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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

Expression levels of selected genes from the microarray analyses and validation by qRT-PCR and immunoblotting.Volcano plots depict the significantly differentially regulated probesets in the invading fraction at 72 (a) and 96 hours (b) time-points. Targets, used for microarray verification by qRT-PCR are highlighted in red. Here, the expression ratios derived from the microarray data are shown for a selected group of genes reportedly known to be functionally involved in the invasion of cells. The graph depicts the up-regulated targets in the invading fraction that is MMP13 (9.5x) and TGFβ1 (3.3x), as well as the down-regulated targets Cav1 (0.8x), Pten (0.3x) and Sfrp1 (0.5x) (n = 5). Data are shown with Benjamini-Hochberg (BH)-adjusted p-values. Genewise testing for differential expression employed the limma t-test and Benjamini-Hochberg multiple testing correction (FDR < 10%) (c). qRT-PCR analyses confirming the differential expression of a selected group of genes (MMP13, TGFβ1, Cav1, and Pten) with the differential expression data derived from the microarrays. The data represent the fold induction values of the ratio between invading and non-invading fibroblasts. Data are shown as mean values from four independent experiments (n = 4). Statistical analysis: paired t-test. *p < 0.05 and ***p < 0.001 (d). Representative immunoblots of TGFβ1, Cav1, Sfrp1 and Pten in invading (inv.) and non-invading (non-inv.) mouse lung fibroblasts on protein level. Immunoblots were cropped to improve clarity. Uncut blots are depicted in supplementary Fig. S2(e) in invading (inv.) and non-invading (non-inv.) mouse lung fibroblasts. qRT-PCR analyses in inv. primary human fibroblasts (phF) reveals a similar deregulation of MMP13, TGFβ1, Cav1, Sfrp1 and Pten as found in the MLg fibroblasts (f). Data shown represent mean values from four independent experiments (n = 4). Statistical analysis: paired t-test. *p < 0.05 and **p < 0.01.
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f4: Expression levels of selected genes from the microarray analyses and validation by qRT-PCR and immunoblotting.Volcano plots depict the significantly differentially regulated probesets in the invading fraction at 72 (a) and 96 hours (b) time-points. Targets, used for microarray verification by qRT-PCR are highlighted in red. Here, the expression ratios derived from the microarray data are shown for a selected group of genes reportedly known to be functionally involved in the invasion of cells. The graph depicts the up-regulated targets in the invading fraction that is MMP13 (9.5x) and TGFβ1 (3.3x), as well as the down-regulated targets Cav1 (0.8x), Pten (0.3x) and Sfrp1 (0.5x) (n = 5). Data are shown with Benjamini-Hochberg (BH)-adjusted p-values. Genewise testing for differential expression employed the limma t-test and Benjamini-Hochberg multiple testing correction (FDR < 10%) (c). qRT-PCR analyses confirming the differential expression of a selected group of genes (MMP13, TGFβ1, Cav1, and Pten) with the differential expression data derived from the microarrays. The data represent the fold induction values of the ratio between invading and non-invading fibroblasts. Data are shown as mean values from four independent experiments (n = 4). Statistical analysis: paired t-test. *p < 0.05 and ***p < 0.001 (d). Representative immunoblots of TGFβ1, Cav1, Sfrp1 and Pten in invading (inv.) and non-invading (non-inv.) mouse lung fibroblasts on protein level. Immunoblots were cropped to improve clarity. Uncut blots are depicted in supplementary Fig. S2(e) in invading (inv.) and non-invading (non-inv.) mouse lung fibroblasts. qRT-PCR analyses in inv. primary human fibroblasts (phF) reveals a similar deregulation of MMP13, TGFβ1, Cav1, Sfrp1 and Pten as found in the MLg fibroblasts (f). Data shown represent mean values from four independent experiments (n = 4). Statistical analysis: paired t-test. *p < 0.05 and **p < 0.01.

