Implementation of a basement membrane invasion assay using mesenteric tissue.

Metastasis accounts for nearly 90% of all cancer associated mortalities. A hallmark of metastasis in malignancies of epithelial origin such as in the pancreas and breast, is invasion of the basement membrane (BM). While various in vitro assays have been developed to address questions regarding the invasiveness of tumors with relation to the BM, most fail to recapitulate a physiologically accurate cell-membrane interface. Here, we introduce a new 3D in vitro assay that uses the mouse mesenteric tissue as a mimic for the epithelial BM. We describe a simple, cost-effective protocol for extraction and setup of the assay, and show that the mesentery is a physiologically accurate model of the BM in its key components-type IV collagen, laminin-1 and perlecan. Furthermore, we introduce a user-friendly quantification tool, Q-Pi, which allows the 3D reconstruction, visualization and quantification of invasion at a cellular level. Overall, we demonstrate that this invasion assay provides a physiologically accurate tool to investigate BM invasion.

Syndecan-4 tunes cell mechanics by activating the kindlin-integrin-RhoA pathway.

Extensive research over the past decades has identified integrins to be the primary transmembrane receptors that enable cells to respond to external mechanical cues. We reveal here a mechanism whereby syndecan-4 tunes cell mechanics in response to localized tension via a coordinated mechanochemical signalling response that involves activation of two other receptors: epidermal growth factor receptor and ß1 integrin. Tension on syndecan-4 induces cell-wide activation of the kindlin-2/ß1 integrin/RhoA axis in a PI3K-dependent manner. Furthermore, syndecan-4-mediated tension at the cell-extracellular matrix interface is required for yes-associated protein activation. Extracellular tension on syndecan-4 triggers a conformational change in the cytoplasmic domain, the variable region of which is indispensable for the mechanical adaptation to force, facilitating the assembly of a syndecan-4/α-actinin/F-actin molecular scaffold at the bead adhesion. This mechanotransduction pathway for syndecan-4 should have immediate implications for the broader field of mechanobiology.

Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells.

Extracellular matrix (ECM) remodelling is integral to numerous physiological and pathological processes in biology, such as embryogenesis, wound healing, fibrosis and cancer. Until recently, most cellular studies have been conducted on 2D environments where mechanical cues significantly differ from physiologically relevant 3D environments, impacting cellular behaviour and masking the interpretation of cellular function in health and disease. We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling. Monitoring and quantification of collagen-I structure in remodelled matrices, through designated algorithms, show that 3D matrices can be used to correlate remodelling with increased ECM stiffness observed in fibrosis. Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer. We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and ß1 integrin-mediated cell-ECM attachment.