Extracellular Matrix Ligands Research Articles

Photoactivatable substrates for systematic study of the impact of an extracellular matrix ligand on appearance of leader cells in collective cell migration

Epithelial cells migrate as multicellular units. The directionality and speed of these units are determined by actively moving leader cells. It is important to understand how external cues affect the appearance of these leader cells in physiological and pathological processes. However, the impact of extracellular matrices (ECMs) is still controversial, because physically-adsorbed ECM proteins are amenable to protein remodeling, and uncontrolled cluster geometry can vary migration phenotypes. Here, we demonstrate a photoactivatable substrate, which we used to study the impact of a cyclic Arg-Gly-Asp (cRGD) ligand on leader cell formation in MDCK cells. This robust platform allowed us to investigate the effect of cRGD density on leader cell formation, in any given cluster geometry, with minimized ECM remodeling. Our results show a biphasic response of leader cell appearance upon reducing the surface cRGD density. The increase, in leader cell appearance, within the higher density range, is not only associated with the weakening of circumferential actomyosin belts, but also reduction of cellular mechanical tension and intercellular junctional E-cadherin. These results indicate that cRGD-mediated cell-ECM interactions positively regulate mechanical and biochemical coupling within cell clusters; both are critical for the coordination of cell collectives and eventual reduction in the appearance of leader cells.

Resident murine macrophage migration and phagocytosis are modulated by growth hormone.

Growth hormone (GH) plays a physiological role in the immune system. In macrophages, GH enhances the production of hydrogen peroxide, superoxide anions, nitric oxide, cytokines, and chemokines, including interferon-γ and macrophage inflammatory protein-1α. However, some of the effects of GH stimulation on the biological functions of macrophages remain to be elucidated. Herein, we showed that in vivo GH treatment resulted in decreased expression of VLA-5 and VLA-6 integrins on the macrophage surface, accompanied by a reduction in macrophage adhesion to extracellular matrix (ECM) ligands, fibronectin, and laminin. Additionally, a decrease in macrophage adhesion to laminin was observed when the cells were treated in vitro with GH. In transwell migration assays, GH-treated macrophages showed increased migration after 6 h. Although in vitro GH treatment did not influence the phagocytic activity of macrophages, when the treatment was performed in vivo, peritoneal macrophages from GH-treated mice showed a higher percentage of phagocytosis and higher phagocytic capacity than cells from control animals. These results led us to analyse the role of insulin-like growth factor-1 (IGF-1), a GH stimulated factor, on macrophage phagocytosis. We observed an increase in phagocytic activity when J774 murine macrophages were treated with IGF-1 for 24 h. Our results revealed an important role for GH in resident macrophage migration and phagocytic activity. Specifically, we demonstrate that IGF-1 may be the GH stimulated factor that induces macrophage phagocytosis in vivo.

Abstract TMP28: Inhibition of Integrin α 5 β 1 With the Small Peptide Atn-161 Reduces Infarct Volume and Improves Functional Recovery Through Reduction of Blood-brain Barrier Permeability

Reperfusion-induced injury after recanalization is exacerbated by blood-brain barrier (BBB) dysfunction after ischemic stroke. Endothelial cell integrin receptors, specifically the β 1 subtype, play a direct role in BBB dysfunction through regulation of barrier-forming tight junction (TJ) proteins. We hypothesize that inhibition of an endothelial specific β 1 integrin, α 5 β 1 , after oxygen-glucose deprivation (OGD) in vitro and also after experimental stroke in vivo will stabilize the BBB through the TJ claudin-5, reducing infarct volumes and improving functional recovery. First, stroke was simulated in vitro by OGD or TNF- α treatment of brain endothelial cell monolayers. Treatment with the small peptide α 5 β 1 inhibitor ATN-161 (10 μM) resulted in decreased barrier permeability as measured by trans-endothelial cell electrical resistance and FITC-dextran permeability. Immunocytochemistry showed extracellular localization and upregulation of claudin-5 with ATN-161 treatment. Next, transient tandem middle cerebral artery occlusion was performed on 12-week-old male mice for 1 hour. On confocal and SEM imaging, post-stroke α 5 β 1 expression was upregulated primarily in the luminal vascular surface by post-stroke day (PSD) 2 rather than at the basement membrane (next to its extracellular matrix ligands). IP injection of ATN-161 (1 mg/kg) immediately after reperfusion and on PSD 1, and PSD2 caused a significant, but transient and acute (after the initial dose only) change in animal pulse distension, but no change in body temperature or heart rate. Infarct volumes, measured by TTC stain, were significantly reduced in ATN-161 treated animals. Furthermore, BBB integrity was improved by ATN-161 as determined by IgG staining and FITC-Dextran injection. Functional recovery, assessed by a 5-item Neuroscore, was also significantly improved. In conclusion, inhibition of α 5 β 1 by ATN-161 was well tolerated, reduced infarct volume, and improved behavioral recovery. Additional studies showed this result was likely due to reduced BBB permeability through the TJ claudin-5. Therefore, inhibition of α 5 β 1 with ATN-161 could represent a novel therapeutic approach for stroke worthy of further investigation.

