain probes or transcripts deviated from the model to a set extent, they were excluded from the quantification process. Normalization of the arrays was done using the invariant set approach. Comparative analysis of the samples was done based on Dchip-generated fold changes and unpaired sample t test. Typically, we considered a P of less than 0.05, a fold change greater than 1.5 or less than –1.5, and a signal intensity difference of greater than 100 or less than –100 as indications of significant change in gene expression.

To aid in discovery of the potential biological processes represented by the differentially expressed genes identified from the microarray data, the MAPPFinder program (19) was used in conjunction with GenMAPP program (20). These programs were developed to reveal global gene expression profiles across all areas of biology by integrating the annotation of the Gene Ontology Project (The Gene Ontology Consortium, 2000). Briefly, the GeneChip data were reformatted to use Genbank accession numbers as the gene identification to query the GenMAPP database. Search terms included the possible pathways and the gene ontology terms. A Z score is generated with each hit to indicate the strength of the association of the cluster of genes to the gene ontology terms or pathways discovered. For our data, we reported the genes with of a Z score equal to or greater than 1.

Quantitative real-time PCR. Total RNA samples used in qRT-PCR were from the same preparations as described for the Affymetrix GeneChip experiments. Reverse transcription was done with MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA) and random hexamers as per the manufacturer's instruction. The resulting cDNA was then subjected to qRT-PCR. For each of the transcripts of interest identified from the GeneChip, primers were designed from the target sequences retrieved from the Affymetrix Probe Sequence Database using the Primer Express 2.0 software (Applied Biosystems). Quantitative PCRs were carried out in triplicates using equal amounts of each cDNA sample equivalent to 50 ng of starting total RNA. Each reaction contained the fluorescent indicator SYBR Green I dye and 6 µL of each respective forward and reverse primer (5 µmol/L) in a total volume of 50 µL. Amplification PCR and monitoring of the fluorescent emission in real-time were done in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems) as recommended by the manufacturer (ABI SYBR Green Protocol). The data collected from these quantitative PCRs defined a threshold cycle (Ct) of detection for the target or the housekeeping genes in each cDNA sample.

To convert the Ct value into a relative abundance of target and housekeeping gene per sample, a standard curve was generated for the housekeeping gene using serial dilutions of cDNA sample: an arbitrary value of template was first assigned to the highest standard and then corresponding values were assigned to the subsequent dilutions, and these relative values were plotted against the Ct value determined for each dilution, resulting in the generation of the standard curve. The relative amount of target and housekeeping genes in each sample was then determined using the comparative Ct method (Applied Biosystems). The relative quantity of target, normalized to an endogenous reference (usually a housekeeping gene) and relative to a calibrator (the Rox reference dye), is given by: relative quantity = 2 – {Delta}{Delta}Ct, where {Delta}{Delta}Ct represents the difference in Ct between the transcript and the housekeeping gene for the same RNA sample. The ratio of the relative quantities for the treated sample and the experiment sample was used to derive the fold change. ANOVA was then used to determine the mean and SE for each comparison.

Immunohistochemistry. Cryosections (8 µm thick) were fixed with cold acetone for 5 minutes and rinsed for 10 minutes in TBS. Paraffin sections used for CD68 detection were first deparaffinized by xylol and ethanol incubations and washed in TBS. Sections were blocked for 1 hour with 10% FCS in TBS before applying the primary antibodies diluted in TBS-FCS for 16 hours at 4°C. The sections incubated with a primary goat antibody were also incubated with a bridging mouse anti-goat antibody (1:100, DAKO Envision, Hamburg, Germany) for 30 minutes. After 3 x 15–minute washes, bound antibodies were detected with alkaline phosphatase–labeled anti-mouse/anti-rabbit polymer (DAKO Envision) and neofuchsin as substrate. Nuclei were counterstained with hematoxylin solution for 1 minute (Shandon, Pittsburgh, PA).

