A microtubule stability switch alters isolated vascular smooth muscle calcium flux in response to matrix rigidity.

During ageing, the extracellular matrix of the aortic wall becomes more rigid. In response, VSMCs generate enhanced contractile forces. Our previous findings demonstrate that VSMC volume is enhanced in response to increased matrix rigidity, but our understanding of mechanisms regulating this process remain incomplete. In this current study, we show that microtubule stability in VSMCs is reduced in response to enhanced matrix rigidity via piezo1-mediated Ca2+ influx. Moreover, VSMC volume and Ca2+ flux was regulated by microtubule dynamics; microtubule stabilising agents reduced both VSMC volume and Ca2+ flux on rigid hydrogels, whereas microtubule destabilising agents increased VSMC volume and Ca2+ flux on pliable hydrogels. Finally, we show that disruption of the microtubule deacetylase HDAC6 uncoupled these processes and increased K40 alpha tubulin acetylation, VSMC volume and Ca2+ flux on pliable hydrogels, but did not alter VSMC microtubule stability. These findings uncover a microtubule stability switch that controls VSMC volume by regulating Ca2+ flux. Together, these data demonstrate that manipulation of microtubule stability can modify VSMC response to matrix stiffness.

Formation of an invasion-permissive matrix requires TGFß/SNAIL1-regulated alternative splicing of fibronectin.

BACKGROUND: As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFß, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFß target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses. METHODS: We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis. RESULTS: In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFß-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix  that does not sustain TGFß-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis. CONCLUSIONS: Our results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.

Molecular clutch drives cell response to surface viscosity.

Cell response to matrix rigidity has been explained by the mechanical properties of the actin-talin-integrin-fibronectin clutch. Here the molecular clutch model is extended to account for cell interactions with purely viscous surfaces (i.e., without an elastic component). Supported lipid bilayers present an idealized and controllable system through which to study this concept. Using lipids of different diffusion coefficients, the mobility (i.e., surface viscosity) of the presented ligands (in this case RGD) was altered by an order of magnitude. Cell size and cytoskeletal organization were proportional to viscosity. Furthermore, there was a higher number of focal adhesions and a higher phosphorylation of FAK on less-mobile (more-viscous) surfaces. Actin retrograde flow, an indicator of the force exerted on surfaces, was also seen to be faster on more mobile surfaces. This has consequential effects on downstream molecules; the mechanosensitive YAP protein localized to the nucleus more on less-mobile (more-viscous) surfaces and differentiation of myoblast cells was enhanced on higher viscosity. This behavior was explained within the framework of the molecular clutch model, with lower viscosity leading to a low force loading rate, preventing the exposure of mechanosensitive proteins, and with a higher viscosity causing a higher force loading rate exposing these sites, activating downstream pathways. Consequently, the understanding of how viscosity (regardless of matrix stiffness) influences cell response adds a further tool to engineer materials that control cell behavior.

Stromal Tissue Rigidity Promotes Mesenchymal Stem Cell-Mediated Corneal Wound Healing Through the Transforming Growth Factor ß Signaling Pathway.

The healing of a corneal epithelial defect is essential for preventing infectious corneal ulcers and subsequent blindness. We previously demonstrated that mesenchymal stem cells (MSCs) in the corneal stroma, through a paracrine mechanism, yield a more favorable therapeutic benefit for corneal wound re-epithelialization than do MSCs in the corneal epithelium. In this study, MSCs were grown on a matrix with the rigidity of the physiological human vitreous (1 kPa), corneal epithelium (8 kPa), or corneal stroma (25 kPa) for investigating the role of corneal tissue rigidity in MSC functions regarding re-epithelialization promotion. MSC growth on a 25-kPa dish significantly promoted the wound healing of human corneal epithelial (HCE-T) cells. Among growth factors contributing to corneal epithelial wound healing, corneal stromal rigidity selectively enhanced transforming growth factor-beta (TGF-ß) secretion from MSCs. Inhibitors of TGF-ß pan receptor, TGF-ß receptor 1, and Smad2 dose dependently abrogated MSC-mediated HCE-T wound healing. Furthermore, MSCs growth on a matrix with corneal stromal rigidity enhanced the ability of themselves to promote corneal re-epithelialization by activating matrix metalloproteinase (MMP) expression and integrin ß1 production in HCE-T cells through TGF-ß signaling pathway activation. Smad2 activation resulted in the upregulation of MMP-2 and -13 expression in HCE-T cells, whereas integrin ß1 production favored a Smad2-independent TGF-ß pathway. Altogether, we conclude that corneal stromal rigidity is a critical factor for MSC-induced promotion of corneal re-epithelialization. The activation of the TGF-ß signaling pathway, which maintains the balance between integrin and MMP expression, in HCE-T cells is the major pathway responsible for MSC-mediated wound healing. Stem Cells 2016;34:2525-2535.

