Increased Extracellular Matrix Stiffness Research Articles

Intracellular pH dynamics respond to microenvironment stiffening and mediate vasculogenic mimicry through β-catenin.

Dysregulated intracellular pH (pHi) dynamics and an altered tumor microenvironment have emerged as drivers of cancer cell phenotypes. However, the molecular integration between the physical properties of the microenvironment and dynamic intracellular signaling responses remains unclear. Here, we use two metastatic cell models, one breast and one lung, to assess pHi response to varying extracellular matrix (ECM) stiffness. To experimentally model ECM stiffening, we use two tunable-stiffness hydrogel systems: Matrigel and hyaluronic acid (HA) gels, which mimic the increased protein secretion and crosslinking associated with ECM stiffening. We find that single-cell pHi decreases with increased ECM stiffness in both hydrogel systems and both metastatic cell types. We also observed that stiff ECM promotes vasculogenic mimicry (VM), a phenotype associated with metastasis and resistance. Importantly, we show that decreased pHi is both a necessary and sufficient mediator of VM, as raising pHi on stiff ECM reduces VM phenotypes and lowering pHi on soft ECM drives VM. We characterize β-catenin as a pH-dependent molecular mediator of pH-dependent VM, where stiffness-driven changes in β-catenin abundance can be overridden by increased pHi. We uncover a dynamic relationship between matrix stiffness and pHi, thus suggesting pHi dynamics can override mechanosensitive cell responses to the extracellular microenvironment.

Investigating the Role of Extracellular Matrix Stiffness in Modulating the Ferroptosis Process in Hepatocellular Carcinoma Cells via Scanning Electrochemical Microscopy.

Extracellular matrix (ECM) stiffness modulates a variety of cellular processes, including ferroptosis, a process with significant potential implications for hepatocellular carcinoma (HCC) fibrosis and cirrhosis. However, the exact relationship between ECM stiffness and HCC ferroptosis is yet unclarified, partially due to the lack of in situ information on key parameters of the ferroptosis process of living HCC cells. This study pioneers the use of in vitro mechanical microenvironment models of HCC and the scanning electrochemical microscopy (SECM) technique for understanding this interplay. We first cultured HuH7 cells on 4.0, 18.0, and 44.0 kPa polyacrylamide (PA) gels to simulate early, intermediate, and advanced HCC ECM stiffness, respectively. Then, we used SECM to in situ monitor changes in cell membrane permeability, respiratory activity, and reactive oxygen species (ROS) levels of erastin-induced HuH7 cells on PA gels, finding that increasing ECM stiffness potentiates ferroptosis, including increased membrane permeabilization and H2O2 release as well as reduced respiratory activity. Through further transcriptome sequencing and molecular biology measurements, we identified a critical role for focal adhesion kinase (FAK)-mediated yes-associated protein (YAP) in regulating the ferroptosis process dependent on ECM stiffness, which provides novel insights into the mechanical regulation of ferroptosis in HCC cells and may pave the way for innovative therapeutic strategies.

The Effect of Substrate Stiffness on Elastic Force Transmission in the Epithelial Monolayers over Short Timescales

PurposeThe importance of mechanical forces and microenvironment in guiding cellular behavior has been widely accepted. Together with the extracellular matrix (ECM), epithelial cells form a highly connected mechanical system subjected to various mechanical cues from their environment, such as ECM stiffness, and tensile and compressive forces. ECM stiffness has been linked to many pathologies, including tumor formation. However, our understanding of the effect of ECM stiffness and its heterogeneities on rapid force transduction in multicellular systems has not been fully addressed.MethodsWe used experimental and computational methods. Epithelial cells were cultured on elastic hydrogels with fluorescent nanoparticles. Single cells were moved by a micromanipulator, and epithelium and substrate deformation were recorded. We developed a computational model to replicate our experiments and quantify the force distribution in the epithelium. Our model further enabled simulations with local stiffness gradients.ResultsWe found that substrate stiffness affects the force transduction and the cellular deformation following an external force. Also, our results indicate that the heterogeneities, e.g., gradients, in the stiffness can substantially influence the strain redistribution in the cell monolayers. Furthermore, we found that the cells’ apico-basal elasticity provides a level of mechanical isolation between the apical cell–cell junctions and the basal focal adhesions.ConclusionsOur simulation results show that increased ECM stiffness, e.g., due to a tumor, can mechanically isolate cells and modulate rapid mechanical signaling between cells over distances. Furthermore, the developed model has the potential to facilitate future studies on the interactions between epithelial monolayers and elastic substrates.

