Material Strategies for Modulating Epithelial to Mesenchymal Transitions

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Material strategies for modulating epithelial to mesenchymal transitions Emily Mihalko, and Ashley C. Brown ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00751 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 15, 2017

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ACS Biomaterials Science & Engineering

Material strategies for modulating epithelial to mesenchymal transitions Emily P. Mihalko1,2 and Ashley C. Brown*1,2 1

Joint Department of Biomedical Engineering, North Carolina State University and University of North Carolina at Chapel Hill, Raleigh, NC 27695 2 Comparative Medicine Institute, North Carolina State University, Raleigh, NC 27695 *

Corresponding Author Ashley C. Brown, PhD Address: Joint Department of Biomedical Engineering North Carolina State University and University of North Carolina at Chapel-Hill 911 Oval Drive; 4204B Engineering Building III Raleigh, NC 27606 Phone: (919) 513-8231 Email: [email protected] Abstract:

Epithelial to mesenchymal transitions (EMT) involve the phenotypic change of

epithelial cells into fibroblast-like cells. This process is accompanied by the loss of cell-cell contacts, increased extracellular matrix (ECM) production, stress fiber alignment and an increase in cell mobility. While essential for development and wound repair, EMT has also been recognized as a contributing factor to fibrotic diseases and cancer. Both chemical and mechanical cues, such as tumor necrosis factor alpha, NF-κΒ, Wnt, Notch, interleukin-8, metalloproteinase3, ECM proteins, and ECM stiffness can determine the degree and duration of EMT events. Additionally, transforming growth factor beta is a primary driver of EMT, and interestingly, can be activated through cell-mediated mechanoactivation. In this review, we highlight recent findings demonstrating the contribution of mechanical stimuli, such as tissue and material stiffness, in driving EMT. We then highlight material strategies for controlling EMT events. Finally, we discuss drivers of the similar process of endothelial to mesenchymal transition (EndoMT) and corresponding material strategies for controlling EndoMT. Keywords:

epithelial to mesenchymal transition, EMT, mechanotransduction, fibrosis,

development, transforming growth factor beta, EndoMT

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I.

Introduction Epithelial to mesenchymal transitions (EMT) are a process by which epithelial cells lose

epithelial characteristics and take on mesenchymal-like properties. During this process, epithelial cells lose cell-cell adhesions, modulate their polarity, alter their cytoskeleton organization, and become more mobile1,2,3. The EMT process is also characterized by an upregulation of α-smooth muscle actin (α-SMA), N-cadherin, and vimentin and increased extracellular matrix (ECM) production4. While many factors play a role in EMT, the pathways are well conserved in different species and represent a potentially controllable process3. The characteristics of EMT are not discrete. EMT exists on a spectrum, as shown in Figure 1A, such that cells can exhibit a range of both epithelial and mesenchymal characteristics. EMT is also reversible and the converse process is known as mesenchymal to epithelial transition (MET)1,2. Both epithelial and mesenchymal cell types were recognized in the late nineteenth century as a result of their different cell shapes5. Mesenchymal cells are solitary and spindle-shaped whereas epithelial cells are closely connected to each other and are bound at their basal surface to the basal lamina. It wasn’t until the early 1980’s that EMT as a process was first described in the primitive streak of chick embryos6. As EMT became more defined and understood, it’s importance during development was recognized. EMT is crucial for the developmental stages of heart morphogenesis and mesoderm and neural crest formation1,3, while MET occurs during somitogenesis, kidney development, and coelomic-cavity formation2. Following development, EMT occurs throughout adulthood, playing an important role in wound healing, tissue regeneration, and controlling inflammation2,7. However, it also has been established that EMT plays a role in the pathological processes of fibrosis and cancer5,8–12. As an example, in human fibrotic lung tissue, epithelial cells are present


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with mesenchymal features, suggesting EMT as a major contributor to fibrotic lung pathology9. Multiple additional studies have provided evidence to indicate that epithelial cells can be activated to produce mesenchymal proteins that induce fibrotic responses8,9,13–15. EMTs play crucial roles in development, wound repair and pathology. In this review, we will focus on the chemical and physical forces contributing to EMT responses. Furthermore, we will discuss how current and future biomaterial applications can target the mesenchymal transition process for improved regeneration and healing. II.

Type I, type II and type III EMT Because EMT leads to different functional consequences in both development and

pathology, EMT has been classified as type 1, type 2 and type 3 to distinguish these different biological consequences2,7,16. As seen in Figure 1B, type 1 EMT is associated with development and embryogenesis. In development, EMT is followed by MET, and this cellular plasticity leads to complex tissue formation. Type 2 EMT is associated with inflammation, wound healing and tissue regeneration, and when not properly regulated, type 2 EMT contributes to fibrotic responses. Type 3 EMT is associated with cancer and metastasis. Despite the wide range of biological outcomes encompassed by these three types of EMT, similar mechanisms are involved in the overarching process. Besides fibrotic disorders, mesenchymal transitions during cancer progression have also been widely examined. EMT is now recognized as a mechanism for epithelial cancer cell metastasis4,7,17. Tumor cells have been characterized by the downregulation of epithelial cell adhesion receptors such as E-cadherins and an upregulation of mesenchymal markers such as Ncadherins and vimentin, characteristics of EMT18. Carcinomas, which are tumors derived from epithelium, have been shown to lose most of their epithelial characteristics during cancer



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progression5. These cellular changes allow decreases in cell-cell attachment which drive tumor cell migration. In human tumors, as well as mouse models of breast cancer, EMT is present during tumor progression and shows a consequent increase in motility that allows the cancer cells to spread1. One study reports that mammary tumor cells undergoing EMT migrate through the lymphatic system similar to dendritic cells in response to inflammation19. Tumor cells also drive mesenchymal cells to deposit matrix, which enhances the assembly and alignment of the ECM, allows for cross-linking of collagen and fibronectin rich matrices, and further contributes to EMT18. Overall, the process of EMT in cancer is a powerful modulator of cellular responses and their environments.