The following antibodies were used: monoclonal antibody directed against human fibroblasts (anti-Thy-1, 1:50, Dianova, Hamburg, Germany), monoclonal antibody to human IL-1ß (1:100, BioSource/Laboserv, Giessen, Germany), goat anti-human GRO{alpha} (1:50, R&D Systems, Wiesbaden, Germany), IL-8 (1:50, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), and mouse anti-human CD68 (1:25, DAKO Envision).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gene expression analysis. Within the 16 HGU95AV2 arrays used for the study, the percentage of genes determined to be present ranged from 49.6% to 55.4% with a median of 54.3%. The average correlation coefficient between biological duplicates was >0.99. In a global view, the two different cell lines display quite different expression profiles as evidenced by the correlation coefficients extracted from the individual expression values for the ~12,000 genes for each sample under various conditions (see Supplementary Data for details). The correlation coefficients within the A2058 cells have a mean of 0.985152 and a SD of 0.007936, and those within the human fibroblast HS-68 cells have a mean of 0.9763 and a SD of 0.01493. The correlation coefficients between the two cell lines have a mean of 0.876462 and a SD of 0.008629.

Effect of coculture on the gene expression profiles of melanoma A2058 cells. The change in the gene expression profile of the melanoma A2058 cells grown in coculture with the human HS-68 fibroblasts compared with growth as a monoculture alone was relativity modest. About 21 genes were determined to have significant fold changes compared with the gene expression profile of the melanoma A2058 cells cultured alone (Table 1).


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  		Table 1. Genes with altered expression levels in A2058 melanoma cells when grown in coculture with HS-68 fibroblasts

 
Dopachrome tautomerase, an enzyme involved in melanin synthesis and melanocyte cell proliferation (21), was observed to be up-regulated in A2058 cells cocultured with fibroblasts, indicating a growth stimulatory effect of fibroblast coculture on the A2058 cells.

The gene showing the greatest down-regulation in A2058 melanoma cells when cocultured with fibroblasts was IL-11. This cytokine has been shown to be anti-inflammatory, functioning by inhibiting the secretion of proinflammatory cytokines by macrophages (22, 23). Another gene that was down-regulated in the cocultured A2058 cells is the inhibitor of DNA-binding domain-1. In mammary epithelial cell culture, inhibitor of DNA-binding domain-1 expression induces apoptosis (24); hence, in coculture with fibroblasts, the A2058 cells with a decreased level of inhibitor of DNA-binding domain-1 may gain some resistance to apoptosis.

The results of analysis of the gene expression data by the MAPPFinder and GenMAPP programs to detect expression patterns associated with biological ontology and pathways, respectively, are shown in Table 2. These data identified nine ontology classes and six pathways as being significantly populated with up-regulated genes in the cocultured melanoma cells. Interestingly, two pathways associated with chemokine signal transduction, the G13 signaling pathway (guanine nucleotide-binding protein) and small ligand G protein-coupled receptors (GPCR), were identified. The inflammatory response pathway was also identified as being active. This suggests involvement of the proinflammatory process as a result of coculture of A2058 melanoma cells with fibroblasts. In the up-regulated biological process class, melanin biosynthesis had the greatest Z score. Up-regulation of this process class suggests an enhanced growth of the melanoma cells grown in coculture with the fibroblasts possibly due to a response to GRO{alpha} (CXCL1) production by cocultured HS-68 fibroblasts (see below). In addition, the defense response biological process class was up-regulated.


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  		Table 2. Pathways and biological process classes associated with differentially expressed genes in cocultured A2058 melanoma cells

 
As seen in Table 2, eight pathway classifications and five biological process classes were identified to be populated by a significant number of down-regulated genes in the cocultured cells.