Preparation of Matrices of Variable Stiffness for the Study of Mechanotransduction in Schwann Cell Development.

Extracellular matrix (ECM) elasticity may direct cellular differentiation and can be modeled in vitro using synthetic ECM-like substrates with defined elastic properties. However, the effectiveness of such approaches depends on the selection of a range of elasticity and ECM ligands that accurately model the relevant tissue. Here, we present a cell culture system than can be used to study Schwann cell differentiation on substrates which model the changes in mechanical ECM properties that occur during sciatic nerve development.

Force loading explains spatial sensing of ligands by cells.

Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin-ligand bonds are separated by more than a few tens of nanometres. It has thus been suggested that a crosslinking 'adaptor' protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model, in which individual integrin-ECM bonds-the molecular clutches-respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.

Extracellular Matrix Induction of Intracellular Reactive Oxygen Species.

The extracellular matrix (ECM) is the noncellular component secreted by cells and is present within all tissues and organs. The ECM provides the structural support required for tissue integrity and also contributes to diseases, including cancer. Many diseases rich in ECM are characterized by changes in reactive oxygen species (ROS) levels that have been shown to have important context-dependent functions. Recent Advances: Many studies have found that the ECM affects ROS production through integrins. The activation of integrins by ECM ligands results in stimulation of multiple pathways that can generate ROS. Furthermore, control of ECM-integrin interaction by matricellular proteins is an underappreciated pathway that functions as an ROS rheostat in remodeling tissues. A better understanding of how the ECM affects the generation of intracellular ROS is required for advances in the development of therapeutic strategies that affect or exploit oxidative stress. Targeting ROS generation can be therapeutic or can promote disease progression in a context-dependent manner. Many ECM proteins can impact ROS generation. However, given the breadth of different proteins that constitute the ECM and the cell surface receptors that interact with ECM proteins, there are likely many tissue and microenvironmental-specific ROS-generating pathways that have yet to be investigated in depth. Identifying canonical pathways of ECM-induced ROS generation should be a priority for the field. Antioxid. Redox Signal. 27, 774-784.

Modeling the Kinetics of Integrin Receptor Binding to Hepatic Extracellular Matrix Proteins

The composition of the extracellular matrix (ECM) proteins and the expression of their cognate receptors dictate cell behavior and dynamics. In particular, the interactions of ECM proteins with integrin receptors are key mediators of these cellular processes, playing a crucial role in the progression of several diseases of the liver, including inflammation, fibrosis/cirrhosis and cancer. This study establishes a modeling approach combining computation and experiments to evaluate the kinetics of integrin receptor binding to hepatic ECM proteins. ECM ligand concentration was derived from LC-MS/MS quantification of the hepatic ECM from mice exposed to chronic carbon tetrachloride (CCl4); receptor density was derived from published literature. Mathematical models for ECM-integrin binding kinetics that were developed incorporate receptor divalence and an aggregation scheme to represent clustering. The computer simulations reproduced positive cooperativity in the receptor aggregation model when the aggregation equilibrium constant (Ka) was positive and greater than Keq for divalent complex formation. Importantly, the modeling projected an increase in integrin binding for several receptors for which signaling is known to be increased after CCl4 exposure in the liver. The proposed modeling approach may be of use to elucidate the kinetics of integrin receptor binding to ECM proteins for homeostatic and diseased livers.