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Similar to epithelial cells, endothelial cells can also undergo a phenotypic transition that mirrors EMT. This process, known as endothelial to mesenchymal transition (EndoMT), is characterized by a loss of endothelial markers such as CD31 and vascular endothelial cadherin, as well as the upregulation of α-SMA and ECM production20,21. Similar to EMT, EndoMT plays an important role in development, especially in the formation of cardiac heart valves22. The occurrence of EndoMT in cardiac development is marked by expression of both the endothelial marker CD31 and the mesenchymal marker α-SMA in the cardiac valve2. However, just as EMT has been associated with fibrotic responses, EndoMT similarly contributes to cardiac fibrosis as it contributes to the excessive matrix production characteristic to the disorder20,23. Studies have also associated cardiac fibrosis with the emergence of fibroblasts that have originated from endothelial cells, implicating EndoMT during disease progression20. As a result of cardiac fibrosis, the heart muscle and ventricular walls stiffen, causing impaired cardiac function. EndoMT has also recently been shown to occur in vein grafts and contributes to ~50% of the developing neointimal cells that lead to vein graft stenosis24. Other fibrotic responses that have shown to incorporate EndoMT include pulmonary fibrosis, liver fibrosis, diabetic renal fibrosis, and corneal fibrosis21,23. The accumulation of α-SMA expressing mesenchymal cells, which play a large role in the vascular remodeling of pulmonary arterial hypertension (PAH), is thought to largely be caused by EndoMTs25. EndoMT will be discussed in more detail in section XII. It should be noted that the contribution of EMT to fibrotic disorders in vivo has been a source of controversy in the field, especially in the area of renal fibrosis. Several outstanding reviews by Andras Kápus, Jeremy Duffield, and Michael Zeisberg26,27 detail evidence supporting and/or refuting the supposed contribution of EMT in renal fibrosis. We point the reader to those articles for greater detail, but in short, while the evidence of EMT in vitro is robust and generally



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agreed upon, in vivo cell fate-mapping studies using a unilateral ureteral obstruction (UUO) model of renal fibrosis have produced conflicting outcomes. While Iwano et al. identified that ~36% of fibroblasts in UUO were derived from epithelial origin28, studies by Humphreys et al. using an identical model of UUO failed to identify any epithelial derived fibroblasts29. Similarly, in various models of liver fibrosis, epithelial fate mapping studies have produced conflicting results30–32.

There are certainly nuances to each of these studies that could explain the

conflicting results, for example differential selection of mesenchymal markers. None-the-less, these conflicting reports also highlight the complexity of the EMTs and the multifactorial nature of the process. In the aforementioned review by Andras Kápus, an important point is raised with regards to these conflicting studies; EMT is a plastic response which exists not in a simple “on” or “off” state, but rather as a spectrum of phenotypes that can vary in degree and duration and that can be altered rapidly in response to the combinatorial cues of multiple inputs from the ECM microenvironment. Biomaterials, especially those with dynamic properties that closely mimic in vivo environments, offer unique experimental modalities for understanding the highly complex biological responses in EMT. III.

Triggers of EMT A wide range of soluble and physical cues have been demonstrated to contribute to the

EMT process. In particular, transforming growth factor beta (TGFβ) has been identified as a potent inducer of EMT.

Interestingly, TGFβ can be mechanically activated through cell

contractile forces, which are upregulated upon increased mechanical stimulation, thereby linking physical forces to soluble cues involved in EMT. The physical forces in EMT, including tissue stiffness, and the critical role of TGFβ will be discussed in detail in sections IV, V, and VI of this review. Other soluble factors that trigger EMT include epidermal growth factor (EGF),


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hepatocyte growth factor (HGF), fibroblast growth factor (FGF), tumor necrosis factor-α (TNFα), nuclear factor-κB (NF-κB), Wnt, Notch, and interleukin-8 (IL-8)2,11,33,34. Many extracellular matrix (ECM) components also play a role in inducing EMT by stimulating cellular responses through integrin-mediated signaling. Also, matrix metalloproteinase-3 (MMP-3), an enzyme that breaks down ECM proteins, has been identified as an EMT inducer for mammary epithelial cells4. Hypoxia and reactive oxygen species (ROS) have been found to trigger an EMT response as well2,35. Specifically, MMP-3 induced EMT occurs via a pathway dependent upon the production of ROS, as shown in Figure 2A36,37. When EMT-inducing cues are received by the cell, it triggers Snail, a zinc-finger transcription factor, to repress E-cadherin expression, which is usually expressed at high levels in epithelial cells. Studies have shown that GSK-3β will bind to and phosphorylate Snail to function as a molecular switch for many signaling pathways that lead to EMT38. If GSK-3β is inhibited, this results in an upregulation of Snail and downregulation of E-cadherin, signaling a phenotypic switch. Furthermore, mechanical stimulation of cells, through external application of force or increased tissue stiffness, has been linked to EMT processes in a range of conditions. IV.

Physical forces in EMT Cells are able to sense and respond to mechanical forces in their microenvironment

through mechanotransduction signaling pathways. After a cell senses mechanical stress, cells can convert the physical cues into biochemical signals that translates into a cellular response. These mechanical cues arise from a variety of sources including external mechanical sources, cellgenerated sources, and ECM derived forces. External sources can include tissue compression or stretching, osmotic pressure and fluid flow. Cell-generated physical forces arise from cellular contractility. This cell-mediated force generation can be transmitted to neighboring cells directly