Effect of matrix rigidity on organ-specific capture of tumor cells by flow.

Accumulating evidences have suggested that tumor metastasis exists prominent organ discrepancy. In this progression, the capture of intravascular tumor cells (TCs) to endothelium in distant tissues and organs plays a decisive role in the organ-specific metastasis formation. However, the mechanism of tumor cells preferentially arrest and adhere to endothelial cells (ECs) of target organ still remains elusive. By using the parallel plate flow chamber and the polyacrylamide gels with different matrix stiffness, we here explored the combined effects of matrix rigidity, shear stress, and chemokine SDF-1 on the capture of circulating tumor cells to ECs in the bloodstream. In addition, the expression and the role of integrin ß1 on the tumor cells surface were also detected by SDF-1 treatment. The results show that breast tumor cells MDA-MB-231 display an increasing number of adherent cells on the preferred substrate, which is similar to the matrix rigidity of breast cancer tissue (about 5kPa), under a certain shear stress. Moreover, ECs exacerbates the preferred capture of tumor cells compared with the FN-coated substrate alone. Besides, SDF-1 upregulates the number of adherent tumor cells by responding to matrix stiffness via promoting the expression of integrin ß1, which is abolished by blocking of integrin ß1. These results may provide a novel point of view for the mechanism of "organ specificity" phenomenon in tumor metastasis, which in turn contribute to a rational development of new drugs for cancer.

Dysregulated Collagen Homeostasis by Matrix Stiffening and TGF-ß1 in Fibroblasts from Idiopathic Pulmonary Fibrosis Patients: Role of FAK/Akt.

Idiopathic pulmonary fibrosis (IPF) is an aggressive disease in which normal lung parenchyma is replaced by a stiff dysfunctional scar rich in activated fibroblasts and collagen-I. We examined how the mechanochemical pro-fibrotic microenvironment provided by matrix stiffening and TGF-ß1 cooperates in the transcriptional control of collagen homeostasis in normal and fibrotic conditions. For this purpose we cultured fibroblasts from IPF patients or control donors on hydrogels with tunable elasticity, including 3D collagen-I gels and 2D polyacrylamide (PAA) gels. We found that TGF-ß1 consistently increased COL1A1 while decreasing MMP1 mRNA levels in hydrogels exhibiting pre-fibrotic or fibrotic-like rigidities concomitantly with an enhanced activation of the FAK/Akt pathway, whereas FAK depletion was sufficient to abrogate these effects. We also demonstrate a synergy between matrix stiffening and TGF-ß1 that was positive for COL1A1 and negative for MMP1. Remarkably, the COL1A1 expression upregulation elicited by TGF-ß1 alone or synergistically with matrix stiffening were higher in IPF-fibroblasts compared to control fibroblasts in association with larger FAK and Akt activities in the former cells. These findings provide new insights on how matrix stiffening and TGF-ß1 cooperate to elicit excessive collagen-I deposition in IPF, and support a major role of the FAK/Akt pathway in this cooperation.

The role of cell-matrix adhesion and cell migration in breast tumor growth and progression.