Extracellular matrix remodelling and stiffening contributes to tumorigenesis of salivary carcinoma ex pleomorphic adenoma——A study based on patient-derived organoids

BackgroundSalivary carcinoma ex pleomorphic adenoma (CXPA) is defined as a carcinoma that develops from benign pleomorphic adenoma (PA). Abnormally activated Androgen signaling pathway and amplification of HER-2/neu(ERBB-2) gene are known to be involved in CXPA tumorigenesis. Recent progress in tumour microenvironment research has led to identification that extracellular matrix (ECM) remodelling and increased stiffness act as critical contributing role in tumour carcinogenesis. This study examined ECM modifications to elucidate the mechanism underlying CXPA tumorigenesis.ResultsPA and CXPA organoids were successfully established. Histological observation, immunohistochemistry (IHC), and whole-exome sequencing demonstrated that organoids recapitulated phenotypic and molecular characteristics of their parental tumours. RNA-sequencing and bioinformatic analysis of organoids showed that differentially expressed genes are highly enriched in ECM-associated terms, implying that ECM alternations may be involved in carcinogenesis. Microscopical examination for surgical samples revealed that excessive hyalinized tissues were deposited in tumour during CXPA tumorigenesis. Transmission electron microscopy confirmed that these hyalinized tissues were tumour ECM in nature. Subsequently, examination by picrosirius red staining, liquid chromatography with tandem mass spectrometry, and cross-linking analysis indicated that tumour ECM was predominantly composed of type I collagen fibers, with dense collagen alignment and an increased level of collagen cross-linking. IHC revealed the overexpression of COL1A1 protein and collagen-synthesis-related genes, DCN and IGFBP5 (p < 0.05). Higher stiffness of CXPA than PA was demonstrated by atomic force microscopy and elastic imaging analysis. We utilized hydrogels to mimic ECM with varying stiffness degrees in vitro. Compared with softer matrices (5Kpa), CXPA cell line and PA primary cells exhibited more proliferative and invasive phenotypes in stiffer matrices (50Kpa, p < 0.01). Protein-protein interaction (PPI) analysis of RNA-sequencing data revealed that AR and ERBB-2 expression was associated with TWIST1. Moreover, surgical specimens demonstrated a higher TWIST1 expression in CXPA over PA. After knocking down TWIST1 in CXPA cells, cell proliferation, migration, and invasiveness were significantly inhibited (p < 0.01).ConclusionDeveloping CXPA organoids provides a useful model for cancer biology research and drug screening. ECM remodelling, attributed to overproduction of collagen, alternation of collagen alignment, and increased cross-linking, leads to increased ECM stiffness. ECM modification is an important contributor in CXPA tumorigenesis.

Stiff Extracellular Matrix Promotes Invasive Behaviors of Trophoblast Cells.

The effect of extracellular matrix (ECM) stiffness on embryonic trophoblast cells invasion during mammalian embryo implantation remains largely unknown. In this study, we investigated the effects of ECM stiffness on various aspects of human trophoblast cell behaviors during cell-ECM interactions. The mechanical microenvironment of the uterus was simulated by fabricating polyacrylamide (PA) hydrogels with different levels of stiffness. The human choriocarcinoma (JAR) cell lineage was used as the trophoblast model. We found that the spreading area of JAR cells, the formation of focal adhesions, and the polymerization of the F-actin cytoskeleton were all facilitated with increased ECM stiffness. Significantly, JAR cells also exhibited durotactic behavior on ECM with a gradient stiffness. Meanwhile, stiffness of the ECM affects the invasion of multicellular JAR spheroids. These results demonstrated that human trophoblast cells are mechanically sensitive, while the mechanical properties of the uterine microenvironment could play an important role in the implantation process.