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through cell-cell contacts or indirectly through cell-ECM interactions, in which cells apply forces to the surrounding ECM, inducing tension in the ECM, which can be transmitted over long ranges to neighboring cells. ECM-derived forces include the aforementioned ECM tension as well as ECM stiffness. Matrix stiffness directly affects the generation of cellular tension, such that cells cultured on stiffer substrates engage their actin-myosin machinery and become more contractile in an attempt to match intracellular stiffness to that of the external substrate. This concept, known as “tensional homeostasis”, highlights the inter-related nature of external mechanical stimuli and cell-generated forces39. In response to external mechanical stimuli, such as stretching or increased tissue stiffness, cells become more contractile, applying forces to the surrounding ECM, which in turn makes the microenvironment stiffer, thereby further stimulating cell contractility. The feed-forward loop has many implications in EMT, particularly in Type 2 and Type 3, as increased tissue stiffness is a hallmark of fibrotic conditions and cancer11,40–46. Numerous recent studies will be detailed in sections VI and X of this review showing correlated increased tissue stiffness with disease-related EMT events. It has also been shown that increased cell contractility and cell spreading are critical factors that regulate the EMT response47. During EMT, the activity of Rho GTPases, including RhoA, Rac1, and Cdc42, contributes to the modulation of cell contractility through regulation of actin dynamics and cytoskeleton reorganization11,48–51. MMP-3-induced EMT has been shown to interrupt cell-ECM contacts that regulate gene expression of Rac, which leads to MMP-3induced EMT in breast, lung, and pancreas4. In response to increased tissue stiffness, the Rho signaling pathway is activated, leading to Rho-associated kinase (ROCK) regulation of cytoskeletal activities40. In particular, ROCK regulates actin organization and cell migration through phosphorylation of LIM kinase (LIMK), myosin light chain (MLC), and MLC


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phosphatase. Through ROCK’s phosphorylation of LIMK, which subsequently phosphorylates ADF/cofilin and inhibits its actin-depolymerization activity, ROCK indirectly inhibits the depolymerization of actin filaments thereby stabilizing the filaments. ROCK regulation of cell contractility and migration is mediated through its phosphorylation of MLC, which increases myosin II ATPase activity, and through its inactivation of MLC phosphatase, which leads to increased levels of phosphorylated MLC. These regulated pathways form a mechanism that enables cells to contract and demonstrates the potential for cell-mediated physical forces in EMT. Increased cell contractility and mobility is itself a hallmark of EMT, but additionally, increased cell contractility is linked to EMT through the cell-mediated mechanical activation of TGFβ 46,51,52. Other pathways related to the physical cues involved in EMT include Rac and Cdc42, which also participate in the cytoskeleton changes involved in EMT, but more specifically are involved in membrane protrusion formation. These pathways induce cellular response through activation of p21 activated kinase 1 (PAK1) and promote the formation of lamellipodia and filopodia53. PAK1 activation by Rac1 or Cdc42 subsequently induces actin polymerization and membrane protrusion for cell spreading and motility. Rac1 activation at the leading edge of the cell also stimulates PI3K, which is critical in initiating front–rear polarity and further participates in the recruitment of Cdc42 and Rac guanine nucleotide exchange factors (GEFs) to the leading edge to create a positive feedback mechanism participating in the reorganization of the microtubule cytoskeleton53,54. Additionally, Rac1 will promote the clustering of integrins towards the front of the cell. PI3K is involved in another EMT regulatory pathway through activation of AKT. During EMT, this PI3K-AKT pathway results in activation of mammalian TOR (mTOR) complex 1 and 2, which contributes to cell size, protein translation, cell motility,



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and the transition from an epithelial to mesenchymal phenotype55,56. In addition, AKT can phosphorylate GSK-3β to inhibit its activity and therefore upregulate Snail to signal a phenotypic switch38. Overall, Rho GTPase signaling plays a pivotal role in EMT responses and material strategies that aim to enhance or inhibit activation of these pathways, such as through modulating substrate stiffness or topography, offer a mechanism by which EMT can be tuned. Specific examples of using materials to modulate these pathways will be discussed in section X. V.

Crucial Role of TGFβ As mentioned previously, TGFβ is one of the primary inducers of EMT, and has been

shown to promote EMT in epithelial cells derived from a multitude of tissues including breast, lung and kidney13,47,57–62. Depicted in Figure 2B(i), TGFβ signaling can occur through canonical SMAD signaling pathways as well as non-canonical pathways including the MAP kinase, JNK and PI3K pathways, and all of these pathways have been implicated in EMT2,11,63. In canonical SMAD signaling, following binding of TGFβ to the TGFβ receptor I, the TGFβ receptor II transphosphorylates and forms a complex with the growth factor bound TGFβ receptor I. This subsequently leads to phosphorylation of serine residues in cytoplasmic R-Smads, Smad2 and Smad 3. The Smad 2 and 3 complex in turn bind to the common-partner Smad, co-Smad 4. This R-Smad-co-Smad complex subsequently translocates to the nucleus and then interacts with transcription factors and transcriptional co-activators/co-repressors to regulate gene expression. Smad 7, an inhibitory Smad, negatively regulates this pathway through multiple mechanisms including binding to activated TGFβ Type I receptors. Smad7 binding to TGFβ Type I receptors competes Smad2/3 binding, thereby downregulating TGFβ signaling. TGFβ must be activated, however, prior to binding to its receptors and affecting downstream signaling. In its inactive form, TGFβ forms a non-covalent association with the


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latency-associated peptide (LAP), and this complex is known as the small latent complex52. The latent TGFβ binding protein (LTBP) can then bind the small latent complex, and this larger assembly is known as the large latent complex. The LTBP binds multiple ECM molecules, including fibrillin-1, fibronectin and vitronectin, and facilitates immobilization of inactive TGFβ within the ECM, thereby providing a reservoir of latent TGFβ in the ECM microenvironment. The latent complex can be activated through several pathways including proteolytic cleavage, heat, integrin-mediated activation through cell contractility, illustrated in Figure 2B(ii)11. In the context of mechanical regulation of EMT, integrin-mediated activation of TGFβ is an important pathway. Cells can bind to the LAP through integrin interactions with the RGD sequences on the LAP. Though many integrins are known to interact with RGD sequences, this mechanism of TGFβ activation has been attributed to αvβ6 and αvβ8 integrin interactions with the LAP9,11. Following binding to the LAP, application of cell contractile forces results in a “clam-shell” like mechanism whereby the LAP complex is pried away from TGFβ. The growth factor is then either released in a soluble form (if signaling occurs through αvβ8 integrin interactions) or is exposed temporally to neighboring cells (if signaling occurs through αvβ6 integrin interactions)64.