Fra-2 negatively regulates postnatal alveolar septation by modulating myofibroblast function.

Mice that globally overexpress the transcription factor Fos-related antigen-2 (Fra-2) develop extensive pulmonary fibrosis and pulmonary vascular remodeling. To determine if these phenotypes are a consequence of ectopic Fra-2 expression in vascular smooth muscle cells and myofibroblasts, we generated mice that overexpress Fra-2 specifically in these cell types (α-SMA-rtTA;tetO-Fra-2). Surprisingly, these mice did not develop vascular remodeling or pulmonary fibrosis but did develop a spontaneous emphysema-like phenotype characterized by alveolar enlargement. Secondary septa formation is an important step in the normal development of lung alveoli, and α-smooth muscle actin (SMA)-expressing fibroblasts (myofibroblasts) play a crucial role in this process. The mutant mice showed reduced numbers of secondary septa at postnatal day 7 and enlarged alveolae starting at postnatal day 12, suggesting impairment of secondary septa formation. Lineage tracing using α-SMA-rtTA mice crossed to a floxed TdTomato reporter revealed that embryonic expression of α-SMA Cre marked a population of cells that gave rise to nearly all alveolar myofibroblasts. Comprehensive transcriptome analyses (RNA sequencing) demonstrated that the overwhelming majority of genes whose expression was significantly altered by overexpression of Fra-2 in myofibroblasts encoded secreted proteins, components of the extracellular matrix (ECM), and cell adhesion-associated genes, including coordinate upregulation of pairs of integrins and their principal ECM ligands. In addition, primary myofibroblasts isolated from the mutant mice showed reduced migration capacity. These findings suggest that Fra-2 overexpression might impair myofibroblast functions crucial for secondary septation, such as myofibroblast migration across alveoli, by perturbing interactions between integrins and locally produced components of the ECM.

Tug of War at the Cell-Matrix Interface

Mechanical interactions between a cell and its environment, or between cells, influence key developmental and physiologic processes as well as many aspect of disease (1,2). Indeed the ability of a cell to sense, produce, and respond to mechanical cues has emerged as a fundamental regulator of cell behaviors such as differentiation, proliferation, survival, and migration. In mechanical terms the interactions governing these behaviors are regulated by intracellular and extracellular physical events that are orchestrated by complex biochemical and mechanical signals.

Mechanical Strain Promotes Oligodendrocyte Differentiation by Global Changes of Gene Expression.

Differentiation of oligodendrocyte progenitor cells (OPC) to oligodendrocytes and subsequent axon myelination are critical steps in vertebrate central nervous system (CNS) development and regeneration. Growing evidence supports the significance of mechanical factors in oligodendrocyte biology. Here, we explore the effect of mechanical strains within physiological range on OPC proliferation and differentiation, and strain-associated changes in chromatin structure, epigenetics, and gene expression. Sustained tensile strain of 10–15% inhibited OPC proliferation and promoted differentiation into oligodendrocytes. This response to strain required specific interactions of OPCs with extracellular matrix ligands. Applied strain induced changes in nuclear shape, chromatin organization, and resulted in enhanced histone deacetylation, consistent with increased oligodendrocyte differentiation. This response was concurrent with increased mRNA levels of the epigenetic modifier histone deacetylase Hdac11. Inhibition of HDAC proteins eliminated the strain-mediated increase of OPC differentiation, demonstrating a role of HDACs in mechanotransduction of strain to chromatin. RNA sequencing revealed global changes in gene expression associated with strain. Specifically, expression of multiple genes associated with oligodendrocyte differentiation and axon-oligodendrocyte interactions was increased, including cell surface ligands (Ncam, ephrins), cyto- and nucleo-skeleton genes (Fyn, actinins, myosin, nesprin, Sun1), transcription factors (Sox10, Zfp191, Nkx2.2), and myelin genes (Cnp, Plp, Mag). These findings show how mechanical strain can be transmitted to the nucleus to promote oligodendrocyte differentiation, and identify the global landscape of signaling pathways involved in mechanotransduction. These data provide a source of potential new therapeutic avenues to enhance OPC differentiation in vivo.