Effect of coculture on the gene expression profiles of human HS-68 fibroblasts. Quantitatively and qualitatively, the change in the gene expression profile of the HS-68 fibroblast cells grown in coculture with the human A2058 melanoma cells was in sharp contrast to that observed for the cocultured melanoma cells. From the gene expression data of cocultured HS-68 fibroblasts, 85 genes were determined to have significant fold changes compared with the gene expression profile of the HS-68 fibroblasts cultured alone (Table 3). From the literature, 23 of the 85 genes that were altered have been implicated in processes associated with proinflammatory response, cell growth, proteolysis of ECM, and tumor invasion and metastasis.


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  		Table 3. Genes with altered expression levels in HS-68 fibroblasts grown in coculture with A2058 melanoma cells

 
The GenMAPP and MAPPFinder programs identified 8 biological pathways and 11 biological process classes up-regulated. In the up-regulated pathways, the blood clotting cascade had the greatest Z score (5.770). Eighteen genes were identified in that pathway from the data set representing 90% of members of the pathway.

The inflammatory response biological process class had the greatest Z score (9.996) in the up-regulated group, with 132 genes in the class being identified from the data set (82% of the class members).

Nine biological pathways and 25 biological process ontology classes were associated with the down-regulated gene expression data. Interestingly, the inflammatory response pathway was identified from the down-regulated gene set as having the greatest Z score (3.975) populated by 29 genes representing 94% of the pathway members, whereas the humoral immune response biological process class had the greatest Z score (5.075) of this category with 23 genes (85% of the members) identified in this class.

Based on the data, it seems that there is a significant response in HS-68 fibroblasts to coculture with A2058 melanoma cells, much more so than that observed with the A2058 cells cocultured with fibroblasts. As noted from the list of genes whose expression was significantly altered in the HS-68 fibroblasts and the results from the pathway and biological process class surveys, the fibroblasts respond to coculture with a proinflammatory response accompanied by other changes that have typically been associated with promotion of tumor invasion and metastasis.

Quantitative real-time PCR. Table 4 shows the results of qRT-PCR on six genes based on identification from the gene analysis data of cocultured HS-68 fibroblasts, which we determined to be of particular interest due to their role in the proinflammatory pathways and matrix degradation. These results, in terms of fold changes, show relatively good concordance with the microarray data in terms of fold change of gene expression.


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  		Table 4. Comparisons of expression levels measured for selected genes from HS-68 fibroblasts grown in coculture with A2058, BLM, WM-164, and SK-Mel-28 melanoma cells

 
qRT-PCR results are also shown in Table 4 for HS-68 fibroblasts cocultured with three additional human melanoma cell lines: BLM, WM-164, and SK-Mel-28. The human melanoma cell line BLM is a highly invasive melanoma that readily forms metastases in nude mice (25). The human melanoma lines WM-164 and SK-Mel-28 are only modestly invasive (26, 27). The BLM melanoma cells exhibited significant fold changes for the six genes analyzed, whereas the fold changes for the WM-164 and SK-Mel-28 cell lines were modest compared with the BLM and A2058 cells.

Immunohistochemistry. To corroborate the in vitro gene expression results, immunohistochemical studies on invasive human melanoma specimens were done. Nevus and melanoma biopsies of primary tumors were analyzed for the presence of selected genes (e.g., GRO{alpha}, IL-1ß, and IL-8) that were surprisingly up-regulated in fibroblasts in cocultures. Antibodies against CD68 and fibroblast-specific antigen (Thy-1) were used to identify macrophages and fibroblasts in the tissue, respectively. Figure 1 is a representative of several biopsies that were examined. In a nodular melanoma with a maximal tumor thickness of 2.3 mm, CD68-positive macrophages were detected between the tumor cells, whereas Thy-1-positive, spindle-shaped fibroblasts embedded in a spare stroma surrounded the melanoma cell nests. GRO{alpha} staining was mainly associated with the stromal fibroblasts located close to the tumor cells. IL-8 and IL-1ß staining was detected in the stromal fibroblasts adjacent to the tumor cells but also in some melanoma cells. In the congenital nevus, some macrophages were depicted in the vicinity to the nevus cell nests at the junctional zone. Thy-1-positive fibroblasts, however, were not stained for IL-8, IL-1ß, and GRO{alpha}.