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This integrin-mediated activation pathway relates both integrin expression patterns and cell contractility, which can be altered in EMT promoting microenvironments. αv integrins are known to be upregulated in epithelial cells following injury and in cancer11. Also, increased


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tissue stiffness, such as that seen in fibrosis and cancer, leads to increased cell contractility. The combination of increased αv integrin expression and cell contractility contribute to increased TGFβ activation and EMT. This paradigm of increased cell contractility leading to cell-mediated TGFβ activation has been demonstrated in both fibroblasts and epithelial cells11,52,65,66. In vitro studies with pulmonary fibroblasts demonstrate that on stiff surfaces, cells are able to generate intracellular tension, thereby facilitating integrin-mediated activation of TGFβ, which in turn promotes differentiation of the cells into highly contractile myofibroblasts52. Similarly, experiments with type II alveolar epithelial cells demonstrate similar responses, such that on stiff surfaces, intracellular tension can be generated, leading to integrin-mediated activation of TGFβ and EMT65. These responses are dependent on the cells’ ability to generate cell contractile forces, which is dependent on the cell’s ECM-microenvironment. Critical contributing factors to this process, and subsequent EMT, include ECM stiffness, composition, external application of force, and tissue geometry. These contributing factors to increase TGFβ activation can be incorporated into material design to, in turn, pattern EMT responses. VI.

Tissue stiffness and EMT Increased tissue stiffness is a hallmark of fibrotic disease and cancer, and numerous

recent studies have begun to link specific disease-related changes in tissue mechanics to the promotion of EMT2,44,51,60,67–70. Elastic (Young’s) modulus is an intrinsic stiffness value that varies among different tissues and organs. ECM composition, fat content, and cell types will all contribute to variable Young’s moduli. For example, bone marrow exhibits a ranging Young’s modulus with a maximum value of approximately 25 kPA71, while blood plasma is approximately 50 Pa44. Healthy epithelial tissues are very compliant, and typically have Young’s



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moduli in the single digit kPA range44. Fibrotic conditions and cancer can increase Young’s moduli values ten to a hundred-fold. During pulmonary fibrosis for example, living lung slices obtained from either healthy or bleomycin-induced fibrotic mice demonstrate an increase in Young’s Modulus from ~2kPA to ~20kPa51,67. Likewise, biopsy samples collected from human IPF patients demonstrate an increase in tissue stiffness from ~2kPa in normal tissue to ~17kPA in fibrotic tissue68. When alveolar type II epithelial cells are cultured in vitro on polyacrylamide gels of varying stiffness spanning this range, cells begin to increasingly decrease expression of epithelial markers, such as E-cadherin, and begin to increasingly express mesenchymal markers, such as α–SMA51. In these studies, stiffness-mediated EMT events were found to correlate with increased cell contractility and increased TGFβ activation, so stiff substrates promote EMT. Similar correlations have been made in numerous other fibrotic conditions. Studies have found that stiffened tissue is a major promoter of myofibroblasts during pulmonary fibrosis, and the stiffness of fibrotic scar tissue can promote myofibroblast contraction and differentiation, which ultimately destroys lung function12. In liver fibrosis, it has been found that liver stiffness precedes fibrosis and potentially myofibroblast activation in rats, suggesting the tissue stiffness, resulting from matrix accumulation, plays a crucial role in the early stages disease onset72. Recently, matrix rigidity has been associated with the subcellular location of myocardin related transcription factor (MRTF-A), illustrating how biophysical triggers, such as tissue stiffness, can regulate protein expression in the development of myofibroblasts during EMT70. Tissue stiffness enhances EMT pathways which have been implicated in tumor invasion and metastasis as well. Tumors are stiffer than normal tissue. Two dimensional substrates and three dimensional culture studies have demonstrated that increasing matrix stiffness can activate the Rho pathway and enhance force generation and cytoskeleton tension39. Therefore, during


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tumor formation where tumors show increased stiffness in a tissue environment, enhanced cytoskeleton tension and cell contractility will follow to regulate cell phenotype39. Additionally, Rac1b, a highly activated form of Rac1 found in tumors, was found to localize to cell membranes when on stiff substrates or collagen-rich regions of human breast tumors, where it can promote the production of reactive oxygen species, expression of Snail, and activation of EMT. However, on soft substrates, these effects were reduced through the inhibition of Rac1b membrane localization73. Recently, high tissue stiffness in human breast tumors has been correlated with nuclear translocation of EMT transcription factor twist-1 after reduced G3BP2 expression69. Through this mechanotransduction pathway that responds to increased tissue stiffness, EMT and tumor metastasis are promoted. These studies involving fibrotic conditions and cancer show a correlated increase in tissue stiffness with disease-related EMT events. The knowledge from these fundamental mechanistic studies can inform methods for designing materials to promote or inhibit EMT in a predictable manner. VII.

Biomaterial Strategies for Understanding and Controlling EMT

Because of its role in development, wound healing, and multiple pathologies, interest has arisen in the EMT field in devising strategies to control EMT in a finely controlled manner. The ability to control development-associated EMT (i.e. Type I EMT) would facilitate the ability to both control the gastrulation process for regenerative medicine applications and design cell instructive microenvironments for investigating the underlying mechanisms of developmental biology. Likewise, controlling Type II EMT responses could allow for the promotion of wound healing while mitigating scar tissue formation. Type II and type III EMT are present in a multitude of pathologies including cancer, pulmonary fibrosis, cardiac fibrosis, and kidney



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fibrosis. Therefore, modulating EMT responses represents a critical therapeutic target to prevent the progression of these diseases. Because EMT-associated phenotypic changes can arise from various gene regulators, ECM compositions, as well as mechanical properties of the surrounding microenvironment, biomaterials which present such cues in a finely-controlled manner present the ability to induce or prevent EMT events in a highly regulated fashion, thereby allowing for control over the overall coordination of the EMT process. Potential strategies include designing materials with controlled moduli, gradients of ECM proteins and/or soluble factors, combinatorial approaches presenting various mechanics, ECM components, and soluble factors, and patterned substrates which spatially control these elements. A summary of these strategies, which will be detailed in sections VIII, IX, X, and XI, is seen in Figure 3.


