Controlling matrix stiffness and topography for the study of tumor cell migration

Cellular studies have long been performed on the bench top, within Petri dishes and flasks that expose cells to surroundings that differ greatly from their native environment. The complexity of a human tissue is such that to truly replicate a cell’s physiologic microenvironment in vitro is currently impossible. It is nevertheless important to determine how various factors of the microenvironment interact to drive cell behavior, particularly with regard to disease states, such as cancer. Here we focus on two key elements of the cellular microenvironment, matrix stiffness and architecture, in the context of tumor cell behavior. We discuss recent work focusing on the effects of these individual properties on cancer cell migration and describe one technique developed by our lab that could be applied to dissect the effects of specific structural and mechanical cues, and which may lead to useful insights into the potentially synergistic effects of these properties on tumor cell behavior.

While the vast majority of cell biology research has been performed on rigid glass or tissue culture plastic (Fig. 1A), these conditions do little to replicate the physiological environment in which cells are naturally found. The majority of mammalian cell types exist in a fully 3D environment, where they are exposed to any number of chemical, 1 mechanical 2 and structural 3 cues, many of which will change over time in response to stimuli 1,4 or as a result of disease progression. [5][6][7][8] In this review, we will briefly highlight previous and current studies that strive to elucidate the effects of two key matrix properties, stiffness and topography, on cell behavior, with a specific focus on their role in cancer progression, and we will discuss the necessity of moving toward novel systems in which two or more of these properties can be independently controlled.

The Role of Matrix Stiffness in Disease States
To elucidate the effects of the complex cellular microenvironment, studies have traditionally taken a deconstructionist approach, focusing on a single characteristic or cue at a time rather than on the integrated effects of multiple elements. One of the more frequently studied characteristics is matrix stiffness. Stiffness within Cellular studies have long been performed on the bench top, within Petri dishes and flasks that expose cells to surroundings that differ greatly from their native environment. The complexity of a human tissue is such that to truly replicate a cell's physiologic microenvironment in vitro is currently impossible. it is nevertheless important to determine how various factors of the microenvironment interact to drive cell behavior, particularly with regard to disease states, such as cancer. Here we focus on two key elements of the cellular microenvironment, matrix stiffness and architecture, in the context of tumor cell behavior. we discuss recent work focusing on the effects of these individual properties on cancer cell migration and describe one technique developed by our lab that could be applied to dissect the effects of specific structural and mechanical cues, and which may lead to useful insights into the potentially synergistic effects of these properties on tumor cell behavior.
the human body varies greatly, ranging from very soft environments, such as those found in the brain (Young's modulus, E = ~250-500 Pa) 9 or in adipose tissue (E = ~20 Pa), 10 to significantly more rigid environments, such as articular cartilage (E = ~950 kPa) 11 or rigid bone (E = ~GPa). 12 Cells are able to detect and respond to changes in mechanical properties through a network of mechanosensory proteins 13 that link the actin cytoskeleton to the extracellular matrix (ECM). 13 For additional details on the mechanisms cells use to sense these changes, see reference 13.
Matrix stiffness can be altered during the progression of multiple diseases, including cancer 5,14 and cardiovascular disease, 8 which together account for over 50% of fatalities in people over the age of 65. During neoplastic progression, increased ECM deposition and crosslinking lead to a significant increase in tumor stiffness. 5,15 For example, in breast cancer, while a healthy mammary gland is highly compliant (E = ~200 Pa), the average tumor is over an order of magnitude stiffer (E = ~4,000 Pa). 5 Additionally, both the stroma surrounding the tumor and the tumor vasculature exhibit increased stiffness (E = ~800-1,000 Pa and ~450 Pa, respectively) compared with healthy mammary tissue and vasculature (E = ~200 Pa). 5,16 Interestingly, recent work by Lopez et al. has also shown that the tumor epithelium is significantly stiffer than isolated tumor epithelial cells, 16 suggesting that the stromal microenvironment plays a critical role in maintaining the stiff mechanical phenotype of the tumor. This increase in matrix stiffness has been shown to cluster integrins, upregulate focal adhesions, increase invasion, alter cell growth and morphology and increase cell contractility. 5,14,15,17 Several of these effects have recently been shown to be mediated by increased phosphorylation of the focal adhesion kinase (FAK)/Src-signaling pathway resulting from enhanced enzymatic crosslinking of the tumor's extracellular matrix. 15 One significant challenge that remains in the study of the effects of matrix stiffening on tumor progression is difficulty in decoupling the effects of chemical and structural changes (e.g., ECM fiber crosslinking, reorganization and bundling) from the effects of the intrinsic changes in stiffness that have been shown to accompany these changes.
In another example of the effects of matrix stiffening on disease progression, during both aging and atherosclerosis, blood vessels stiffen dramatically, a phenomenon that has been shown by Huynh et al. to destabilize endothelial cell-cell contacts 18 and increase vascular permeability, further promoting atherogenesis. 8,19 While this study focused on the mechanisms of atherosclerotic progression, one could speculate from their results that the observed increase in permeability of blood vessels on stiff substrates could also have profound implications for tumor vasculature. It is well established that tumor vasculature is generally highly disorganized and leaky, which causes difficulties in effectively delivering chemotherapeutics to the tumor site. 20 One possible cause of this deviance from normal vasculature could be the increased stiffness of the tumor microenvironment, which could drive an increase in endothelial cell branching and permeability, in a manner parallel to that previously observed in atherogenesis. 8 Further studies will be required to determine if this is indeed the case. Additionally, changes in matrix stiffness have been linked to many other disorders such as diabetes, 21 liver fibrosis and cirrhosis, 22 emphysema 23 and scleroderma. 24 Therefore, understanding the effects of substrate stiffness on cell behavior is essential to understanding and combating a wide variety of disease types that affect millions of people worldwide.

