Integrated Biophysical Characterization of Fibrillar Collagen-Based Hydrogels.

This paper describes an experimental characterization scheme of the biophysical properties of reconstituted hydrogel matrices based on indentation testing, quantification of transport via microfluidics, and confocal reflectance microscopy analysis. While methods for characterizing hydrogels exist and are widely used, they often do not measure diffusive and convective transport concurrently, determine the relationship between microstructure and transport properties, and decouple matrix mechanics and transport properties. Our integrated approach enabled independent and quantitative measurements of the structural, mechanical, and transport properties of hydrogels in a single study. We used fibrillar type I collagen as the base matrix and investigated the effects of two different matrix modifications: (1) cross-linking with human recombinant tissue transglutaminase II (hrTGII) and (2) supplementation with the nonfibrillar matrix constituent hyaluronic acid (HA). hrTGII modified the matrix structure and transport but not mechanical parameters. Furthermore, changes in the matrix structure due to hrTGII were seen to be dependent on the concentration of collagen. In contrast, supplementation of HA at different collagen concentrations altered the matrix microstructure and mechanical indentation behavior but not transport parameters. These experimental observations reveal the important relationship between extracellular matrix (ECM) composition and biophysical properties. The integrated techniques are versatile, robust, and accessible; and as matrix-cell interactions are instrumental for many biological processes, the methods and findings described here should be broadly applicable for characterizing hydrogel materials used for three-dimensional (3-D) tissue-engineered culture models.

Disruption of stromal hedgehog signaling initiates RNF5-mediated proteasomal degradation of PTEN and accelerates pancreatic tumor growth.

The contribution of the tumor microenvironment to pancreatic ductal adenocarcinoma (PDAC) development is currently unclear. We therefore examined the consequences of disrupting paracrine Hedgehog (HH) signaling in PDAC stroma. Herein, we show that ablation of the key HH signaling gene Smoothened (Smo) in stromal fibroblasts led to increased proliferation of pancreatic tumor cells. Furthermore, Smo deletion resulted in proteasomal degradation of the tumor suppressor PTEN and activation of oncogenic protein kinase B (AKT) in fibroblasts. An unbiased proteomic screen identified RNF5 as a novel E3 ubiquitin ligase responsible for degradation of phosphatase and tensin homolog (PTEN) in Smo-null fibroblasts. Ring Finger Protein 5 (Rnf5) knockdown or pharmacological inhibition of glycogen synthase kinase 3ß (GSKß), the kinase that marks PTEN for ubiquitination, rescued PTEN levels and reversed the oncogenic phenotype, identifying a new node of PTEN regulation. In PDAC patients, low stromal PTEN correlated with reduced overall survival. Mechanistically, PTEN loss decreased hydraulic permeability of the extracellular matrix, which was reversed by hyaluronidase treatment. These results define non-cell autonomous tumor-promoting mechanisms activated by disruption of the HH/PTEN axis and identifies new targets for restoring stromal tumor-suppressive functions.

Stromal PDGFR-α Activation Enhances Matrix Stiffness, Impedes Mammary Ductal Development, and Accelerates Tumor Growth.

The extracellular matrix (ECM) is critical for mammary ductal development and differentiation, but how mammary fibroblasts regulate ECM remodeling remains to be elucidated. Herein, we used a mouse genetic model to activate platelet derived growth factor receptor-alpha (PDGFRα) specifically in the stroma. Hyperactivation of PDGFRα in the mammary stroma severely hindered pubertal mammary ductal morphogenesis, but did not interrupt the lobuloalveolar differentiation program. Increased stromal PDGFRα signaling induced mammary fat pad fibrosis with a corresponding increase in interstitial hyaluronic acid (HA) and collagen deposition. Mammary fibroblasts with PDGFRα hyperactivation also decreased hydraulic permeability of a collagen substrate in an in vitro microfluidic device assay, which was mitigated by inhibition of either PDGFRα or HA. Fibrosis seen in this model significantly increased the overall stiffness of the mammary gland as measured by atomic force microscopy. Further, mammary tumor cells injected orthotopically in the fat pads of mice with stromal activation of PDGFRα grew larger tumors compared to controls. Taken together, our data establish that aberrant stromal PDGFRα signaling disrupts ECM homeostasis during mammary gland development, resulting in increased mammary stiffness and increased potential for tumor growth.