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  		Figure 1. Immunohistologic staining of Thy-1 (fibroblast-specific antigen), CD68 (macrophage antigen), IL-8, GRO{alpha}, and IL-1ß in specimens of a representative sample of human congenital nevus and a primary nodular melanoma. Serial sections (8 µm) of paraffin-embedded tissue were stained using monoclonal or polyclonal antibodies raised against Thy-1, CD68, IL-8, GRO{alpha}, and IL-1ß. Bound antibodies were detected by alkaline phosphatase and neofuchsin as a substrate and counterstained with hematoxylin. Magnification, x20. Bar, 50 µm.

 
Table 5 presents a summary of an immunohistochemical survey for the expression of GRO{alpha}, IL-1ß, and IL-8 in biopsies from nine human malignant melanomas. In all sections, strong positive staining associated with fibroblasts was observed for IL-1ß and IL-8, with modest staining observed for GRO{alpha}. These data corroborate the representative immunohistochemistry shown in Fig. 1. Interestingly, there did not seem to be any significant correlation of staining intensity to whether the biopsies were from nodular malignant melanomas or superficial spreading melanomas.


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  		Table 5. Expression of GRO{alpha}, IL-1ß, and IL-8 in human malignant melanomas

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The relationship between host and tumor has recently been appreciated to be a dynamic one, whereby the environment of host as represented by the stroma, tissues, organs, and ECM affects the behavior of the tumor and similarly the presence of the tumor has a reciprocal effect on the host. This interplay between the host and the tumor may in fact be a critical factor in terms of whether the host eliminates the tumor or the tumor continues to progress and metastasize (28). As early as 1863, cancer was noted to often be associated with chronic inflammation and subsequent cell proliferation, and over the years, anti-inflammatory therapies have been suggested as an approach for cancer prevention and treatment (13, 29). Thus, the host-tumor interaction can result in a variety of proinflammatory processes that may lead to the successful immunologic abatement of the tumor by the host (13). However, if the proinflammatory response resulting from host-tumor interaction is blunted, such that a full, effective immune response does not occur, then an environment may occur that could promote tumorigenesis, tumor progression, and metastasis (1).

Effect of coculture of A2058 melanoma cells and HS-68 fibroblasts. The design of the experimental system for the coculture of melanoma cells with fibroblasts in the context of fibrillar collagen allowed for the exchange of soluble mediators in the culture medium yet segregated the cells. The pore size of the membrane separating the two cell lines was such that physical interaction of invadopods from the cells could occur but not cellular passage through the membrane. As seen from Table 1, there is relatively little change in the gene expression profile of A2058 cells grown in coculture with fibroblasts compared with growth alone in fibrillar collagen. However, there seems to be a significant effect of coculture on the gene expression profile of HS-68 fibroblasts in the presence of A2058 cells (Table 3). This suggests the possibility that the fibroblasts are more responsive to the pool of soluble mediators produced by the two cell lines than the melanoma cells. It could also be interpreted that the melanoma cells are constitutively activated and not responsive to factors released by fibroblasts.

Proinflammatory environment in cocultured A2058 cells and HS-68 fibroblasts. From the gene expression profile of the cocultured fibroblasts, there are a variety of changes in gene expression that could be considered to be of a proinflammatory nature (Tables 3 and 6). For example, the proinflammatory chemokine IL-8 (CXCL8) and the inflammatory cytokine IL-1ß were observed to increase 6.8- and 2.4-fold, respectively. The up-regulation of these genes is indicative of the development of a proinflammatory/inflammatory environment resulting from the coculture of melanoma cells and fibroblasts. IL-8 is produced by many cell types, and in addition to being a neutrophil chemoattractant, it has been shown to have other activities that can be considered prometastatic (30, 31). However, in a recent report, Li et al. (32) showed a direct role of IL-8 in angiogenesis by inducing MMP-2 synthesis, inhibiting endothelial cell apoptosis, and enhancing antiapoptotic gene expression. It is well established that melanoma cells constitutively express increased amounts of IL-8. Melanoma cell–induced expression of this cytokine by adjacent stromal fibroblast therefore would amplify enhanced neovascularization of the tumor.