Polyacrylamide Hydrogels for the Study of 2D Matrix Stiffness
The most common tool used for culturing cells on substrates of variable stiffness is polyacrylamide (PA) hydrogels. PA gels have been utilized to study the effects of stiffness on a wide variety of cell behaviors, including, but not limited to, proliferation, 25,26 adhesion, 27,28 migration, 29-31 differentiation, 32,33 matrix degradation, 34 network formation, 18 cell-cell and cell-matrix interactions 18,35 and force generation. 17,35,36 PA gels are ideal for these studies because stiffness can be precisely tuned simply by varying the ratio of monomer (acrylamide) to cross-linker (bisacrylamide) to achieve a Young's modulus (E) in the range of tens of pascals to hundreds of kilopascals. 37,38 Additionally, PA gels can be functionalized using any protein or amino acid fragment of interest, and the concentration of this protein can be held constant, independent of the gel stiffness. These qualities allow the user to distinctly separate the relative effects of stiffness and ligand density in one system. It should be noted that there remains some controversy over whether PA gels can be considered a "blank slate" for cell adhesion, as protein adsorption may occur. Additionally, different methods have been adapted to create and functionalize PA gels, and the method of functionalization has been shown in some cases to affect the mechanical properties of the underlying gel. 39 On the other hand, one of the primary benefits of PA gels is that they can be easily fabricated using standard chemical techniques. 37,40,41 As such, they have been used by hundreds of labs and have been widely published. Of particular note for this review, PA gels have been utilized in several studies of cancer cell behavior. 5,17,36,[42][43][44] For example, Ulrich et al. have shown that increasing the Young's modulus (E) of the PA gel substrate from 0.08-119 kPa increases the cell area, migration speed and proliferation of several glioma cell lines. 42 Additionally, recent work in our own lab has shown that a more moderate increase in matrix stiffness (E = 1-10 kPa) promotes increased traction force generation independently of cell area in a number of metastatic and non-metastatic breast, lung and prostate cancer cell lines. 17 Despite the popularity of PA gels, however, one obvious limitation of PA gels is that they create only a 2D environment (Fig. 1B). While there are a few cell types which exist physiologically in an environment in which they primarily sense one-dimensional features (e.g., cells migrating along individual ECM fibers) 45 or exist within a planar, two-dimensional environment (e.g., endothelial cells within the wall of a blood vessel), 8 the majority of cell types exist in a native 3D microenvironment which provides a unique set of architectural cues that are lacking in a 2D microenvironment (Fig. 1C). 36