During breast cancer progression, there is typically increased collagen deposition resulting in elevated extracellular matrix rigidity. This results in changes to cell-matrix adhesion and cell migration, impacting processes such as the epithelial-mesenchymal transition (EMT) and metastasis. We aim to investigate the roles of cell-matrix adhesion and cell migration on breast tumor growth and progression by studying the impacts of different types of extracellular matrices and their rigidities. We embedded MCF7 spheroids within three-dimensional (3D) collagen matrices and agarose matrices. MCF7 cells adhere to collagen but not agarose. Contrasting the results between these two matrices allows us to infer the role of cell-matrix adhesion. We found that MCF7 spheroids exhibited the fastest growth rate when embedded in a collagen matrix with a rigidity of 5.1 kPa (0.5 mg/mL collagen), whereas, for the agarose matrix, the rigidity for the fastest growth rate is 15 kPa (1.0% agarose) instead. This discrepancy is attributable to the presence of cell adhesion molecules in the collagen matrix, which initiates collagen matrix remodeling and facilitates cell migration from the tumor through the EMT. As breast tumors do not adhere to agarose matrices, it is suitable to simulate the cell-cell interactions during the early stage of breast tumor growth. We conducted further analysis to characterize the stresses exerted by the expanding spheroid on the agarose matrix. We identified two distinct MCF7 cell populations, namely, those that are non-dividing and those that are dividing, which exerted low and high expansion stresses on the agarose matrix, respectively. We confirmed this using Western blot which showed the upregulation of proliferating cell nuclear antigen, a proliferation marker, in spheroids grown in the 1.0% agarose (≈13 kPa). By treating the embedded MCF7 spheroids with an inhibitor or activator of myosin contractility, we showed that the optimum spheroids' growth can be increased or decreased, respectively. This finding suggests that tumor growth in the early stage, where cell-cell interaction is more prominent, is determined by actomyosin tension, which alters cell rounding pressure during cell division. However, when breast tumors begin generating collagen into the surrounding matrix, collagen remodeling triggers EMT to promote cell migration and invasion, ultimately leading to metastasis.

Piezo1-mediated regulation of smooth muscle cell volume in response to enhanced extracellular matrix rigidity.

BACKGROUND AND PURPOSE: Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined. EXPERIMENTAL APPROACH: Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation. KEY RESULTS: On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic-like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin-1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin-1 gene expression were up-regulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation. CONCLUSIONS AND IMPLICATIONS: In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin-1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin-1 gene expression is observed in disease relevant VSMC phenotypes.

Adjustable extracellular matrix rigidity tumor model for studying stiffness dependent pancreatic ductal adenocarcinomas progression and tumor immunosuppression.

Pancreatic ductal adenocarcinomas (PDAC) is one of the stiffest malignancies with strong solid stresses. Increasing stiffness could alter cellular behavior and trigger internal signaling pathways and is strongly associated with a poor prognosis in PDAC. So far, there has been no report on of an experimental model that can rapidly construct and stably maintain a stiffness gradient dimension in both vitro and in vivo. In this study, a gelatin methacryloyl (GelMA)-based hydrogel was designed for in vitro and in vivo PDAC experiments. The GelMA-based hydrogel has porous, adjustable mechanical properties and excellent in vitro and in vivo biocompatibility. The GelMA-based in vitro 3D culture method can effectively form a gradient and stable extracellular matrix stiffness, affecting cell morphology, cytoskeleton remodeling, and malignant biological behaviors such as proliferation and metastasis. This model is suitable for in vivo studies with long-term maintenance of matrix stiffness and no significant toxicity. High matrix stiffness can significantly promote PDAC progression and tumor immunosuppression. This novel adaptive extracellular matrix rigidity tumor model is an excellent candidate for further development as an in vitro and in vivo biomechanical study model of PDAC or other tumors with strong solid stresses.

Materials and extracellular matrix rigidity highlighted in tissue damages and diseases: Implication for biomaterials design and therapeutic targets.

Rigidity (or stiffness) of materials and extracellular matrix has proven to be one of the most significant extracellular physicochemical cues that can control diverse cell behaviors, such as contractility, motility, and spreading, and the resultant pathophysiological phenomena. Many 2D materials engineered with tunable rigidity have enabled researchers to elucidate the roles of matrix biophysical cues in diverse cellular events, including migration, lineage specification, and mechanical memory. Moreover, the recent findings accumulated under 3D environments with viscoelastic and remodeling properties pointed to the importance of dynamically changing rigidity in cell fate control, tissue repair, and disease progression. Thus, here we aim to highlight the works related with material/matrix-rigidity-mediated cell and tissue behaviors, with a brief outlook into the studies on the effects of material/matrix rigidity on cell behaviors in 2D systems, further discussion of the events and considerations in tissue-mimicking 3D conditions, and then examination of the in vivo findings that concern material/matrix rigidity. The current discussion will help understand the material/matrix-rigidity-mediated biological phenomena and further leverage the concepts to find therapeutic targets and to design implantable materials for the treatment of damaged and diseased tissues.