Static Compression Induces ECM Remodeling and Integrin α2β1 Expression and Signaling in a Rat Tail Caudal Intervertebral Disc Degeneration Model

A three-level rat tail caudal intervertebral disc (IVD) degeneration (IVDD) model was established to study effects of static compression on extracellular matrix (ECM) remodeling and integrin signaling in IVDs during IVDD. The aim of this study was to investigate the effect of compression force on ECM remodeling and integrin signaling in IVDs during IVDD. Integrins sense mechanical environment alteration via binding to ECM ligands and trigger intracellular signaling for pathological ECM remodeling during IVDD. However, the role of compression force in ECM remodeling and integrin signaling during IVDD remains elusive. Compared with the classical one-level rat tail IVDD model that exerts axial stress on the 8th to 9th caudal vertebral bodies, a three-level model was established by using an Ilizarov-type apparatus to exert stress on the 7th to 10th caudal vertebral bodies in rat tails for four weeks. To exclude side effects from surgical stab injury on manipulated discs, intact coccygeal (Co) disc Co8-9 was analyzed. In three-level IVDD model, significant degeneration of the Co8-9 disc was observed. Quantitative real-time polymerase chain reaction (qRT-PCR) showed elevated mRNA expression of collagen types I, III, and V; matrix metalloproteinases (MMPs) 2, 3, 9, 13, 14; and decreased mRNA expression of collagen type II in Co8-9 disc. Compression loading altered the expression of integrin α2β1 (upregulated) and α10β1 (downregulated) in NP cells, and activated integrin downstream signaling. By contrast, one-level model showed more severe disc degeneration and ECM remodeling. Integrin α1, α2, α11, and β1 were upregulated, whereas α10 was downregulated. Similar activation of integrin signaling was observed. Static compression altered collagen and MMP expression, and promoted β1 integrin expression and signaling in IVD. Compared with one-level rat tail IVDD model, three-level model showed milder effects on disc degeneration, ECM remodeling, and integrin expression, suggesting one-level model might involve other causes that induce IVDD via mechanisms independent of compression force. N/A.

CD44: A Multifunctional Cell Surface Adhesion Receptor Is a Regulator of Progression and Metastasis of Cancer Cells.

CD44 is a cell surface adhesion receptor that is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface. Its interaction with appropriate extracellular matrix ligands promotes the migration and invasion processes involved in metastases. It was originally identified as a receptor for hyaluronan or hyaluronic acid and later to several other ligands including, osteopontin (OPN), collagens, and matrix metalloproteinases. CD44 has also been identified as a marker for stem cells of several types. Beside standard CD44 (sCD44), variant (vCD44) isoforms of CD44 have been shown to be created by alternate splicing of the mRNA in several cancer. Addition of new exons into the extracellular domain near the transmembrane of sCD44 increases the tendency for expressing larger size vCD44 isoforms. Expression of certain vCD44 isoforms was linked with progression and metastasis of cancer cells as well as patient prognosis. The expression of CD44 isoforms can be correlated with tumor subtypes and be a marker of cancer stem cells. CD44 cleavage, shedding, and elevated levels of soluble CD44 in the serum of patients is a marker of tumor burden and metastasis in several cancers including colon and gastric cancer. Recent observations have shown that CD44 intracellular domain (CD44-ICD) is related to the metastatic potential of breast cancer cells. However, the underlying mechanisms need further elucidation.

Binding of the chemokine CXCL12α to its natural extracellular matrix ligand heparan sulfate enables myoblast adhesion and facilitates cell motility.

The chemokine CXCL12α is a potent chemoattractant that guides the migration of muscle precursor cells (myoblasts) during myogenesis and muscle regeneration. To study how the molecular presentation of chemokines influences myoblast adhesion and motility, we designed multifunctional biomimetic surfaces as a tuneable signalling platform that enabled the response of myoblasts to selected extracellular cues to be studied in a well-defined environment. Using this platform, we demonstrate that CXCL12α, when presented by its natural extracellular matrix ligand heparan sulfate (HS), enables the adhesion and spreading of myoblasts and facilitates their active migration. In contrast, myoblasts also adhered and spread on CXCL12α that was quasi-irreversibly surface-bound in the absence of HS, but were essentially immotile. Moreover, co-presentation of the cyclic RGD peptide as integrin ligand along with HS-bound CXCL12α led to enhanced spreading and motility, in a way that indicates cooperation between CXCR4 (the CXCL12α receptor) and integrins (the RGD receptors). Our findings reveal the critical role of HS in CXCL12α induced myoblast adhesion and migration. The biomimetic surfaces developed here hold promise for mechanistic studies of cellular responses to different presentations of biomolecules. They may be broadly applicable for dissecting the signalling pathways underlying receptor cross-talks, and thus may guide the development of novel biomaterials that promote highly specific cellular responses.