Mentions: The gene expression profiles of the invading fibroblasts at 72 hours and 96 hours (>1.5-fold) greatly overlapped: among the differentially regulated genes in the invading subtype at 72 and 96 hours, 621 genes overlapped in total: 166 in the up- and 455 in the down-regulated group (Fig. 3A). Of note, there were more than twice as many overlapping down-regulated genes than overlapping up-regulated genes. This comparative approach allowed us to enrich for those targets that are commonly regulated after 72 and 96 hours of invasion and to define the invasion signature of fibroblasts. Enrichment analyses using IPA’s ‘disease and function’ ontology revealed that ‘invasion of cells’, ‘idiopathic pulmonary fibrosis (IPF)’, and ‘metastasis’ ranked as the top three most significantly over-represented ‘disease processes’ and ‘biological functions’ within the invasion signature (Fig. 3B). In agreement with the well-known role of TGFβ1 in invasion and fibrosis, TGFβ1 associated with all three key networks of invasion (Fig. 3C). These data clearly corroborate our experimental approach. In order to further validate the profiling approach used, several known invasion-promoting genes were chosen for confirmative expression analysis by qRT-PCR. These targets included matrix metalloprotease 13 (MMP13)2021, TGFβ11022, Caveolin 1 (Cav1)23, secreted frizzled-related protein (Sfrp) 1 24, and Phosphatase and Tensin Homolog (Pten)2526 (Fig. 4A,B). Consistent with our microarray data (Fig. 4C), qRT-PCR analysis demonstrated a significant up-regulation of MMP13 and TGFβ1 in the invading fibroblasts by 6- and 1.8-fold, respectively (Fig. 4D). By contrast, Cav1, Sfrp1, and Pten were significantly down-regulated by 3.5-, 2.9-, and 2.6-fold, respectively (Fig. 3D). Reduced Sfrp1 and Cav1 protein expression was further corroborated by immunoblot analysis (Fig. 4E). Furthermore, TGFβ1 and MMP1319 were up-regulated on protein level as well (Fig. 4E). Interestingly, the protein expression of Pten, which was strongly diminished on transcript level, was found to be largely unchanged. Additionally, in primary human lung fibroblasts we identified a corresponding regulation of the same target genes in the invading subpopulation (Fig. 4F). The qRT-PCR analyses of selected target genes thus validated the robustness of our invasion assay and the identified gene signature for invading fibroblasts.


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)