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VIII. Controlling EMT through soluble factors Many soluble factors have been identified to induce EMT responses. Therefore, the use of soluble factors represents a primary method for controlling EMT. Soluble signaling can be controlled by either delivering EMT-inducing factors or by inhibiting their activity. EMTinducing soluble cues include TGFβ and various other growth factors such as tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), Wnt, Notch, interleukin-8 (IL-8), and plasminogen activator inhibitor-1 (PAI-1)2,11,33,34. As described previously, the addition of soluble, active TGFβ to epithelial cells robustly induces EMT responses. However, inhibition of TGFβ signaling via function blocking antibodies or siRNA results in the maintenance of epithelial



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phenotypes. Recent studies have demonstrated that inhibition of PAI-1 through small interfering RNA (siRNA) targeting PAI-1 (PAI-1-siRNA), which has been implicated in pulmonary fibrosis associated EMT, has the potential to limit the development of pulmonary fibrosis. One study examined limiting the development of bleomycin-induced pulmonary fibrosis in a mouse model after distributing PAI-1-siRNA in the lung, and studied the effect of PAI-1-siRNA on the EMT using a mouse lung epithelial cell line74. It was found that the transfection of lung epithelial cells with PAI-1-siRNA inhibited TGFβ-induced EMT, and the fibrotic response was attenuated in vivo. Likewise, microRNA based approaches can be used to control growth factor-induced EMTs75. In particular, Madin-Darby canine kidney epithelial (MDCK) cells underwent EMT after inhibition of microRNA-200, and mesenchymal derived cells reverted to an epithelial-like phenotype after enforced microRNA-200 expression. Double-negative feedback mechanisms have been identified by controlling microRNA-200 family expression with ZEB1-SIP1, a key transcription factor that represses epithelial genes such as E-cadherin, which expands understanding of microRNA regulation of EMT76. Beyond simply introducing soluble factors into the media, materials which incorporate growth factors or signaling molecules into a matrix, either covalently or non-covalently, can be used to modulate EMT outcomes with spatiotemporal control. In a study by Li et al. that examined signaling molecule presentation strategies to control EMT, cell-fate was modulated through precise spatial control over TGFβ signaling77. In this study, peptide ligands to TGFβ receptors were spatially controlled on a surface, and cells that adhered to this ligand increased TGFβ-responsive gene expression as they underwent EMT. While the use of signaling molecule/growth factor presentation in 3D microenvironments specifically applied to controlling EMT has only been shown in a limited number of studies, including the one described above, a


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number of synthetic three-dimensional ECM mimics have been developed that can be used to regulate ECM-bound growth factor presentation77–81. Such materials could easily be applied in future applications to regulate EMT dynamics in a spatially controlled manner through presentation of known EMT-inducing growth factors, such as TGFβ or TNFα. In general, these materials can either bind or release soluble factors similar to natural ECMs to regulate tissue dynamics. One study in particular described an injectable multi-protein delivery mechanism to deliver VEGF and PDGF through enzymatic release using enantiomerically engineered protein nanocapsules79. To modulate the binding of growth factors in a synthetic matrix to regulate their activity, one study has shown that fibrin(ogen) can promiscuously bind growth factors from the PDGF, FGF, TGFβ, and neurotrophin families through a heparin binding domain80. This binding can be used in a synthetic fibrin-mimetic matrix to create an ECM-mimetic material that matches the ability of the natural ECM to tightly regulate growth factor activity and support growth factor presentation for controlling cellular responses. For example, immobilizing FGF or TGFβ within such a matrix could be useful for patterned induction of EMT. Designing other scaffolds such as hyaluronic acid-based hydrogel scaffolds, instead of fibrin, could take advantage of the same highly controllable signaling molecule presentation to direct cellular behavior due to translatable chemistry between fibrin and hyaluronic acid scaffolds81. These or similar approaches could be applied using EMT promoting and/or inhibiting growth factors to spatiotemporally control EMT. IX.

Controlling EMT through ECM proteins Beyond the control of soluble signals in regulating EMT, ECM proteins are key

regulators of the mesenchymal transition from epithelial cells. Epithelial tissues are defined by tightly interacting cells on top of a basement membrane such as a collagen or laminin-rich ECM that connects the cells to the underlying tissue18. Conversely, mesenchymal cells are



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characterized by cell-ECM interactions with connective ECM rich in collagen, proteoglycans, and fibronectin. Perhaps not surprisingly, epithelial cells cultured on mesenchymal-associated ECM proteins tend to spontaneously undergo EMT9. Additionally, disrupting cell-cell contacts, such as through treatment with matrix metalloproteinases (MMPs), can promote EMT. Such cues can be used in a combinatorial method for controlling EMT. For example, when examining EMT of mouse mammary epithelial cells, it was found that the basement membrane protein laminin suppresses the EMT response in MMP-3-treated cells, whereas fibronectin promotes EMT82. In a different study utilizing alveolar epithelial type II (ATII) cells, it was found that cells cultured on laminin matrices and exposed to active TGFβ exhibit an apoptosis-prone phenotype9,83, while cells cultured on fibronectin surfaces in the presence of TGFβ exhibited EMT. This is an example of a strategy that incorporates several material strategies and highlights the synergistic signaling of adhesive and soluble cues in directing EMT responses. These studies were performed using a polydimethylsiloxane (PDMS) microfluidic device that was fabricated to analyze the regulated EMT responses of epithelial cells on laminin and fibronectin coated surfaces in the presence or absence of soluble cues, such as TGFβ, in a single platform83. This approach is an attractive strategy in screening potential synergistic signaling between soluble factors and ECM cues in EMT in a high throughput manner and could be used in future studies to address unanswered questions regarding the synergistic activity between various soluble and adhesive cues in EMT. Cells interact with underlying ECM proteins through integrin receptors that attach the cell cytoskeleton to the ECM and specific integrin receptors can lead to differential EMT responses. For example, RGD-binding integrins such as αvβ3, αvβ5, αvβ6, and αvβ8 have been associated with induction of EMT through activation of cell contractile machinery and enhanced