Characterization of the Phospho-Adhesome by Mass Spectrometry-Based Proteomics.

Integrin adhesion receptors engage with their extracellular matrix (ECM) ligands, initiating intracellular signaling pathways that regulate a range of fundamental cell functions. Protein kinases and phosphatases play an integral role in integrin adhesion-mediated signaling. However, until recently, knowledge of the phosphorylation sites regulated downstream of integrin ligation was limited to candidate-based approaches and did not support a system-level understanding of the molecular mechanisms through which ECM engagement influences cell behavior. Here, we describe a mass spectrometry (MS)-based phosphoproteomic protocol that enables the global characterization of phosphorylation-based signaling networks activated by integrin-mediated adhesion. To analyze specifically integrin-proximal signaling, the phosphoproteomic workflow involves the affinity-based isolation and analysis of integrin-associated complexes (IACs) rather than proteins solubilized from whole-cell lysates , which are typically used for global phosphoproteomic studies. The detection of phosphorylation sites from IAC proteins was optimized at various stages of the workflow, including IAC isolation, proteolytic digestion, and MS-based data acquisition strategies. The protocol permits the identification and quantification of IAC components by both Western blotting and MS. Notably, compared to phosphoproteomic analyses of cell lysates, the workflow described here enables an improved detection of phosphorylation sites from well-defined IAC proteins, including many known components of the signaling pathways activated by adhesion to the ECM.

The Muscular Dystrophy Gene TMEM5 Encodes a Ribitol β1,4-Xylosyltransferase Required for the Functional Glycosylation of Dystroglycan

A defect in O-mannosyl glycan is the cause of α-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant α-dystroglycan (α-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAβ1-3Xylα1]n-3GlcAβ1-4Xyl-Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4 (phospho-6)Manα1-, which is required for the binding of α-DG to extracellular matrix ligands, has been proposed. However, the linkage of the first Xyl residue to ribitol 5-phosphate (Rbo5P) is not clear. TMEM5 is a gene product responsible for α-dystroglycanopathy and was reported as a potential enzyme involved in this linkage formation, although the experimental evidence is still incomplete. Here, we report that TMEM5 is a xylosyltransferase that forms the Xylβ1-4Rbo5P linkage on O-mannosyl glycan. The anomeric configuration and linkage position of the product (β1,4 linkage) was determined by NMR analysis. The introduction of two missense mutations in TMEM5 found in α-dystroglycanopathy patients impaired xylosyltransferase activity. Furthermore, the disruption of the TMEM5 gene by CRISPR/Cas9 abrogated the elongation of the (-3GlcAβ1-3Xylα1-) unit on O-mannosyl glycan. Based on these results, we concluded that TMEM5 acts as a UDP-d-xylose:ribitol-5-phosphate β1,4-xylosyltransferase in the biosynthetic pathway of O-mannosyl glycan.

Abstract 302: ECM/Integrin signaling promotes resistance to the combination of HER2 and PI3K inhibitors in HER2+, PIK3CA-mutant breast cancer