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  		Table 6. Pathways and biological process classes associated with differentially expressed genes in cocultured HS-68 fibroblasts

 
IL-1 receptor antagonist (IL-1RA) was observed to be modestly up-regulated (1.6-fold) in cocultured fibroblasts (Table 3). IL-1RA binds to the IL-1 receptor, thereby inhibiting interaction with IL-1. IL-1 is a key, pleiotropic inflammatory cytokine that promotes angiogenesis, tumor growth, and metastasis (33). IL-1RA is often coexpressed with IL-1, and depending on the ratio between cytokine and its antagonist, there is diminution of the biological effects of IL-1. For example, IL-1 is constitutively expressed at high levels in the metastatic melanoma cell line SMEL (34). When this cell line is transduced with IL-1RA, tumor growth resulting from the transduced cells was significantly reduced in athymic nude mice, as was metastasis to the lung, clearly indicating a role for IL-1RA in modulating IL-1 activities. In addition, IL-1 is required for angiogenesis by stimulating the proliferation of endothelial cells and the synthesis of adhesion molecules and proteolytic enzymes on endothelial cells (35). Using IL-1{alpha} and IL-1ß knockout mice, these authors were able to show that local tumor or lung metastases of B16 melanoma cells were not observed compared with wild-type animals. In addition, vascularization of melanoma cell–populated Matrigel plugs by endothelial cells was observed in wild-type and inhibited by the addition of IL-1RA, whereas in-growth of endothelial cells was absent in IL-1ß knockout mice, suggesting that host-derived IL-1 has an important impact on tumor growth and metastasis.

In our studies of the cocultured fibroblasts, we have not examined the protein levels of either IL-1ß or IL-1RA and hence have no real understanding of the relative amounts of these proteins; however, from the perspective of the gene expression data, both the intensities and the fold changes were greater for IL-1ß than IL-1RA. Therefore, we would expect that the net result is that the IL-1RA expression in this system may serve to attenuate the inflammatory response due to IL-1ß, but it is not likely to completely ablate it.

The down-regulation of IL-11 represents another interesting change in the gene expression profile of the cocultured A2058 cells that could play a role in an inflammatory environment. As seen in Table 1, gene expression of IL-11 was down-regulated. IL-11 is involved in the regulation of type I cytokine proinflammatory pathways and has been shown to decrease keratinocyte proliferation and cutaneous inflammation by attenuating the expression of a variety of critical genes associated with inflammation and disease. Therefore, it is reasonable that the down-regulation observed for IL-11 in cocultured A2058 cells could contribute to a proinflammatory state.

From the MAPPFinder and GenMAPP analyses of the gene expression data for pathways and biological process classes that are altered due to the coculture of melanoma cells and fibroblasts, it was seen that the inflammatory response pathway was identified as being up-regulated in the cocultured melanoma cells (Table 2). However, closer consideration of this suggests that this pathway may not be fully engaged. Of the 31 genes associated with the pathway, 28 were detected as "present," although only 2 of these genes were actually changed due to the coculture conditions. The Z score for this pathway was 1.9, and although significant, it is relatively low; hence, it is probable that the up-regulation of this pathway under these conditions is rather modest and does not represent a fully effective inflammatory response.

One interesting consideration whether the conditions for a complete inflammatory response could be developed under these conditions stem from the MAPPFinder and GenMAPP analyses of the gene expression data of cocultured HS-68 fibroblasts. From these data, the inflammatory response biological process class was identified as being up-regulated (Table 6) with a highly significant Z score, yet the inflammatory response pathway was identified as being down-regulated. We interpret these superficially conflicting data to be due to the fact that a sufficient variety of genes associated with proinflammation and inflammation are present and up-regulated and hence identified as an up-regulated biological process class. However, they are not sufficient in number or fold change to be considered as an up-regulated pathway. This, in conjunction with the down-regulation of IL-11 in the A2058 cells, leads us to conclude that it is likely that only a partial, incomplete proinflammatory/inflammatory response is being developed due to the coculture of the A2058 cells with the fibroblasts.