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migration84. When studying laminin and fibronectin matrices using coated protein on polystyrene dishes, and their effect on EMT response, it was found that α6 integrin sequesters Rac1b from the cell membrane and is required to inhibit EMT by laminin82. On the other hand, α5 integrin maintains Rac1b at the cell membrane and is required to promote EMT by fibronectin82. In other studies, primary alveolar epithelial cells that are either deficient in their prominent laminin receptor, α3β1, or already have their integrins pre-engaged on laminin, have significantly reduced EMT responses in the presence of TGFβ85. Also, the α3β1 receptor coordinates SMAD signaling pathways as a function of extracellular cues, demonstrating the complex interactions between the extracellular environment and TGFβ signaling in regulating and controlling the epithelial cell response85. From a materials perspective, engineered ECM variants that display only a fraction of the native proteins, but direct specific integrin binding response, can be used to provide sufficient instruction for directing cell phenotype86. Using these variants represents a critical aspect of precise control and understanding of the role of integrin-specific binding on EMT outcomes. Such studies have shown that epithelial cell integrin binding can be controlled through engineered fibronectin fragments displaying a synergy (Pro-His-Ser-Arg-Asn) and RGD (ArgGly-Asp) sequence or RGD sequence alone87,88. If the synergy sequence is present along with the RGD sequence, epithelial cells bind to fibronectin via α3 and α5 integrins, and epithelial cells exhibit upregulation of E-cadherin and downregulation of α–SMA. However, if only the RGD sequence is present, epithelial cells will bind to fibronectin via αv integrins and will display a shift toward mesenchymal phenotype with downregulation of E-cadherin and upregulation of α– SMA. However, all cells expressed significant α–SMA when in the presence of TGFβ for both substrate compositions, showing TGFβ promotes EMT regardless of underlying ligand87.



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Overall, the fibronectin synergy sequence is shown to be important in regulating the EMT process for engineering material applications to guide cell phenotype. In the future, these variants could also be incorporated on materials or scaffolds to test strategies in controlling EMT responses for therapeutic interventions in fibrotic diseases and cancer. X.

Controlling EMT through substrate stiffness Increased tissue stiffness has been linked to increased EMT in numerous studies.

Therefore, modulating substrate stiffness presents a robust method to regulate the EMT process in regenerative medicine and tissue engineering applications. Additionally, modulating EMT in vitro using materials with finely controlled material mechanics affords scientists a platform for studying molecular mechanisms governing EMT. Polyacrylamide (PA) hydrogels, which are synthetic hydrogel matrices with a tunable stiffness, are widely used for such studies.

In

particular, studies culturing epithelial cells on PA gels have been instrumental in recent years in linking increased tissue stiffness to increased TGFβ activation and downstream EMT. The ability of increased tissue stiffness to modulate TGFβ activation was first demonstrated with mesenchymal cells where it was found that the efficiency of latent TGFβ activation by myofibroblasts increases with increasing ECM stiffness52. A similar process was subsequently revealed in epithelial cells whereby epithelial cells increasingly activate TGFβ on stiff surfaces, which was found to correlate with fibronectin-mediated EMT. These studies utilized alveolar epithelial cells cultured on PA gels coated in fibronectin, and it was found that stiff, but not soft, fibronectin substrates induce EMT51. This response is shown in Figure 4A and was found to also depend on cell contraction-mediated integrin activation of TGFβ51. In other words, there is increased epithelial cell contraction on stiff fibronectin surfaces that leads to integrin-mediated TGFβ activation and spontaneous EMT. These studies also demonstrated that EMT could be


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modulated by a combination of stiffness and soluble cues. In response to exogenously added TGFβ, cells are able to initiate EMT on soft substrates. Interestingly, these responses were found to be dose and time dependent. When cells were exposed to low levels of TGFβ on soft surfaces they underwent EMT, but upon removal of TGFβ from the culture media, cells were able to undergo MET and revert to an epithelial phenotype51. These results demonstrate the plasticity of the EMT response and highlight that matrix stiffness and/or cell contractility are critical targets for controlling EMT. Because matrix stiffness is a critical regulator of the EMT process it is an important parameter to consider for future therapeutic strategies that incorporate materials to modulate EMT. Stiffness-mediated EMT is dependent on integrin specific responses. Therefore, controlling both stiffness and integrin mediated binding represents an additional avenue for modulating EMT responses.

A study analyzing the combinatorial effects of ECM proteins,

specifically different mutants of fibronectin’s cell binding domain, and varying polyacrylamide (PA) gel substrate stiffness on EMT demonstrated that precise control of the cellular response can be obtained using combinatorial approaches65. Alveolar epithelial cells cultured on combinations of fibronectin mutants and varying substrate stiffness demonstrated that while stiff substrates induce spontaneous EMT, the response can be overcome with specific fibronectin components, specifically via fragments that promote α3 and α5 integrin engagement. Additionally, this study also identified that suppressing cell-contractility was sufficient to maintain an epithelial phenotype. This reveals a synergistic relationship between integrin specific binding and ECM mechanics in directing EMT responses. Matrix stiffness and TGFβ have likewise been linked to tumor-associated EMT by clustering integrins that enhances ROCK-generated contractility and focal adhesions39. In these



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studies, normal murine mammary gland epithelial cells and MDCK epithelial cells were cultured on PA gels with elastic modulus ranging from 0.4 to 60 kPa, and treated with TGFβ60. It was found that altering matrix stiffness switched the functional response to TGFβ; decreasing the matrix rigidity increased TGFβ induced apoptosis, while increasing substrate stiffness resulted in EMT responses60. While the matrix stiffness changes did not affect SMAD signaling, it did alter the PI3K signaling pathway such that direct genetic changes could be observed that either induced apoptosis or EMT. Collectively, these findings highlight the robustness of using substrate stiffness as a mechanism to control TGFβ signaling and EMT.

XI.

Controlling EMT through substrate patterning


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Substrate patterning is a useful tool to examine and control cellular processes under finely controlled environments89. For example, micropatterning has previously been utilized to examine organ development, specifically for the branching structure of mammary epithelial tubules, to show that tissue geometry can control organ morphogenesis90. Further studies of this branching pattern using microfabricated tubules to model the mammary duct have led to findings that characterize the transcription factors (Snail1, Snail2, and E47) that can promote branching through regulating EMT91. Epithelial cells reside in environments where topography and cellular confinement can vary, which, similarly to changes in stiffness, can affect activation of mechanotransduction signaling and enhance cell contractility. For example, the basement membrane is comprised of fibrillar ECM components which are often textured and can present cells with topographical cues. Under conditions in which the composition or conformation of matrix proteins are altered, such as in wound healing, topographical cues will likewise be altered. Additionally, cellular confinement can be altered through changes in ECM topography and/or cell-cell contacts. In the context of cell interactions with materials in vivo, cell confinement can occur through modulation of scaffold or matrix pore sizes; increasing degrees of cell confinement, induced by the fabrication of smaller pore sizes, has been shown to correlate with collagen deposition and alignment92. Given the strong link between mechanotransduction and increased cell contractility in EMT, it has been hypothesized that topography and cellular confinement could also influence EMT. Surfaces with defined geometrical parameters are a useful tool to understand and control EMT in response to topography and cellular confinement.