Cancer-associated fibroblasts in a 3D engineered tissue model induce tumor-like matrix stiffening and EMT transition.

There is an increasing need for the use of 3D in vitro models over 2D culture. Yet, most models remain unable to fully reproduce the complexity observed in vivo. Tissue-engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture, composition, and cell heterogeneity. Here, we used the self-assembly method and a bladder tissue model to engineer a tumor-like environment, and validated its relevance as a cancer model. The bioengineered bladder cancer model was reconstituted using healthy primary fibroblasts (HFs) and a sub-population of induced cancer-associated fibroblast-like cells (iCAFs) along with a normal urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be more fibrotic with increased collagen remodeling and altered fibronectin content. Moreover, the average stiffness of the ECM scaffolds produced by the iCAFs was 7.4 kPa compared to 1.3 kPa for those produced by HFs. In line with those results, urothelial cells overlaid on the iCAFs-derived ECM was found to be more contractile. We also observed increased nuclear translocation of the mechanosensor YAP1 in urothelial cells overlaid on these iCAFs-derived ECM. Furthermore, urothelial cells seeded on iCAFs-derived stroma displayed invasive behavior compared to urothelial cells on HFs. As increased ECM stiffness and cell contractility are associated with the epithelial-to-mesenchymal transition (EMT), we showed an increase expression of the mesenchymal EMT markers, vimentin and ZEB1, within the urothelium on the iCAFs-derived ECM. Overall, our tissue-engineered tumor model achieved stiffness levels comparable to those observed in bladder cancers along with CAF-mediated ECM remodeling, while the urothelium displayed the biological responses that would be expected within a tumor-like environment. Future experiments will take advantage of the 3D and in vivo-like properties of our engineered model to investigate tumor cell invasion.

A kidney proximal tubule model to evaluate effects of basement membrane stiffening on renal tubular epithelial cells.

The kidney tubule consists of a single layer of epithelial cells supported by the tubular basement membrane (TBM), a thin layer of specialized extracellular matrix (ECM). The mechanical properties of the ECM are important for regulating a wide range of cell functions including proliferation, differentiation and cell survival. Increased ECM stiffness plays a role in promoting multiple pathological conditions including cancer, fibrosis and heart disease. How changes in TBM mechanics regulate tubular epithelial cell behavior is not fully understood. Here we introduce a cell culture system that utilizes in vivo-derived TBM to investigate cell-matrix interactions in kidney proximal tubule cells. Basement membrane mechanics was controlled using genipin, a biocompatibility crosslinker. Genipin modification resulted in a dose-dependent increase in matrix stiffness. Crosslinking had a marginal but statistically significant impact on the diffusive molecular transport properties of the TBM, likely due to a reduction in pore size. Both native and genipin-modified TBM substrates supported tubular epithelial cell growth. Cells were able to attach and proliferate to form confluent monolayers. Tubular epithelial cells polarized and assembled organized cell-cell junctions. Genipin modification had minimal impact on cell viability and proliferation. Genipin stiffened TBM increased gene expression of pro-fibrotic cytokines and altered gene expression for N-cadherin, a proximal tubular epithelial specific cell-cell junction marker. This work introduces a new cell culture model for cell-basement membrane mechanobiology studies that utilizes in vivo-derived basement membrane. We also demonstrate that TBM stiffening affects tubular epithelial cell function through altered gene expression of cell-specific differentiation markers and induced increased expression of pro-fibrotic growth factors.