Effect of coculture on cell growth, proliferation, matrix degradation, and angiogenesis. There is a close relationship between cell growth, proliferation, matrix degradation, and angiogenesis and the proinflammatory pathway is considered to be central to these processes in melanoma (36). Although we did not assay for cellular growth and proliferation in these studies, it is likely that these events were occurring in the coculture system based on the results from the gene expression profiles. Epiregulin was observed to be up-regulated in the cocultured fibroblasts (Table 3). Epiregulin is a member of the epidermal growth factor family, and in addition to its abilities to stimulate cell growth, it has been implicated in the pathobiology of pancreatic ductal adenocarcinoma (37). The gene for immediate-early response was also seen to be up-regulated in the cocultured fibroblasts. This gene has been shown to function to protect cells from Fas ligand or TNF-{alpha}-induced apoptosis and its up-regulation in fibroblasts therefore may serve to protect these cells from apoptosis under the conditions of inflammation during tumor invasion (38).

Matrix degradation has been shown to play a crucial role during tumor invasion and to involve various classes of proteases, including MMPs. Genes of MMP-1 and MMP-3 were found to be up-regulated in fibroblasts cocultured with melanoma cells. Both proteases play a key role in the degradation of fibrillar collagens and have been immunolocalized at the host-tumor junction at a dermal invasion zone (39).

The primary driver of cell growth, proliferation, matrix degradation, and angiogenesis in invasive melanoma is likely IL-1ß (40). Stimulation of melanoma cells by IL-1ß causes the expression of the chemokine IL-8, which can result in a wide variety of biological responses, including proliferation of keratinocytes and melanoma cells, haptotatic migration of the melanoma cells, and induction of angiogenesis (41–44). Interestingly, these activities seem to have some dependence on the environment of the tumor cells. IL-8 production in A375 melanoma cells is high when these cells are cocultured with human keratinocytes that produce IL-1, whereas when A375 cells are cocultured with hepatocytes that do not produce IL-1 their IL-8 level of expression was decreased (45). These studies indicate the role of the tumor environment in terms of promoting processes associated with tumor progression and metastasis. In our studies of the coculture of A2058 human melanoma cells with HS-68 primary human fibroblasts in fibrillar collagen, no up-regulation of chemokine or cytokine expression was observed in the A2058 cells; however, in the cocultured fibroblasts, IL-1ß, IL-8, GROß, GRO{alpha}, and CCL2 (monocyte chemoattractant protein-1) were observed to be up-regulated (Table 3). Overexpression of CXCL1-3 by stimulated melanoma cells has been shown to function in an autocrine fashion to promote melanoma cell growth and proliferation (46, 47). Based on our data, it is quite possible that host stroma, in this case represented by melanoma-stimulated fibroblasts, can also express these chemokines to promote melanoma growth and proliferation.

As noted above, the chemokine CCL2 was also up-regulated in fibroblasts in coculture with melanoma cells. This chemokine has been shown to be important in attracting macrophage infiltrates to tumors, a process that has been linked to tumor progression, invasion, and angiogenesis (48, 49). As observed in our experiments, the stimulation of cocultured fibroblasts by the A2058 cells promotes an environment rich in chemokines known to stimulate melanoma proliferation, synthesis of proteolytic enzymes, tumor cell invasion, and angiogenesis associated with macrophage invasion, underscoring the potential for a central role of host stroma in melanoma metastasis.

