Cells in vivo
are constantly exposed to an array of biophysical forces such as hydrostatic pressure, shear stress, compression loading, and tensional forces. Cells rely on these physical cues to maintain homeostasis and adapt to them by altering cell signaling and gene expression and by remodeling their local microenvironment [1
]. From an organismal point of view, ECM compliance directs the development of tissues [1
] and influences the onset of many pathological conditions, including cardiovascular disease [4
], arthritis [5
] , and neural degenerative diseases [6
]. The ECM also progressively stiffens in tumors and recent work suggests this phenotype has functional significance because increasing ECM rigidity promotes malignant transformation, while inhibiting ECM stiffening reduces tumor incidence [8
]. Accordingly, clarifying the role by which ECM compliance influences diverse cellular and tissue level functions is central to understanding the molecular basis for development and organ homeostasis. Nevertheless, the molecular mechanisms whereby ECM compliance regulates cellular behavior and tissue phenotype remain poorly understood.
One frequently employed simplified model system used to study the effect of ECM stiffness on cell behavior is protein-conjugated polyacrylamide gels (PA gels) [11
]. These nearly elastic 2D gels permit the systematic and predictable modulation of ECM compliance by changing cross-linker concentration while maintaining ligand density and growth factor milieu constant. PA gels have proved quite useful in exploring fundamental links between ECM stiffness and cell behavior, and when used in conjunction with a matrix overlay assay, they have illustrated a role for ECM tension in epithelial morphogenesis [3
]. These PA gels have also been used to identify molecular mechanisms by which ECM stiffness modulates cell phenotype including highlighting how ECM compliance can regulate cell behavior by influencing integrin adhesions and growth factor receptor signaling [10
]. Indeed, studies using PA gels have proved instrumental in illustrating how physical cues from the ECM are sensed and propagated and how ECM tension can alter membrane receptor function and nuclear morphology to modify gene expression [25
]. Yet, most cells exist within the context of a three dimensional (3D) tissue and it is now recognized that dimensionality per se is a profound regulator of cell and tissue phenotype [28
]. In this regard, PA gels represent a pseudo 3D rigidity assay system because only the basal domain of the cell remains in contact with, and therefore responds to, the elasticity of the protein-laminated PA gel. Moreover, while animal studies have yielded important insight regarding the interplay between ECM topology, and rigidity within a 3D context [10
] in vivo
tissues are inherently complex and hence do not lend themselves as readily to rigorous mechanistic manipulations and quantitative analysis. Accordingly, tractable in vitro
systems are needed with which to study the molecular basis by which ECM stiffness influences cellular fate in the context of a 3D ECM.
A variety of natural matrices, such as Matrigel (rBM), collagen I (col I), and fibrin gels have been exploited with varying degrees of success to explore the effect of ECM stiffness and topology in vitro
on cellular behavior and fate in a 3D context [39
]. Using these hydrogel systems gel stiffness has been routinely modulated by altering the concentration or composition of the gel constituents or by varying cross-link density. Such approaches as these however, simultaneously alter gel pore size, fiber architecture, and/or the number or availability of adhesion sites [44
]. Further, these natural ECM systems frequently display inconsistencies and batch to batch variation. By contrast, synthetic biomaterials promise greater control of mechanical and adhesive properties. In this regard, a variety of approaches have been undertaken to design 3D scaffolds that combine biological functionality and the architecture of natural ECM materials with the robust controllability of synthetic materials. These scaffolds include agarose-stiffened collagen I gels [46
], polyethylene glycol (PEG) gels with tethered adhesion and degradation sites [47
], as well as a variety of systems with dynamic biophysical and biochemical properties [51
]. Unfortunately however, many of these gel systems lack the appropriate ECM-like fiber architecture and display limited pore size with increased ECM stiffness.
Self-assembling peptides (SAPs) are a family of 8–32 amino acid peptides that, when exposed to physiological salt solutions, self-assemble into fibrils [53
]. SAPs are chemically defined and biologically compatible biomaterials that mimic the architectural features observed in some natural matrices such as type I collagen gels [55
]. Moreover, SAP family members support cell adhesion and can direct the differentiated behavior of neural stem cells [56
], osteoblasts [57
], hepatocytes [58
], and endothelial cells [55
]. Motivated by these results we decided to explore the applicability of PuraMatrix, one type of commercially available SAP, to study the interplay between ECM stiffness and MEC morphogenesis in 3D. We determined that laminin-adsorbed (ligation of laminin receptors promotes MEC tissue polarity and differentiation) PuraMatrix SAPs not only support MEC acinar morphogenesis but that stiff SAPs promote an invasive epithelial tumor-like phenotype and do so without significantly changing pore size or gel architecture. Accordingly, we contend that these studies represent the first demonstration of a tractable, well defined hydrogel system that is able to recapitulate the biochemical and micro architectural features of the native normal tissue ECM so that the interplay between ECM compliance and multi-cellular tissue behavior can be studied in a 3D “tissue-like” context.