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A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis

ABSTRACT: Extracellular matrix interactions play essential roles in normal physiology and many pathological processes. Here, we report a novel screening platform capable of measuring phenotypic responses to combinations of ECM molecules. While the importance of ECM interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Using a genetic mouse model of lung adenocarcinoma, we measured the ECM-dependent adhesion of tumor-derived cells. Hierarchical clustering of adhesion profiles generated using this platform differentially segregated metastatic cell lines from primary tumor lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8, or laminin. These interactions appear to be mediated in part by α3β1 integrin both in vitro and in vivo. We show that these galectins also correlate with human disease at both a transcriptional and histological level. Thus, our in vitro platform allowed us to interrogate the interactions of metastatic cells with their surrounding environment, and identified ECM and integrin interactions that could lead to therapeutic targets for metastasis prevention.

ORGANISM(S): Mus musculus

PROVIDER: GSE40222 | GEO | 2012/10/11

SECONDARY ACCESSION(S): PRJNA173238

REPOSITORIES: GEO

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Project description:Background: The differentiation of pericytes into myofibroblasts causes microvascular degeneration, extracellular matrix (ECM) accumulation, and tissue stiffening, characteristics of fibrotic diseases. It is unclear how pericyte-myofibroblast differentiation is regulated in the microvascular environment. Our previous study established a novel two-dimensional platform for coculturing microvascular endothelial cells (ECs) and pericytes derived from the same tissue. This study investigated how ECM stiffness regulated microvascular ECs, pericytes, and their interactions. Methods: Primary microvessels were cultured in the TGM2D medium. Stiff ECM was prepared by incubating ECM solution in regular culture dishes for one hour followed by PBS wash. Soft ECM with Young’s modulus of approximately 6 kPa was used unless otherwise noted. Bone grafts were prepared from the rat skull. Immunostaining, RNA sequencing, qRT-PCR, western blotting, and knockdown experiments were performed on the cells. Results: Primary microvascular pericytes differentiated into myofibroblasts (NG2+αSMA+) on stiff ECM, even with the TGFβ signaling inhibitor A83-01. Soft ECM and A83-01 cooperatively maintained microvascular stability while inhibiting pericyte-myofibroblast differentiation (NG2+αSMA-/low). We thus defined two pericyte subpopulations: primary (NG2+αSMA-/low) and activated (NG2+αSMA+) pericytes. Soft ECM promoted microvascular regeneration and inhibited fibrosis in bone graft transplantation in vivo. As Integrins are the major mechanosensor, we performed qRT-PCR screening of Integrin family members selected from RNA sequencing data. We found that Integrin β1 (Itgb1) was the major subunit downregulated by soft ECM and A83-01 treatment. Knocking down Itgb1 suppressed myofibroblast differentiation on stiff ECM. Interestingly, ITGB1 phosphorylation (Y783) was mainly located on microvascular ECs on stiff ECM, which promoted EC secretion of paracrine factors, including CTGF, to induce pericyte-myofibroblast differentiation. CTGF knockdown or monoclonal antibody treatment partially reduced myofibroblast differentiation, implying the participation of multiple pathways in fibrosis formation. Conclusions: Microvascular ECs mediate ECM stiffness-induced pericyte-myofibroblast differentiation through paracrine signaling.

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A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis

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