ECM Fiber Architecture Affects Cell Behavior in 3D
Cells in the body experience a rich 3D topographical environment resulting from the various structural proteins of the ECM, which have proven difficult to effectively replicate in vitro. Collagen is the dominant structural protein in the human body, and as such gels consisting of polymerized collagen fibers have become one of the most popular in vitro systems for studying cellular response to physiological topographical cues. 3,5,7,36,37 Notably, increased stromal collagen deposition and crosslinking have been linked to poor prognosis in cancer patients. 14,46-48 While these factors contribute to the increased matrix stiffness of the tumor microenvironment, as discussed above, they also significantly alter the architecture of the ECM, which has been shown to directly mediate tumor cell migration. Recent work has shown that cancer cells actively remodel the ECM fibers surrounding a tumor, using contractile force to align the fibers perpendicularly to the tumor. 7,49 The cancer cells have been shown to then persistently migrate along these aligned fibers in a largely one-dimensional fashion both in vitro and in vivo. 6,7 Indeed, Conklin et al. recently linked the presence of bundled and aligned collagen fibers to poor longterm survival in breast cancer patients. 6 There are several limitations of using collagen gels in vitro to specifically study the effects of matrix architecture on cancer cell behavior. The most important limitation is the inability to decouple structure and matrix stiffness within this system. Collagen gels can be readily stiffened using exogenous crosslinking agents, 14 but this process typically alters their structure and the structure that is sensed by the cell and, depending on the crosslinking agent, it can be cytotoxic. Alternatively, the structure of the collagen can be changed by manipulating the density of collagen fibers, 3,5 but this too is accompanied by corresponding changes in stiffness. The use of collagen gels to study cancer cell migration is further complicated by ongoing controversy stemming from the use of different collagen extraction techniques. 50 Cancer cell migration has been traditionally defined in two main categories: protease-dependent and protease-independent. [50][51][52][53] Extensive work has been done in the last decade to show that when one of these pathways is inhibited, cancer cells are able switch their behavior and continue the invasion process. 52,54 However, these results have recently been called into question. 50 New evidence suggests that many of these experiments utilized collagen that had been extracted with pepsin, 52,55 a process that removes the nonhelical telopeptides at the ends of the collagen molecules and prevents the fibrils from forming the covalent cross-links that are found in vivo. 50 This lack of covalent cross-links has been shown to allow protease-independent cancer cell migration that is not recapitulated in the more physiologically relevant, non-pepsinized collagen, which retains robust cross-links. 50 These data would suggest that cancer cells must rely on protease activity to invade and metastasize, and perhaps have a smaller requirement for force generation. However, work has also been done on tumor cells in vivo showing that contractile force generation, not protease activity, is required for cancer cell motility. 53 Moreover, protease inhibitors to date have largely failed in clinical trials, 56 suggesting that more studies need to be performed to validate this hypothesis. Overall, improved control over the structural, mechanical and chemical properties of in vitro systems and novel methodologies will be critical to delineating the true mechanisms which guide tumor cell migration in vivo.

Microfabricated Structures for the Study of Topographical Effects on Cell Behavior
There has also been a considerable amount of research using rigid micro-and nano-fabricated substrates such as polydimethylsiloxane (PDMS), poly(methylmethacrylate) (PMMA), silicon and others to specifically assess the effects of tightly controlled topographical features on cell adhesion, 57 morphology, 57-59 migration behavior, 58,60,61 stem cell differentiation [62][63][64] and other cell functions. Cell behavior has been extensively analyzed on substrates containing features such as grooves, ridges, posts, and wells that are fabricated at the sub-cellular, cellular and supercellular scales (for detailed reviews, see refs. 58, 59 and 61). Nanogratings or ridges in particular have been used by many labs to induce cytoskeletal alignment and guided migration in different cell types, including smooth muscle cells, macrophages, corneal epithelial cells, fibroblasts, endothelial cells and neurons, with optimal ridge width varying significantly with cell type. 57,60,65,66 Interestingly, in a seminal study by Doyle et al., fibroblast migration on micropatterned 1D substrates was found to closely mimic 3D migration along ECM fibers. 67 They also found that the mechanisms of migration employed by the fibroblasts were similar between 1D and 3D environments, but were in distinct contrast to 2D migration mechanisms, 67 thus highlighting the ongoing need for improved 3D and 3D-like topographical model systems.
Additionally, several groups have used microfabricated structures to study cancer cell migration under conditions of mechanical confinement within narrow PDMS channels. [68][69][70][71][72] When introduced to channels that are of average cell-width (approximately 3-10 mm), cancer cells have been shown to preferentially and persistently invade through these channels, with 70 or without 68,69 the application of the chemical gradients often required to induce persistent cell migration on planar 2D substrates. Interestingly, this mechanism of migration appears to resemble the migration in 1D and 3D fibrillar environments by Doyle et al., discussed above, 67 particularly in its dependence on intact microtubule dynamics. 69 In another example of using rigid substrates to study cancer cell migration, Liu et al. have used microfabricated silicon posts with height of several hundred microns, nicknamed Tepuis, to study metastatic prostate cancer invasion behavior. 73 They found that highly metastatic PC-3 prostate cancer cells efficiently invade the Tepui tops and proceed to confluency, while less metastatic LNCaP cells are not able to achieve similar levels of invasion. 73 While these systems have been used to elegantly describe the effects of different micro-and nano-topographical features on several key cell functions, it is difficult to interpret their physiological relevance, as no surface within the body (with the exception of bone) shares such a rigid stiffness. As discussed previously, matrix stiffness has been shown to have a dramatic effect on cell migration, adhesion, force generation and multiple other behaviors. Therefore, to more accurately assess the effects of physiological topographical features on cell behavior, there is a great need to adapt methods to create topography on softer substrates, ideally with defined and controllable stiffness.

