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Validated prediction of pro-invasive growth factors using a transcriptome-wide invasion signature derived from a complex 3D invasion assay.

Oehrle B, Burgstaller G, Irmler M, Dehmel S, Grün J, Hwang T, Krauss-Etschmann S, Beckers J, Meiners S, Eickelberg O - Sci Rep (2015)

Bottom Line: Unbiased pathway analysis (Ingenuity) identified significant enrichment for the functional clusters 'invasion of cells', 'idiopathic pulmonary fibrosis', and 'metastasis'.Matrix metalloprotease 13 (MMP13), transforming growth factor (TGF)-β1, Caveolin (Cav) 1, Phosphatase and Tensin Homolog (Pten), and secreted frizzled-related protein (Sfrp) 1 were among the highest regulated genes, confirmed by qRT-PCR and Western Blotting.We next performed in silico analysis (Ingenuity Pathway Analysis) to predict mediators that induced fibroblast invasion.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Pneumology Center, University Hospital of the Ludwig-Maximilians-University Munich and Helmholtz Zentrum München, Member of the German Center for Lung Research, 81377 Munich, Germany.

ABSTRACT
The invasion of activated fibroblasts represents a key pathomechanism in fibrotic diseases, carcinogenesis and metastasis. Invading fibroblasts contribute to fibrotic extracellular matrix (ECM) formation and the initiation, progression, or resistance of cancer. To construct transcriptome-wide signatures of fibroblast invasion, we used a multiplex phenotypic 3D invasion assay using lung fibroblasts. Microarray-based gene expression profiles of invading and non-invading fibroblasts demonstrated that 1,049 genes were differentially regulated (>1.5-fold). Unbiased pathway analysis (Ingenuity) identified significant enrichment for the functional clusters 'invasion of cells', 'idiopathic pulmonary fibrosis', and 'metastasis'. Matrix metalloprotease 13 (MMP13), transforming growth factor (TGF)-β1, Caveolin (Cav) 1, Phosphatase and Tensin Homolog (Pten), and secreted frizzled-related protein (Sfrp) 1 were among the highest regulated genes, confirmed by qRT-PCR and Western Blotting. We next performed in silico analysis (Ingenuity Pathway Analysis) to predict mediators that induced fibroblast invasion. Of these, TGFβ1, epidermal growth factor (EGF), fibroblast growth factor (FGF) 2, and platelet-derived growth factor (PDGF)-BB were tested in our 3D invasion assay and found to significantly induce invasion, thus validating the transcriptome profile. Accordingly, our transcriptomic invasion signature describes the invading fibroblast phenotype in unprecedented detail and provides a tool for future functional studies of cell invasion and therapeutic modulation thereof using complex phenotypic assays.

No MeSH data available.


Related in: MedlinePlus

Analysis of differentially regulated targets of invading vs non-invading fibroblasts and hierarchical clustering.After 72 hours of invasion, 1,086 targets with expression ratios greater than 1.5-fold and 163 targets regulated greater than 2-fold were identified in the invading fraction. After 96 hours, an altered regulation of 1,049 probes with expression ratios greater than 1.5-fold and 182 greater than 2-fold were identified. The FDR for all of these targets was lower than 10%. The proportion of genes with expression ratios of 1 <> 1.5 between invading (inv.) and non-invading (non-inv.) phenotypes are depicted in light grey. The amount of targets with an expression ratio of >1.5 and >2 is represented in dark grey and black, respectively (a). Hierarchical clustering distinguished two main clusters: invading (inv.) and non-invading (non-inv.) and two sub-clusters: 72 and 96 hours. The cluster dendrogram was conducted with R using the script hclust, on RMA data filtered for expression values >100 in at least one sample (b).
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f1: Analysis of differentially regulated targets of invading vs non-invading fibroblasts and hierarchical clustering.After 72 hours of invasion, 1,086 targets with expression ratios greater than 1.5-fold and 163 targets regulated greater than 2-fold were identified in the invading fraction. After 96 hours, an altered regulation of 1,049 probes with expression ratios greater than 1.5-fold and 182 greater than 2-fold were identified. The FDR for all of these targets was lower than 10%. The proportion of genes with expression ratios of 1 <> 1.5 between invading (inv.) and non-invading (non-inv.) phenotypes are depicted in light grey. The amount of targets with an expression ratio of >1.5 and >2 is represented in dark grey and black, respectively (a). Hierarchical clustering distinguished two main clusters: invading (inv.) and non-invading (non-inv.) and two sub-clusters: 72 and 96 hours. The cluster dendrogram was conducted with R using the script hclust, on RMA data filtered for expression values >100 in at least one sample (b).