Taken together, there appears from our gene expression data to be an activation of a pathway(s) initiated with IL-1ß. IL-1 has been shown to activate nuclear factor-kB, which in turn can up-regulate the expression of MMPs, urokinase-type plasminogen activator, CXCL1-3, chemokines, and TNF-{alpha} (49). TNF-{alpha} has also been shown to up-regulate IL-8 and CCL2 (50). In the cocultured fibroblasts, we observed the up-regulation of IL-1ß, nuclear factor-kB, MMP-1, MMP-3, tissue plasminogen activator, CXCL1, and CXCL2. Furthermore, we also saw IL-8 and CCL2 up-regulated, but not TNF-{alpha}. Nevertheless, it could be that TNF-{alpha} protein levels, but not gene expression, was increased in the coculture system because the gene for TNF-{alpha}-induced protein 6 was up-regulated, suggesting an effect of TNF-{alpha} on the system.

Relevance of gene expression data to invasive human melanoma. Over the years, many experimental and clinical efforts have aimed to improve the criteria for melanoma diagnosis and treatment. To improve the knowledge in molecular pathology of melanoma, particularly in discriminating between very early melanoma lesions and benign nevi, cDNA/oligonucleotide array technology is increasingly being used to identify new biological markers for malignancy or for invasive potential. However, analysis of tumor tissue alone may not identify small changes in gene expression occurring in the microenvironment of tumor-stroma. Identification of regulated genes resulting from cross-talk and activation due to coculture of melanoma cells and fibroblasts from our studies have provided insight into the molecular mechanisms involved in tumor-stroma interactions.

Our studies have shown that coculture of melanoma cells and fibroblasts has a profound reciprocal effect on gene expression profiles, with dramatic alterations in fibroblasts compared with a more modest effect on the melanoma cells. The fibroblasts were found to be more easily activated and responded to coculture by up-regulation of a large number of genes associated with the proinflammatory pathway, matrix proteolysis, and cellular proliferation. Interestingly, cytokines and chemokines (e.g., IL-1{alpha}, CXCL1/GRO{alpha}, and CXCL8/IL-8) that were thought to be produced by inflammatory cells or by melanoma cells were identified to be up-regulated in fibroblasts, suggesting that these cells are likely to also contribute to the inflammatory reaction.

In this study, we extended the coculture experiments of A2058 cells, a modestly invasive human melanoma cell line with HS-68 fibroblasts to other human melanoma cell lines. The human melanoma BLM cell line has been shown to be highly metastatic with early and frequent formation of metastasis in nude mice after s.c. inoculation (25). SK-Mel-28 and WM-164 melanoma cells are a low invasive cell lines (26) and WM-164 melanoma cells show no organ metastasis unless injected i.v. in mice (29). Interestingly, we observed that the fibroblasts grown in coculture with the highly metastatic BLM melanoma cell lines showed the significantly greater fold changes in the six relevant gene transcripts compared with the low invasive A2058, SK-Mel-28, and WM-164 melanoma cell lines. It is interesting to speculate that melanoma with a greater invasive potential may elicit a greater "proinvasion" effect on the gene expression profiles of stromal cells.

Therefore, we conclude that stroma, due to signals originating from the tumor, by responding to the presence of transformed cells is primarily responsible for the generation of a microenvironment that is proinflammatory, proproliferation, proinvasion, and prometastatic. Studies directed at disrupting the cross-talk between host and tumor may define new strategies for therapeutic intervention.


   Acknowledgments
 
Grant support: Polifarma S.p.A (J.W. Fox), University of Virginia Cancer Center (J.W. Fox), Alexander von Humboldt Stiftung (J.W. Fox), Wilhelm Sander Foundation grant 1999.093.2 (R. Nischt and C. Mauch), Koeln Fortune Program of the Faculty of Medicine grant 10/2004 (P. Zigrino), Deutsche Forschungsgemeinschaft through the SFB 587 at the University of Cologne (C. Mauch), and German Research Foundation grant Ni 304/9-1 (R. Nischt).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 2/ 6/04. Revised 1/ 6/05. Accepted 3/ 1/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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