Patterning 3D Topographical Features on 2D PA Hydrogels
Recently, our lab has developed a technique in which 3D topographical features can be reproducibly patterned onto a 2D PA hydrogel where stiffness can be precisely controlled over a physiological range with negligible differences in swelling. 74 By using this method, we retain the benefits of the well-characterized 2D PA system-most notably the independent control over stiffness and ligand density-and we gain the ability to expose cells to a more 3D-like environment with features in the orthogonal plane (Fig. 1D). While still seeded in a 2D fashion, after spreading cells are able to contact and migrate along the patterned 3D topographical features. We patterned plateaus with heights on the order of 20-50 mm and examined the effect of stiffness on contact guidance behavior of endothelial cells as they interacted with the plateaus. 74 Although cell migration behavior was similar across the stiffness range we tested (~1-42 kPa), the cells that interacted with the stiffer plateaus had a significantly higher average contact length independent of feature height (Fig. 2), which is consistent with previous experiments by our lab and others, 35,75 indicating that endothelial cells have a larger spreading area on stiffer 2D substrates. We chose to quantify contact length, rather than cell spread area, to specifically focus on the interaction of the cells with the patterned 3D features. While PA hydrogel swelling is typically not problematic in traditional 2D experiments, we found that it can significantly distort the topographical features patterned in 3D, such that different stiffness hydrogels can exhibit different degrees of swelling. 74 Differences in topography caused by this swelling can be sensed by cells and cause their responses to become difficult to interpret. Because similar stiffness hydrogels can be created using different ratios of acrylamide to bis-acrylamide, we quantitatively determined guiding principles that can be used to minimize swelling differences. Gel composition is conventionally described using the parameters T and C. The concentration (w/v) of total acrylamide (i.e., both acrylamide and bis-acrylamide) is known as T, and the percentage of this total that is bis-acrylamide (crosslinker) is referred to as C. Because C has been implicated previously as an effector of PA hydrogel swelling, 76,77 we held T constant and varied C from 1.15-7.00%. We quantified PA gel swelling in the vertical direction using a swelling ratio parameter (SR Z ), which we defined as the ratio of the feature height to the corresponding mold dimension. A value of 1 indicates that the molded feature is identical to the master mold used to create it, while a value greater than 1 indicates an increase in feature size compared with the mold caused by swelling. We found that differences in SR Z were negligible once the percentage of bisacrylamide was > 5%, independent of the concentration of total acrylamide. 74 To more precisely examine how the composition of the gel influences the amount of swelling, we recently modeled the relationship between gel formulation (T and C) and vertical feature swelling (SR Z ) using a 6 parameter linear regression analysis fit (Eq. 1), where b i are constants. 78 The corresponding fit to our empirical data points yields an R 2 value of 0.94 (Fig. 3). We found this model to correspond well with previous studies of PA gel swelling published by other groups. 76,77 As we determined previously, to minimize swelling C must be kept higher than 5%, limiting the stiffness range that can be acquired when T is held constant. In order to obtain a broader stiffness range, the value of T can also be modulated accordingly.
The model presented here can be used to guide the selection of T and C ratios for experiments in which it is important to minimize any differences in swelling in order to achieve nearly identical topographical landscapes for cell behavior. While the work done so far with this system has been limited to endothelial cells, it would be readily applicable to the study of tumor biomechanics. As our knowledge of cancer cell behavior grows, it will be critical to study these behaviors in environments that mimic the native tumor environment as closely as possible. This is particularly important given the key role of the microenvironment in dictating cancer behavior, which we are only just now beginning to fully comprehend. To this end, one could envision using the  technique described here to specifically elucidate the mechanisms governing stiffness-mediated contact guidance in cancer cells. However, it should be noted that while this system significantly advances the study of synergistic effects on cell behavior by creating a novel method for simultaneously controlling matrix stiffness, ligand density and topography, more work needs to be done to move toward a thorough understanding of these effects in a fully-rendered physiological 3D environment. In particular, the development of in vitro 3D models whose matrix properties can be independently and precisely manipulated will be key to furthering our understanding of true cell behavior and could lead to dramatic advances in our ability to comprehend and treat cancer and other diseases.