Effects of extracellular matrix rigidity on sonoporation facilitated by targeted microbubbles: Bubble attachment, bubble dynamics, and cell membrane permeabilization.

In this study, we investigated the effects of extracellular matrix rigidity, an important physical property of microenvironments regulating cell morphology and functions, on sonoporation facilitated by targeted microbubbles, highlighting the role of microbubbles. We conducted mechanistic studies at the cellular level on physiologically relevant soft and rigid substrates. By developing a unique imaging strategy, we first resolved details of the 3D attachment configurations between targeted microbubbles and cell membrane. High-speed video microscopy then unveiled bubble dynamics driven by a single ultrasound pulse. Finally, we evaluated the cell membrane permeabilization using a small molecule model drug. Our results demonstrate that: (1) stronger targeted microbubble attachment was formed for cells cultured on the rigid substrate, while six different attachment configurations were revealed in total; (2) more violent bubble oscillation was observed for cells cultured on the rigid substrate, while one third of bubbles attached to cells on the soft substrate exhibited deformation shortly after ultrasound was turned off; (3) higher acoustic pressure was needed to permeabilize the cell membrane for cells on the soft substrate, while under the same ultrasound condition, acoustically-activated microbubbles generated larger pores as compared to cells cultured on the soft substrate. The current findings provide new insights to understand the underlying mechanisms of sonoporation in a physiologically relevant context and may be useful for the clinical translation of sonoporation.

The effects of extracellular matrix rigidity on 3-dimensional cultures of amnion membrane cells.

INTRODUCTION: To determine 3D growth of amnion membrane cells using soft substrate plates of various rigidities. METHODS: Amnion epithelial (AEC) and mesenchymal cells (AMC) were cultured on 6-well soft substrate plates coated with matrigel and elastomer with rigidities of 0.5, 2, 8, 16, and 64 kPa (n = 3 each). Controls were cells in standard culture conditions. Cell morphology, spheroids' and sheets' formations and viability (bright field microscopy and crystal violet staining), and cellular transitions (vimentin/cytokeratin-18 [CK-18] ratios) were analyzed. A Student t-test was used for statistical analyses. RESULTS: AECs in substrate rigidities between 2 and 8 kPa formed 3D features (spheroids and sheets) while retaining viability. Two kPa produced spheroids with epithelial characteristics (decrease in vimentin), and 8 kPa favored sheets. Transplantation and culture of AEC sheets with no matrix or elastomers, retained AECs' viability and maintained their epithelial characteristics. Optimum AMC growth was also between 2 and 8 kP A, with predominance of vimentin; however, AMCs did not form 3D structures. Lower and higher rigidities transitioned AMCs into AECs (decrease in vimentin). DISCUSSION: Matrix rigidities between 2 and 8 kPa produced 3D structures of AECs (spheroids and sheets), resembling amnion membranes' morphology and exhibiting regenerative capacity in utero. Although AMCs grew in similar rigidities, a lack of 3D structures support their dispersed character in the membrane matrix. Extreme rigidities transitioned AMCs into AECs, suggesting that AMCs are transient cells (reservoirs) in the matrix required for remodeling. Compromises in matrix rigidity can cause membrane dysfunction and lead to adverse pregnancy outcomes.

The role of exogenous lipids in starch and protein mediated sponge cake structure setting during baking.

While it is well established that using exogenous lipids (ELs) such as monoacylglycerols and polyglycerolesters of fatty acids improves gas cell incorporation and stability in sponge cake batter (SCB) and allows producing sponge cakes (SCs) with very high volume, fine grained crumb and soft texture, their impact on starch gelatinization and protein polymerization remained unknown. Here, differential scanning calorimetry and size-exclusion high performance liquid chromatography were performed on SC(B) samples prepared with or without ELs. Starch gelatinization and protein denaturation and polymerization started at temperatures exceeding 67 °C and mostly occurred up to a temperature of 96 °C. During further isothermal treatment at 96 °C the rigidity of the cake matrix (for which temperature-controlled time domain 1H NMR T2 relaxation times are a predictor) further increased mainly because of protein polymerization. While the temperature range of starch crystal melting was not affected by the use of ELs, protein polymerized more intensively in an 88 to 94 °C temperature range when SCB contained ELs. The more intense protein polymerization and the high water binding capacity of ELs presumably made the cake matrix more rigid at that point in time. The present results allow concluding that ELs not only impact air-liquid interface stability but also cake structure setting. Hence, both aspects most likely contribute to the superior quality of SCs containing ELs.