Mentions: In order to systematically identify the molecular signature of invading fibroblasts we applied a whole transcriptome analysis (Affymetrix Mouse Gene 1.0 ST array), using our recently established 3D invasion assay19. A subpopulation of murine MLg 2908 (MLg) lung fibroblasts spontaneously invaded a 3D collagen matrix when plated on top of it19. Separation of invading from non-invading MLg fibroblasts was carried out 72 or 96 hours after plating. RNA isolation and subsequent whole transcriptome analysis provided gene expression profiles of invading and non-invading fibroblasts. Transcripts with a false discovery rate <10% were considered as being statistically significant regulated and were used for subsequent analyses. After 72 hours of invasion, 1,086 targets with expression ratios greater than 1.5-fold and 163 targets regulated greater than 2-fold were identified in the invading fraction as compared to the non-invading fraction. In fibroblasts that invaded the matrix for 96 hours, an altered regulation of 1,049 probes with expression ratios greater than 1.5-fold and 182 greater than 2-fold were identified (Fig. 1A). Hierarchical clustering of the different fractions is depicted in Fig. 1B. Heatmaps of up- and down-regulated target genes (>2-fold) in the invading fractions at 72 hours and 96 hours validate the reproducibility of our replicate analyses (Fig. 2A and Fig. 2B). The whole array data have been submitted to GEO (GSE55322).


Validated prediction of pro-invasive growth factors using a transcriptome-wide invasion signature derived from a complex 3D invasion assay.

Oehrle B, Burgstaller G, Irmler M, Dehmel S, Grün J, Hwang T, Krauss-Etschmann S, Beckers J, Meiners S, Eickelberg O - Sci Rep (2015)

Analysis of differentially regulated targets of invading vs non-invading fibroblasts and hierarchical clustering.After 72 hours of invasion, 1,086 targets with expression ratios greater than 1.5-fold and 163 targets regulated greater than 2-fold were identified in the invading fraction. After 96 hours, an altered regulation of 1,049 probes with expression ratios greater than 1.5-fold and 182 greater than 2-fold were identified. The FDR for all of these targets was lower than 10%. The proportion of genes with expression ratios of 1 <> 1.5 between invading (inv.) and non-invading (non-inv.) phenotypes are depicted in light grey. The amount of targets with an expression ratio of >1.5 and >2 is represented in dark grey and black, respectively (a). Hierarchical clustering distinguished two main clusters: invading (inv.) and non-invading (non-inv.) and two sub-clusters: 72 and 96 hours. The cluster dendrogram was conducted with R using the script hclust, on RMA data filtered for expression values >100 in at least one sample (b).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4525140&req=5

