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

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.

3D pulmospheres serve as a personalized and predictive multicellular model for assessment of antifibrotic drugs

“…Other investigators have used lung cells to develop 3D cultures of invading fibroblasts or stem cells obtained from lung tissue from human or mice lung (22,39). Henry et al used lung tissue pieces in culture to recoup "outgrowth cells" to form lung pulmospheres, which were found to be enriched for stem cells and protected from bleomycin lung injury in mice (22).…”

“…Henry et al used lung tissue pieces in culture to recoup "outgrowth cells" to form lung pulmospheres, which were found to be enriched for stem cells and protected from bleomycin lung injury in mice (22). A group from Germany performed a pathway analysis on the invading fibroblasts, which has provided important information on the signaling pathways, which mediate the process of invasion in lung fibroblasts (39). A key difference in the pulmospheres obtained in our studies is that they were grown immediately following resection of lung tissue and were developed within 24 hours from lung cell suspensions and adherent cells following the initial passage through filter and removal of red blood cells.…”

Mechanosensing by the α6-integrin confers an invasive fibroblast phenotype and mediates lung fibrosis

“…Fibrotic lung myofibroblasts isolated from patients with IPF are characterized by an invasive phenotype 20 21 22 23 24 . To determine whether the mechanical properties of the ECM may regulate the ability of IPF lung myofibroblasts to invade the BM, we pre-cultured primary lung myofibroblasts isolated from patients with IPF on soft (1 kPa) and stiff (20 kPa) PA hydrogel substrates.…”

Therapeutic and pathological roles of fibroblast growth factors in pulmonary diseases

“…FGF2 also binds to fibrinogen that enhances its proliferative effect on cancer cells (Sahni et al, ). A recent study has identified FGF2 as part of a transcriptome‐wide invasion signature in lung fibroblasts (Oehrle et al, ).…”