Indeed, recent studies have

demonstrated that surface topography and cellular confinement can lead to differential EMT responses compared to responses on bulk materials of identical moduli. In one study, PA



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hydrogels with varying levels of stiffness were fabricated to have patterns of defined topography and confinement, and the responses of mammary epithelial cell clusters were examined93. It was found that highly confined patterns induced an EMT response even if the cell clusters were on a soft matrix, which would otherwise protect against EMT93. Here, stiffness-induced EMT was linked to increased cell-matrix adhesions and confinement-induced EMT was linked to cytoskeleton polarization. These studies demonstrate that surface patterns can also be a useful technique in controlling EMT phenotypes. Separate studies have examined cell spreading as a requirement of MMP-3/Rac1b induced EMT62. In these studies, if cell spreading was limited by increasing cell density or by culturing cells on precisely defined micropatterned substrates of fibronectin islands, EMT marker expressions were reduced62. This observation however is not seen with TGFβ-induced EMT, demonstrating distinct differences in EMT-inducing agents. It should be noted that this study focused on regulation of vimentin expression as a function of cell shape. Additional studies by O’Connor et al, demonstrated that cell shape can also regulate the expression of additional mesenchymal markers during TGFβ-induced EMT, including α-SMA. Furthermore, a recent study on TGFβ-induced EMT also used micropatterned substrates of fibronectin islands to show that even when cell spreading was limited, a reduction to partial cellcell contacts can actually promote TGFβ-induced expression of α-SMA and can control the subcellular localization of Notch1 and MRTF-A.94. Other surface modification techniques have been utilized to investigate the effect of patterning on EMT. Spatial patterning of proteins on PA hydrogels fabricated with lines containing fibronectin, laminin, or collagen type I and varying in widths from 5-400nm95 demonstrated that differential cell attachment and proliferation of cells is obtained with these patterns. Using this strategy in combination with various ECM proteins can be employed to


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elucidate the underlying mechanisms of EMT in response to different ECM components18,95. Studies culturing epithelial cells on patterns presenting gradients of mechanical stress demonstrated altered EMT events at distinct spatial locations, which can be visualized in Figure 4B. For example, cells at the corners and edges of square mammary epithelial sheets express EMT markers when treated with TGFβ, whereas cell in the center of the square do not57. Traction force microscopy and finite element modeling demonstrated that areas marked by an EMT response correspond to areas with the highest mechanical stress, suggesting critical effects of tissue geometry and mechanical stress on the spatial patterning of EMT57. The results from this study also identify cell contractility as a critical target for novel EMT therapeutics because inhibiting cell contractility was able to reverse the EMT responses by reducing the levels of α– SMA expression, shown in Figure 4B, panel G. Micropatterned surfaces have also been utilized in studies that demonstrated that epithelial cell migration into interstitial tissues is driven by a reversal of cell polarity which results from centrosome displacement from the cell periphery to the cell center96. Recent studies performed by Lee et al. expand findings of the effects of geometric confinement on EMT to the promotion of stem cell fractions in cancer cells97. Their studies utilizing patterned geometries indicate that interfacial geometry modulates cell shape, enhances α5β1 adhesion, and promotes MAPK and STAT signaling to promote transformation of cancer stem cells. Interestingly, in these studies, tumorigenicity was maintained after cells were removed from the patterns and injected into mice. While these studies are focused on cancer phenotypes, this finding indicates that patterns of high interfacial boundaries can be used to induce an EMT program with the potential to persist even following removal from the pattern. Such an approach could be useful for programing cells for cell therapies. Taken together, the use



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of spatial patterning offers the ability to control EMT responses for many future material applications.

XII.

The impact of biochemical and mechanical cues on EndoMT and biomaterial strategies for controlling EndoMT

As mentioned earlier, EndoMT and EMT share some key characteristics, such as their importance in the developmental process22 and their presence in fibrotic diseases. Many of the molecular events governing EMT likewise govern EndoMT. In this section, we briefly highlight recent studies demonstrating the role of EndoMT in development and disease. Here we also highlight key drivers for EndoMT and provide a limited number of examples of material strategies for controlling EndoMT. Like EMT, TGFβ is known to be a potent inducer of EndoMT24,98–101. At the onset of EndoMT during chick embryo cardiogenesis, TGFβ-3 is expressed in the transforming endothelial cells and invading mesenchymal cells, illustrating TGFβ’s critical role during the initial phenotypic changes of EndoMT102. TGFβ-2 specifically has been found to induce the differentiation of mouse embryonic stem cell-derived endothelial cells into mural cells that display decreased expression in endothelial markers such as claudin-5 and increased expression of α-SMA98. The same study found that TGFβ type 1 receptor kinase inhibitor decreases EndoMT, and the transcription factor Snail, also involved in EMT, is induced during EndoMT, whereas knockdown of Snail expression inhibited TGFβ-2-induced EndoMT. Additionally, studies have been conducted that identify that EndoMT occurs in some fibrotic disorders, and that this cellular behavior could be a potential target for therapeutic