Expression levels of selected genes from the microarray analyses and validation by qRT-PCR and immunoblotting.Volcano plots depict the significantly differentially regulated probesets in the invading fraction at 72 (a) and 96 hours (b) time-points. Targets, used for microarray verification by qRT-PCR are highlighted in red. Here, the expression ratios derived from the microarray data are shown for a selected group of genes reportedly known to be functionally involved in the invasion of cells. The graph depicts the up-regulated targets in the invading fraction that is MMP13 (9.5x) and TGFβ1 (3.3x), as well as the down-regulated targets Cav1 (0.8x), Pten (0.3x) and Sfrp1 (0.5x) (n = 5). Data are shown with Benjamini-Hochberg (BH)-adjusted p-values. Genewise testing for differential expression employed the limma t-test and Benjamini-Hochberg multiple testing correction (FDR < 10%) (c). qRT-PCR analyses confirming the differential expression of a selected group of genes (MMP13, TGFβ1, Cav1, and Pten) with the differential expression data derived from the microarrays. The data represent the fold induction values of the ratio between invading and non-invading fibroblasts. Data are shown as mean values from four independent experiments (n = 4). Statistical analysis: paired t-test. *p < 0.05 and ***p < 0.001 (d). Representative immunoblots of TGFβ1, Cav1, Sfrp1 and Pten in invading (inv.) and non-invading (non-inv.) mouse lung fibroblasts on protein level. Immunoblots were cropped to improve clarity. Uncut blots are depicted in supplementary Fig. S2(e) in invading (inv.) and non-invading (non-inv.) mouse lung fibroblasts. qRT-PCR analyses in inv. primary human fibroblasts (phF) reveals a similar deregulation of MMP13, TGFβ1, Cav1, Sfrp1 and Pten as found in the MLg fibroblasts (f). Data shown represent mean values from four independent experiments (n = 4). Statistical analysis: paired t-test. *p < 0.05 and **p < 0.01.
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f4: Expression levels of selected genes from the microarray analyses and validation by qRT-PCR and immunoblotting.Volcano plots depict the significantly differentially regulated probesets in the invading fraction at 72 (a) and 96 hours (b) time-points. Targets, used for microarray verification by qRT-PCR are highlighted in red. Here, the expression ratios derived from the microarray data are shown for a selected group of genes reportedly known to be functionally involved in the invasion of cells. The graph depicts the up-regulated targets in the invading fraction that is MMP13 (9.5x) and TGFβ1 (3.3x), as well as the down-regulated targets Cav1 (0.8x), Pten (0.3x) and Sfrp1 (0.5x) (n = 5). Data are shown with Benjamini-Hochberg (BH)-adjusted p-values. Genewise testing for differential expression employed the limma t-test and Benjamini-Hochberg multiple testing correction (FDR < 10%) (c). qRT-PCR analyses confirming the differential expression of a selected group of genes (MMP13, TGFβ1, Cav1, and Pten) with the differential expression data derived from the microarrays. The data represent the fold induction values of the ratio between invading and non-invading fibroblasts. Data are shown as mean values from four independent experiments (n = 4). Statistical analysis: paired t-test. *p < 0.05 and ***p < 0.001 (d). Representative immunoblots of TGFβ1, Cav1, Sfrp1 and Pten in invading (inv.) and non-invading (non-inv.) mouse lung fibroblasts on protein level. Immunoblots were cropped to improve clarity. Uncut blots are depicted in supplementary Fig. S2(e) in invading (inv.) and non-invading (non-inv.) mouse lung fibroblasts. qRT-PCR analyses in inv. primary human fibroblasts (phF) reveals a similar deregulation of MMP13, TGFβ1, Cav1, Sfrp1 and Pten as found in the MLg fibroblasts (f). Data shown represent mean values from four independent experiments (n = 4). Statistical analysis: paired t-test. *p < 0.05 and **p < 0.01.
Mentions: The gene expression profiles of the invading fibroblasts at 72 hours and 96 hours (>1.5-fold) greatly overlapped: among the differentially regulated genes in the invading subtype at 72 and 96 hours, 621 genes overlapped in total: 166 in the up- and 455 in the down-regulated group (Fig. 3A). Of note, there were more than twice as many overlapping down-regulated genes than overlapping up-regulated genes. This comparative approach allowed us to enrich for those targets that are commonly regulated after 72 and 96 hours of invasion and to define the invasion signature of fibroblasts. Enrichment analyses using IPA’s ‘disease and function’ ontology revealed that ‘invasion of cells’, ‘idiopathic pulmonary fibrosis (IPF)’, and ‘metastasis’ ranked as the top three most significantly over-represented ‘disease processes’ and ‘biological functions’ within the invasion signature (Fig. 3B). In agreement with the well-known role of TGFβ1 in invasion and fibrosis, TGFβ1 associated with all three key networks of invasion (Fig. 3C). These data clearly corroborate our experimental approach. In order to further validate the profiling approach used, several known invasion-promoting genes were chosen for confirmative expression analysis by qRT-PCR. These targets included matrix metalloprotease 13 (MMP13)2021, TGFβ11022, Caveolin 1 (Cav1)23, secreted frizzled-related protein (Sfrp) 1 24, and Phosphatase and Tensin Homolog (Pten)2526 (Fig. 4A,B). Consistent with our microarray data (Fig. 4C), qRT-PCR analysis demonstrated a significant up-regulation of MMP13 and TGFβ1 in the invading fibroblasts by 6- and 1.8-fold, respectively (Fig. 4D). By contrast, Cav1, Sfrp1, and Pten were significantly down-regulated by 3.5-, 2.9-, and 2.6-fold, respectively (Fig. 3D). Reduced Sfrp1 and Cav1 protein expression was further corroborated by immunoblot analysis (Fig. 4E). Furthermore, TGFβ1 and MMP1319 were up-regulated on protein level as well (Fig. 4E). Interestingly, the protein expression of Pten, which was strongly diminished on transcript level, was found to be largely unchanged. Additionally, in primary human lung fibroblasts we identified a corresponding regulation of the same target genes in the invading subpopulation (Fig. 4F). The qRT-PCR analyses of selected target genes thus validated the robustness of our invasion assay and the identified gene signature for invading fibroblasts.

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