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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.
Highlights
In the expression profile of the invading fibroblast, the interstitial collagenase matrix metalloprotease 13 (MMP13) and TGFβ 1 were found to be significantly up-regulated while Cav[1], Phosphatase and Tensin Homolog (Pten), and secreted frizzled-related protein (Sfrp)[1] were down-regulated on mRNA level
In idiopathic pulmonary fibrosis (IPF), down-regulation of Pten in fibroblastic foci was found to be accompanied by decreased Caveolin 1 (Cav1) levels
As the mere expression level of upstream regulators such as TGFβ 1 was found to be significantly higher in invading fibroblasts, we aimed to validate the functional relevance of the generated invasion signature by using a systematic approach (Upstream Regulator Analysis (URA), Ingenuity)
Summary
Repair, and fibrosis, a plethora of soluble mediators are released by different cell types, which activate and attract resident fibroblasts and/or induce transformation of epithelial or pleural mesothelial cells (PMC) into mesenchymal-like cells[10,11,12,13] Such aberrantly activated mesenchymal cells exhibit an exceptional plasticity with respect to proliferation, apoptosis, ECM deposition, activation, and tissue invasion[11]. We hypothesized that pathway analysis of the transcriptome-wide signature of invading lung fibroblasts by comparative microarray analyses, using a collagen-based invasion assay previously established in our lab[19], will identify pro-invasive growth factors with relevance to disease To this end, we generated a specific fibroblast invasion signature, followed by functional validation of in silico-predicted upstream regulators of fibroblast invasion; namely transforming growth factor (TGF)β 1, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), and platelet-derived growth factor (PDGF)-BB. This invasion signature advances the understanding of an activated invasive fibroblast phenotype in particular for fibrotic and metastatic pathologies
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