Engineering microenvironments towards harnessing pro-angiogenic potential of mesenchymal stem cells.

Mesenchymal stem cell (MSC)-based therapy for promoting vascular regeneration is a promising strategy for treating ischemic diseases. However, low engraftment and retention rate of MSCs at the target site highlights the importance of paracrine signaling of MSCs in the reparative process. Thus, harnessing MSC-secretome is essential for rational design of MSC-based therapies. The role of microenvironment in regulating the paracrine signaling of MSCs is not well known. In this study, human bone marrow-derived MSCs were seeded on matrices with varying stiffness or cell adhesive sites, and conditioned media was collected. The concentrations of angiogenic molecules in the media was measured via ELISA. In addition, the bioactivity of the released molecules was investigated via assessing the proliferation and capillary morphogenesis of human umbilical vein endothelial cells (HUVECs) incubated with conditioned media. Our study revealed that secretion of vascular endothelial growth factor (VEGF) is dependent on substrate stiffness. Maximal secretion was observed when MSCs were seeded on hydrogel matrices of 5.0 kPa stiffness. Proliferation and tubulogenesis of HUVECs supported ELISA data. On the other hand, variation of cell adhesive sites while maintaining a uniform optimal stiffness, did not influence the pro-angiogenic activity of MSCs.

Matrix Rigidity-Dependent Regulation of Ca2+ at Plasma Membrane Microdomains by FAK Visualized by Fluorescence Resonance Energy Transfer.

The dynamic regulation of signal transduction at plasma membrane microdomains remains poorly understood due to limitations in current experimental approaches. Genetically encoded biosensors based on fluorescent resonance energy transfer (FRET) can provide high spatiotemporal resolution for imaging cell signaling networks. Here, distinctive regulation of focal adhesion kinase (FAK) and Ca2+ signals are visualized at different membrane microdomains by FRET using membrane-targeting biosensors. It is shown that rigidity-dependent FAK and Ca2+ signals in human mesenchymal stem cells (hMSCs) are selectively activated at detergent-resistant membrane (DRM or rafts) microdomains during the cell-matrix adhesion process, with minimal activities at non-DRM domains. The rigidity-dependent Ca2+ signal at the DRM microdomains is downregulated by either FAK inhibition or lipid raft disruption, suggesting that FAK and lipid raft integrity mediate the in situ Ca2+ activation. It is further revealed that transient receptor potential subfamily M7 (TRPM7) participates in the mobilization of Ca2+ signals within DRM regions. Thus, the findings provide insights into the underlying mechanisms that regulate Ca2+ and FAK signals in hMSCs under different mechanical microenvironments.

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates.

The mechanical properties of living cells reflect their propensity to migrate and respond to external forces. Both cellular and nuclear stiffnesses are strongly influenced by the rigidity of the extracellular matrix (ECM) through reorganization of the cyto- and nucleoskeletal protein connections. Changes in this architectural continuum affect cell mechanics and underlie many pathological conditions. In this context, an accurate and combined quantification of the mechanical properties of both cells and nuclei can contribute to a better understanding of cellular (dys-)function. To address this challenge, we have established a robust method for probing cellular and nuclear deformation during spreading and detachment from micropatterned substrates. We show that (de-)adhesion kinetics of endothelial cells are modulated by substrate stiffness and rely on the actomyosin network. We combined this approach with measurements of cell stiffness by magnetic tweezers to show that relaxation dynamics can be considered as a reliable parameter of cellular pre-stress in adherent cells. During the adhesion stage, large cellular and nuclear deformations occur over a long time span (>60 min). Conversely, nuclear deformation and condensed chromatin are relaxed in a few seconds after detachment. Finally, our results show that accumulation of farnesylated prelamin leads to modifications of the nuclear viscoelastic properties, as reflected by increased nuclear relaxation times. Our method offers an original and non-intrusive way of simultaneously gauging cellular and nuclear mechanics, which can be extended to high-throughput screens of pathological conditions and potential countermeasures.

Force-dependent breaching of the basement membrane.