f1: Analysis of differentially regulated targets of invading vs non-invading fibroblasts and hierarchical clustering.After 72 hours of invasion, 1,086 targets with expression ratios greater than 1.5-fold and 163 targets regulated greater than 2-fold were identified in the invading fraction. After 96 hours, an altered regulation of 1,049 probes with expression ratios greater than 1.5-fold and 182 greater than 2-fold were identified. The FDR for all of these targets was lower than 10%. The proportion of genes with expression ratios of 1 <> 1.5 between invading (inv.) and non-invading (non-inv.) phenotypes are depicted in light grey. The amount of targets with an expression ratio of >1.5 and >2 is represented in dark grey and black, respectively (a). Hierarchical clustering distinguished two main clusters: invading (inv.) and non-invading (non-inv.) and two sub-clusters: 72 and 96 hours. The cluster dendrogram was conducted with R using the script hclust, on RMA data filtered for expression values >100 in at least one sample (b).
Mentions: In order to systematically identify the molecular signature of invading fibroblasts we applied a whole transcriptome analysis (Affymetrix Mouse Gene 1.0 ST array), using our recently established 3D invasion assay19. A subpopulation of murine MLg 2908 (MLg) lung fibroblasts spontaneously invaded a 3D collagen matrix when plated on top of it19. Separation of invading from non-invading MLg fibroblasts was carried out 72 or 96 hours after plating. RNA isolation and subsequent whole transcriptome analysis provided gene expression profiles of invading and non-invading fibroblasts. Transcripts with a false discovery rate <10% were considered as being statistically significant regulated and were used for subsequent analyses. After 72 hours of invasion, 1,086 targets with expression ratios greater than 1.5-fold and 163 targets regulated greater than 2-fold were identified in the invading fraction as compared to the non-invading fraction. In fibroblasts that invaded the matrix for 96 hours, an altered regulation of 1,049 probes with expression ratios greater than 1.5-fold and 182 greater than 2-fold were identified (Fig. 1A). Hierarchical clustering of the different fractions is depicted in Fig. 1B. Heatmaps of up- and down-regulated target genes (>2-fold) in the invading fractions at 72 hours and 96 hours validate the reproducibility of our replicate analyses (Fig. 2A and Fig. 2B). The whole array data have been submitted to GEO (GSE55322).

Bottom Line: Unbiased pathway analysis (Ingenuity) identified significant enrichment for the functional clusters 'invasion of cells', 'idiopathic pulmonary fibrosis', and 'metastasis'.Matrix metalloprotease 13 (MMP13), transforming growth factor (TGF)-β1, Caveolin (Cav) 1, Phosphatase and Tensin Homolog (Pten), and secreted frizzled-related protein (Sfrp) 1 were among the highest regulated genes, confirmed by qRT-PCR and Western Blotting.We next performed in silico analysis (Ingenuity Pathway Analysis) to predict mediators that induced fibroblast invasion.

View Article: PubMed Central - PubMed

Affiliation: Comprehensive Pneumology Center, University Hospital of the Ludwig-Maximilians-University Munich and Helmholtz Zentrum München, Member of the German Center for Lung Research, 81377 Munich, Germany.

ABSTRACT
The invasion of activated fibroblasts represents a key pathomechanism in fibrotic diseases, carcinogenesis and metastasis. Invading fibroblasts contribute to fibrotic extracellular matrix (ECM) formation and the initiation, progression, or resistance of cancer. To construct transcriptome-wide signatures of fibroblast invasion, we used a multiplex phenotypic 3D invasion assay using lung fibroblasts. Microarray-based gene expression profiles of invading and non-invading fibroblasts demonstrated that 1,049 genes were differentially regulated (>1.5-fold). Unbiased pathway analysis (Ingenuity) identified significant enrichment for the functional clusters 'invasion of cells', 'idiopathic pulmonary fibrosis', and 'metastasis'. Matrix metalloprotease 13 (MMP13), transforming growth factor (TGF)-β1, Caveolin (Cav) 1, Phosphatase and Tensin Homolog (Pten), and secreted frizzled-related protein (Sfrp) 1 were among the highest regulated genes, confirmed by qRT-PCR and Western Blotting. We next performed in silico analysis (Ingenuity Pathway Analysis) to predict mediators that induced fibroblast invasion. Of these, TGFβ1, epidermal growth factor (EGF), fibroblast growth factor (FGF) 2, and platelet-derived growth factor (PDGF)-BB were tested in our 3D invasion assay and found to significantly induce invasion, thus validating the transcriptome profile. Accordingly, our transcriptomic invasion signature describes the invading fibroblast phenotype in unprecedented detail and provides a tool for future functional studies of cell invasion and therapeutic modulation thereof using complex phenotypic assays.

No MeSH data available.


Related in: MedlinePlus