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applications for conditions including cardiac fibrosis, kidney fibrosis, and pulmonary fibrosis20,23,25,99,103. For example, it has been demonstrated that endothelial cells can serve as a source of fibroblasts in idiopathic pulmonary fibrosis; these studies found that activated Ras and TGFβ induced EndoMT in a bleomycin-induced lung fibrosis mouse model99. In studies geared towards preventing clinical vein graft stenosis by reducing EndoMT, TGFβ antagonism methods, including the use of a neutralizing antibody, short hairpin RNA-mediated Smad3 or Smad2 knockdown, Smad3 haploinsufficiency, and endothelial cell-specific Smad2 deletion,

were

shown to reduce EndoMT and neointimal formation24. To inhibit the action of TGFβ pathway to decrease EndoMT in renal fibrosis, a specific Smad3 inhibitor SIS3 has been used to abrogate EndoMT and slow the progression of nephropathy104. EndoMT is also inhibited by bone morphogenetic protein (BMP)25,102. In regard to cardiac fibrosis, recombinant human BMP-7 has been shown to significantly inhibit EndoMT and the progression of cardiac fibrosis in mouse models of pressure overload. Furthermore, BMP-7 significantly inhibited the propensity of adult human coronary endothelial cells to undergo EndoMT after TGFβ induction20. Results from this study propose hemodynamic stress as a trigger of pro-inflammatory process that could lead to the stimulation of endothelial cells to induce fibroblast activation and myocardial fibrosis. Investigations into the molecular mechanisms that govern EndoMT have shed light onto the some of the similar pathways activated in EndoMT and EMT including upregulation of snail and twist. For example, a study of mouse pulmonary endothelial cells elucidated intracellular transduction pathways in EndoMT following TGFβ treatment and found upregulation of Snail, independent of Smad2/3 activation, and upregulation of kinases such as c-abl protein kinase, protein kinase C, and GSK-3, which mirrors the crucial role of GSK-3 during EMT100. In another study using a rat model of pulmonary hypertension, genetically modified with BMPR2



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deficiency, EndoMT was linked to changes in BMPR2 signaling and was found to include twist overexpression and vimentin phosphorylation25. In the same study, rapamycin, an inhibitor of mTOR, partially reversed the protein expression pattern of EndoMT and improved experimental PAH by inducing VE-cadherin expression, inhibiting vimentin and twist-1 expression, and decreasing migration of primary PAECs.

Aberrant BMPR2 signaling has also been

demonstrated to contribute to EndoMT in patient lung samples with idiopathic PAH. In these studies it was determined that increased High Mobility Group AT-hook 1 (HMGA1) in pulmonary arterial endothelial cells, resulting from dysfunctional BMPR2 signaling, induced EndoMT and matched EndoMT responses from mice endothelial cells103. Elucidation of the intricacies of EndoMT is an emerging field, and less is known about strategies geared towards controlling the cellular behavior that contributes to EndoMT compared to EMT. Therefore, developing strategies to control the cellular behavior in applications of cardiac fibrosis, kidney fibrosis, pulmonary fibrosis, and other fibrotic pathologies that include EndoMT processes is an area primed for biomaterial scientists. One example of controlling EndoMT through material properties is demonstrated in studies by Zhang et al. in which the authors used a natural compound genipin to fine tune matrix stiffness to promote endothelial differentiation and higher levels of mature endothelial marker expression from human MSCs105. Specifically, these studies demonstrated that cells cultured on 1mM genipin crosslinked materials had more robust vWF expression after three days in cultured compared to uncrosslinked or 2mM crosslinked materials.

Future studies should focus on tailoring other aspects of material

characteristics to fine tune cellular behavior and EndoMT. Similar to the aforementioned presentation of growth factors on materials as a strategy to control EMT, presenting growth factors on substrates in a defined manner could be utilized to spatially control the transition of


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endothelial cells to mesenchymal cells for therapeutic applications. Specifically, disruption of FGF signaling has been shown to induce EndoMT through reductions in let-7 miRNA levels that increases activation of TGFβ signaling101. Therefore, modulating FGF signaling through growth factor presentation techniques on surfaces could begin to elucidate material strategies for spatially controlling EndoMT. XIII. Conclusion EMT is involved in a multitude of physiologically relevant events ranging from development to disease.

Biomaterials with finely controlled mechanical and biological

properties have provided much insight into the cellular processes governing EMT responses. This has been particularly true in the case of understanding the role of increasing tissue stiffness in fibrosis and cancer in initiating EMT programs in disease. Additionally, biomaterial strategies afford scientists and engineers the ability to control EMT responses. Though uncontrolled EMT responses are associated with fibrosis and cancer, EMT is an important process in development and wound healing. While several key drivers of EMT have been identified (i.e. TGFβ, stiff substrates, patterns which induce cellular stress), it is ultimately the combination of different instructional cues (adhesion, stiffness, soluble signaling, and geometry) that work together to specify final outcomes. Surface geometry sets up a context for cells to perceive substrate stiffness and substrate stiffness works in concert with specific adhesive and soluble cues through cell surface receptors. As an example, while increased substrate stiffness has been linked to increased EMT, such responses are ECM ligand specific. Studies with alveolar epithelial cells have demonstrated that stiff laminin surfaces lead to maintenance of epithelial phenotypes while stiff fibronectin surfaces strongly induce EMT. However, even within the framework of the finding that stiff fibronectin surfaces responses lead to EMT, specific cell surface receptors



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dictate the ultimate response to fibronectin. Fibronectin-driven EMT has been linked to αv integrin binding, whereas α5β1 or α3β1 integrin binding to fibronectin promotes more epithelial phenotypes, even on stiff surfaces. Such results highlight the high level of complexity of the microenvironmental cues that ultimately dictate morphogenetic processes like EMT. Biomaterials that present complex combinatorial environments of mechanics and soluble cues offer the potential to begin to address the many unanswered questions in EMT. For example, the factors that affect the degree of plasticity of EMT responses is not well understood. Materials with dynamic mechanical properties may in the future provide detailed insight into these complex questions. Finally, biomaterials presenting patterns of mechanical and soluble cues that promote or inhibit EMT could in the future be used for translational applications to promote EMT responses in a finely controlled manner to direct differentiation responses and/or improve wound healing outcomes. Acknowledgements Funding sources: AHA Scientist Development Grant (16SDG29870005), North Carolina State University Faculty Research and Professional Development Program References (1) (2)

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Table of Contents Graphic Material strategies for modulating epithelial to mesenchymal transitions Emily P. Mihalko and Ashley C. Brown


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Material Strategies for Modulating Epithelial to Mesenchymal Transitions

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