Clinically, non-invasive carcinomas are confined to the epithelial side of the basement membrane and are classified as benign, whereas invasive cancers invade through the basement membrane and thereby acquire the potential to metastasize. Recent findings suggest that, in addition to protease-mediated degradation and chemotaxis-stimulated migration, basement membrane invasion by malignant cells is significantly influenced by the stiffness of the associated interstitial extracellular matrix and the contractility of the tumor cells that is dictated in part by their oncogenic genotype. In this review, we highlight recent findings that illustrate unifying molecular mechanisms whereby these physical cues contribute to tissue fibrosis and malignancy in three epithelial organs: breast, pancreas, and liver. We also discuss the clinical implications of these findings and the biological properties and clinical challenges linked to the unique biology of each of these organs.

Mechanobiology of cell migration in the context of dynamic two-way cell-matrix interactions.

Migration of cells is integral in various physiological processes in all facets of life. These range from embryonic development, morphogenesis, and wound healing, to disease pathology such as cancer metastasis. While cell migratory behavior has been traditionally studied using simple assays on culture dishes, in recent years it has been increasingly realized that the physical, mechanical, and chemical aspects of the matrix are key determinants of the migration mechanism. In this paper, we will describe the mechanobiological changes that accompany the dynamic cell-matrix interactions during cell migration. Furthermore, we will review what is to date known about how these changes feed back to the dynamics and biomechanical properties of the cell and the matrix. Elucidating the role of these intimate cell-matrix interactions will provide not only a better multi-scale understanding of cell motility in its physiological context, but also a more holistic perspective for designing approaches to regulate cell behavior.

Regulation of metastatic ability and drug resistance in pulmonary adenocarcinoma by matrix rigidity via activating c-Met and EGFR.

Lung fibrosis is a poor prognostic factor for pulmonary adenocarcinoma, and the effect of a rigid microenvironment on cancer behavior is unclear. We cultured A549 cells on matrices of 0.2, 2, and 25 kPa to mimic the rigidities of normal lung parenchyma, progressive fibrotic change, and lung fibrosis, respectively. Lung tissue from patients with pulmonary adenocarcinoma was used to confirm the in vitro findings. Increased matrix rigidity promoted cell proliferation and upregulated the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-Met), and Snail expression in A549 cells. A549 cells became more resistant to the EGFR inhibitor (Erlotinib) and c-Met inhibitor (PHA-665752) when matrix rigidity increased; however, a high concentration of PHA-665752 reversed the rigidity-induced morphological pleomorphism. In human lung tissue, expression of type I collagen was more consistent with clinical fibrosis than the expression of alpha-smooth muscle antibody was. c-Met- and Snail-expressing tumor cells, rather than EGFR-experssing cells, were localized with lung parenchyma rich in type I collagen. Our findings suggest that c-Met causes the rigidity-induced biophysical reaction in pulmonary adenocarcinoma. Treatment targeting both EGFR and c-Met should be considered for patients with lung fibrosis and who are abundant type I collagen expression in the tumor mass.

Matrix rigidity regulates the transition of tumor cells to a bone-destructive phenotype through integrin ß3 and TGF-ß receptor type II.

Cancer patients frequently develop skeletal metastases that significantly impact quality of life. Since bone metastases remain incurable, a clearer understanding of molecular mechanisms regulating skeletal metastases is required to develop new therapeutics that block establishment of tumors in bone. While many studies have suggested that the microenvironment contributes to bone metastases, the factors mediating tumors to progress from a quiescent to a bone-destructive state remain unclear. In this study, we hypothesized that the "soil" of the bone microenvironment, specifically the rigid mineralized extracellular matrix, stimulates the transition of the tumor cells to a bone-destructive phenotype. To test this hypothesis, we synthesized 2D polyurethane (PUR) films with elastic moduli ranging from the basement membrane (70 MPa) to cortical bone (3800 MPa) and measured expression of genes associated with mechanotransduction and bone metastases. We found that expression of Integrin ß3 (Iß3), as well as tumor-produced factors associated with bone destruction (Gli2 and parathyroid hormone related protein (PTHrP)), significantly increased with matrix rigidity, and that blocking Iß3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iß3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-ß), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iß3 co-localized with TGF-ß Receptor Type II (TGF-ß RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iß3 shRNA into the tibiae of athymic nude mice significantly reduced PTHrP and Gli2 expression, as well as bone destruction, suggesting a crucial role for tumor-produced Iß3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iß3/TGF-ß-dependent manner, and suggests that Iß3 inhibitors are a potential therapeutic approach for blocking tumor transition to a bone destructive phenotype.