Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome

The Effect of Extracellular Biophysical Cues on Cellular Signal Processing

Cell behavior is regulated by both internal and external signals. In early biological research, studies into cell signaling focused on biochemical cues. However, in recent decades, the scientific community has come to recognize the importance of biophysical cues in determining cell fate. The extracellular matrix (ECM), in particular, has emerged as a major regulator of cell behavior and is a source of both chemical and physical cues. Extracellular matrix makeup, organization, and presentation have been implicated in influencing cell growth, migration, differentiation, and more. My doctoral work focused on dissecting the molecular mechanisms underlying biophysical regulation of cell signaling. Specifically, I examined the effect of two biophysical properties of the extracellular matrix—dimensionality and rigidity—on mammary epithelial cell growth and survival signaling. My thesis details work connecting altered ECM properties to changes in the biophysical properties of the cell, which directly influence the context and dynamics of canonical growth factor receptor and GTPase signaling to reprogram cell behavior

Deconstructing signaling in three dimensions.

Cells in vivo exist within the context of a multicellular tissue, where their behavior is governed by homo- and heterotypic cell-cell interactions, the material properties of the extracellular matrix, and the distribution of various soluble and physical factors. Most methods currently used to study and manipulate cellular behavior in vitro, however, sacrifice physiological relevance for experimental expediency. The fallacy of such approaches has been highlighted by the recent development and application of three-dimensional culture models to cell biology, which has revealed striking phenotypic differences in cell survival, migration, and differentiation in genetically identical cells simply by varying culture conditions. These perplexing findings beg the question of what constitutes a three-dimensional culture and why cells behave so differently in two- and three-dimensional culture formats. In the following review, we dissect the fundamental differences between two- and three-dimensional culture conditions. We begin by establishing a basic definition of what "three dimensions" means at different biological scales and discuss how dimensionality influences cell signaling across different length scales. We identify which three-dimensional features most potently influence intracellular signaling and distinguish between conserved biological principles that are maintained across culture conditions and cellular behaviors that are sensitive to microenvironmental context. Finally, we highlight state-of-the-art molecular tools amenable to the study of signaling in three dimensions under conditions that facilitate deconstruction of signaling in a more physiologically relevant manner

A 3D tension bioreactor platform to study the interplay between ECM stiffness and tumor phenotype

Extracellular matrix (ECM) structure, composition, and stiffness have profound effects on tissue development and pathologies such as cardiovascular disease and cancer. Accordingly, a variety of synthetic hydrogel systems have been designed to study the impact of ECM composition, density, mechanics, and topography on cell and tissue phenotype. However, these synthetic systems fail to accurately recapitulate the biological properties and structure of the native tissue ECM. Natural three dimensional (3D) ECM hydrogels, such as collagen or hyaluronic acid, feature many of the chemical and physical properties of tissue, yet, these systems have limitations including the inability to independently control biophysical properties such as stiffness and pore size. Here, we present a 3D tension bioreactor system that permits precise mechanical tuning of collagen hydrogel stiffness, while maintaining consistent composition and pore size. We achieve this by mechanically loading collagen hydrogels covalently-conjugated to a polydimethylsiloxane (PDMS) membrane to induce hydrogel stiffening. We validated the biological application of this system with oncogenically transformed mammary epithelial cell organoids embedded in a 3D collagen I hydrogel, either uniformly stiffened or calibrated to create a gradient of ECM stiffening, to visually demonstrate the impact of ECM stiffening on transformation and tumor cell invasion. As such, this bioreactor presents the first tunable 3D natural hydrogel system that is capable of independently assessing the role of ECM stiffness on tissue phenotype

Visualizing mechanical modulation of nanoscale organization of cell-matrix adhesions

The mechanical properties of the extracellular matrix influence cell signaling to regulate key cellular processes, including differentiation, apoptosis, and transformation. Understanding the molecular mechanisms underlying mechanotransduction is contingent upon our ability to visualize the effect of altered matrix properties on the nanoscale organization of proteins involved in this signalling. The development of super-resolution imaging techniques has afforded researchers unprecedented ability to probe the organization and localization of proteins within the cell. However, most of these methods require use of substrates like glass or silicon wafers, which are artificially rigid. In light of a growing body of literature demonstrating the importance of mechanical properties of the extracellular matrix in regulating many aspects of cellular behavior and signaling, we have developed a system that allows scanning angle interference microscopy on a mechanically tunable substrate. We describe its implementation in detail and provide examples of how it may be used to aide investigations into the effect of substrate rigidity on intracellular signaling

Autolysis in Crustacean Tissues after Death: A Case Study Using the Procambarus clarkii Hepatopancreas

Autolysis is an internal phenomenon following the death of an organism that leads to the degradation of tissues. In order to explore the initial stages of autolysis and attempt to establish reference standards for tissue changes after death, we studied the rapidly autolyzing tissue of the crayfish hepatopancreas. Samples from the hepatopancreas of crayfish were examined 0, 5, 10, 30, 60, and 120 minutes after death. Histological and ultrapathological examinations and evaluations and apoptotic cell counts were conducted to determine the initiation time and degree of autolysis. The results showed that autolysis in the hepatopancreas of crayfish began within 5 minutes. Initially, autolysis manifested in the swelling of hepatic tubular cells and the widening of mesenchyme. Cells undergoing autolysis showed severe organelle necrolysis. Based on these observations, tissue samples should be collected and preserved within five minutes to avoid interfering with histopathological diagnoses