Abstract HER2 amplification and activating mutations in PIK3CA, the gene encoding the p110α subunit of PI3K, often co-occur in breast cancer. We generated a transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. In these mice, PIK3CAH1047R accelerates HER2-mediated tumor formation and promotes resistance to HER2 inhibitors (Hanker et al. PNAS 2013). HER2+/PIK3CA tumor growth was inhibited by treatment with the HER2 antibodies trastuzumab and pertuzumab in combination with the pan-PI3K inhibitor BKM120 (TPB). We sought to discover mechanisms of acquired resistance to the triple therapy by long-term treatment of established HER2+/PIK3CA tumors. Tumor transplants derived from a transgenic HER2+/PIK3CA tumor were initially growth inhibited by TPB. After several weeks, a subset of transplants (3/11) resumed growth in the presence of continuous TPB therapy. TPB-resistant tumors were cross-resistant to the combination of T + P + BYL719 (a p110α-specific inhibitor). Whole exome sequencing did not identify acquired somatic alterations in TPB-resistant tumors, including in HER2. However, RNA-seq revealed significant transcriptional upregulation of extracellular matrix (ECM) genes and genes involved in cell adhesion, including collagens, tenascins, and thrombospondins. Likewise, trichrome staining revealed a significant increase in collagen fibers and IHC analysis confirmed increased Tenascin expression in the TPB-resistant tumor stroma. In addition, western blot analysis revealed increased expression of an activated form of integrin β1, a substrate for ECM ligands such as collagen, as well as P-SrcY416 (activated by integrins/focal adhesion). We also found that transcription of many of these genes is induced by short-term TPB treatment in human breast cancer cell lines by qRT-PCR. Interestingly, primary tumor cells derived from TPB-resistant tumors no longer displayed resistance when grown in vitro. These cells regained TPB resistance when re-introduced into mice. Plating primary tumor cells on growth factor-reduced Matrigel or on Collagen I-coated plates restored resistance, suggesting that the ECM directly promotes TPB resistance. We are currently investigating whether inhibition of Integrin/Src signaling reverses TPB resistance. We are also exploring whether components of the ECM are altered in residual disease specimens from HER2+ breast cancer patients treated with neoadjuvant anti-HER2 therapies. Our data suggest that upregulation of ECM/integrin/Src signaling contributes to resistance to combinations of HER2 and PI3K inhibitors, and strongly support the growing body of literature indicating that components of the tumor microenvironment promote resistance to targeted therapies. Citation Format: Ariella B. Hanker, Monica Valeria Estrada, Junfei Zhao, Feixiong Cheng, Preston D. Moore, Darren Tyson, Violeta Sanchez, Brent N. Rexer, Melinda Sanders, Zhongming Zhao, Thomas P. Stricker, Carlos L. Arteaga. ECM/Integrin signaling promotes resistance to the combination of HER2 and PI3K inhibitors in HER2+, PIK3CA-mutant breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 302.

Reduced adhesive ligand density in engineered extracellular matrices induces an epithelial-mesenchymal-like transition

A synergistic effect of biochemical and mechanical cues emanating from the extracellular matrix (ECM) on inducing malignant transformation of epithelial cells has been observed recently. However, the effect of quantitative changes in biochemical stimuli on cell phenotype, without changes in ECM component and rigidity, remains unknown. To determine this effect, we grew Madin-Darby canine kidney epithelial cells (MDCK) on gold surfaces immobilized with varying densities of cyclic arginine-glycine-aspartate (cRGD) peptide and analyzed cell morphology, cell migration, cytoskeletal organization, and protein expression. Cells grown on a surface presenting a higher density of cRGD displayed an epithelial morphology and grew in clusters, while those grown on a diluted cRGD surface transformed into an elongated, fibroblast-like form with extensive scattering. Time-lapse imaging of cell clusters grown on the concentrated cRGD surface revealed collective migration with intact cell-cell contacts accompanied by the development of cortical actin. In contrast, cells migrated individually and formed stress fibers on the substrate with sparse cRGD. These data point towards transdifferentiation of epithelial cells to mesenchymal-like cells when plated on a diluted cRGD surface. Supporting this hypothesis, immunofluorescence microscopy and western blot analysis revealed increased membrane localization and total expression of N-cadherin and vimentin in cells undergoing mesenchymal-like transition. Taken together, these results suggest a possible role of decreased biochemical stimuli from the ECM in regulating epithelial phenotype switching. Statement of SignificanceEpithelial-mesenchymal transition (EMT) is a process where adherent epithelial cells convert into individual migratory mesenchymal phenotype. It plays an important role both in physiological and pathological processes. Recent studies demonstrate that the program is not only governed by soluble factors and gene expressions, but also modulated by biochemical and mechanical cues in ECMs. In this study, we developed chemically defined surfaces presenting controlled ECM ligand densities and studied their impact on the EMT progression. Morphological and biochemical analyses of epithelial cells cultured on the surfaces indicate the cells undergo an EMT-like transition on the diluted cRGD surface while retaining epithelial characteristics on the cRGD-rich substrate, suggesting an important role of the ECM ligand density in epithelial phenotype switching.

Surface-Driven Collagen Self-Assembly Affects Early Osteogenic Stem Cell Signaling.

This study reports how extracellular matrix (ECM) ligand self-assembly on biomaterial surfaces and the resulting nanoscale architecture can drive stem cell behavior. To isolate the biological effects of surface wettability on protein deposition, folding, and ligand activity, a polydimethylsiloxane (PDMS)-based platform was developed and characterized with the ability to tune wettability of elastomeric substrates with otherwise equivalent topology, ligand loading, and mechanical properties. Using this platform, markedly different assembly of covalently bound type I collagen monomers was observed depending on wettability, with hydrophobic substrates yielding a relatively rough layer of collagen aggregates compared to a smooth collagen layer on more hydrophilic substrates. Cellular and molecular investigations with human bone marrow stromal cells revealed higher osteogenic differentiation and upregulation of focal adhesion-related components on the resulting smooth collagen layer coated substrates. The initial collagen assembly driven by the PDMS surface directly affected α1β1 integrin/discoidin domain receptor 1 signaling, activation of the extracellular signal-regulated kinase/mitogen activated protein kinase pathway, and ultimately markers of osteogenic stem cell differentiation. We demonstrate for the first time that surface-driven ligand assembly on material surfaces, even on materials with otherwise identical starting topographies and mechanical properties, can dominate the biomaterial surface-driven cell response.

3D extracellular matrix interactions modulate tumour cell growth, invasion and angiogenesis in engineered tumour microenvironments

Interactions between tumour cells and extracellular matrix proteins of the tumour microenvironment play crucial roles in cancer progression. So far, however, there are only a few experimental platforms available that allow us to study these interactions systematically in a mechanically defined three-dimensional (3D) context. Here, we have studied the effect of integrin binding motifs found within common extracellular matrix (ECM) proteins on 3D breast (MCF-7) and prostate (PC-3, LNCaP) cancer cell cultures, and co-cultures with endothelial and mesenchymal stromal cells. For this purpose, matrix metalloproteinase-degradable biohybrid poly(ethylene) glycol-heparin hydrogels were decorated with the peptide motifs RGD, GFOGER (collagen I), or IKVAV (laminin-111). Over 14days, cancer spheroids of 100–200μm formed. While the morphology of poorly invasive MCF-7 and LNCaP cells was not modulated by any of the peptide motifs, the aggressive PC-3 cells exhibited an invasive morphology when cultured in hydrogels comprising IKVAV and GFOGER motifs compared to RGD motifs or nonfunctionalised controls. PC-3 (but not MCF-7 and LNCaP) cell growth and endothelial cell infiltration were also significantly enhanced in IKVAV and GFOGER presenting gels. Taken together, we have established a 3D culture model that allows for dissecting the effect of biochemical cues on processes relevant to early cancer progression. These findings provide a basis for more mechanistic studies that may further advance our understanding of how ECM modulates cancer cell invasion and how to ultimately interfere with this process. Statement of SignificanceThreedimensional in vitro cancer models have generated great interest over the past decade. However, most models are not suitable to systematically study the effects of environmental cues on cancer development and progression. To overcome this limitation, we have developed an innovative hydrogel platform to study the interactions between breast and prostate cancer cells and extracellular matrix ligands relevant to the tumour microenvironment. Our results show that hydrogels with laminin- and collagen-derived adhesive peptides induce a malignant phenotype in a cell-line specific manner. Thus, we have identified a method to control the incorporation of biochemical cues within a three dimensional culture model and anticipate that it will help us in better understanding the effects of the tumour microenvironment on cancer progression.

Promising Biomarkers of Thyroid Cancer: BRAF V600E Mutation, miRNAs, Integrins, and Their Extracellular Matrix Ligands

Promising Biomarkers of Thyroid Cancer: BRAF V600E Mutation, miRNAs, Integrins, and Their Extracellular Matrix Ligands