Combined role for YAP-TEAD and YAP-RUNX2 signalling in substrate-stiffness regulation of cardiac fibroblast proliferation

Cardiac fibrosis is associated with increased stiffness of the myocardial extracellular matrix (ECM) in part mediated by increased cardiac fibroblast proliferation However, our understanding of the mechanisms regulating cardiac fibroblast proliferation are incomplete. Here we characterise a novel mechanism involving a combined activation of Yes-associated protein (YAP) targets RUNX Family Transcription Factor 2 (RUNX2) and TEA Domain Transcription Factor (TEAD).We demonstrate that cardiac fibroblast proliferation is enhanced by interaction with a stiff ECM compared to a soft ECM. This is associated with activation of the transcriptional co-factor, YAP. We demonstrate that this stiffness induced activation of YAP enhances the transcriptional activity of both TEAD and RUNX2 transcription factors. Inhibition of either TEAD or RUNX2, using gene silencing, expression of dominant-negative mutants or pharmacological inhibition, reduces cardiac fibroblast proliferation. Using mutants of YAP, defective in TEAD or RUNX2 activation ability, we demonstrate a dual role of YAP-mediated activation of TEAD and RUNX2 for substrate stiffness induced cardiac fibroblast proliferation.Our data highlights a previously unrecognised role of YAP mediated RUNX2 activation for cardiac fibroblast proliferation in response to increased ECM stiffness.

A Stiff Extracellular Matrix Favors the Mechanical Cell Competition that Leads to Extrusion of Bacterially-Infected Epithelial Cells

Cell competition refers to the mechanism whereby less fit cells (“losers”) are sensed and eliminated by more fit neighboring cells (“winners”) and arises during many processes including intracellular bacterial infection. Extracellular matrix (ECM) stiffness can regulate important cellular functions, such as motility, by modulating the physical forces that cells transduce and could thus modulate the output of cellular competitions. Herein, we employ a computational model to investigate the previously overlooked role of ECM stiffness in modulating the forceful extrusion of infected “loser” cells by uninfected “winner” cells. We find that increasing ECM stiffness promotes the collective squeezing and subsequent extrusion of infected cells due to differential cell displacements and cellular force generation. Moreover, we discover that an increase in the ratio of uninfected to infected cell stiffness as well as a smaller infection focus size, independently promote squeezing of infected cells, and this phenomenon is more prominent on stiffer compared to softer matrices. Our experimental findings validate the computational predictions by demonstrating increased collective cell extrusion on stiff matrices and glass as opposed to softer matrices, which is associated with decreased bacterial spread in the basal cell monolayer in vitro. Collectively, our results suggest that ECM stiffness plays a major role in modulating the competition between infected and uninfected cells, with stiffer matrices promoting this battle through differential modulation of cell mechanics between the two cell populations.

Adipose tissue stiffness in the development of metabolic diseases

Obesity, defined as an expansion of white adipose tissue, is also accompanied by interstitial fibrosis and overexpression of extracellular matrix (ECM) in adipose tissue in recent studies. We hypothesize that obesity induces adipose tissue ECM crosslinking and stiffening, leading to adipocyte dysfunction. First, we applied a collagen gel system to investigate the effect of ECM stiffness on adipogenesis and mature adipocyte functions. Our results showed that increased ECM stiffness impaired adipogenesis; and blunted the sensitivity to insulin and lipolytic cues, as well as secretion of adipokines, in differentiated adipocytes. In contrast, increased ECM stiffness exacerbated the sensitivity to inflammatory stimulation. Next, we found that ob/ob adipose tissue exhibited an increased signal in the polarized view of picrosirius red stained section, as well as increased crosslinks and aggregated collagen in second harmonics generation (SHG) imaging. These results suggest that ECM crosslinking was upregulated in the adipose tissue of obese mice. Ex vivo crosslinking inhibition in ob/ob adipose tissue explants increased insulin sensitivity, adiponectin expression and lipolytic activity. Finally, in vivo crosslinking inhibition reversed metabolic impairments and adipocyte functional signature genes in ob/ob mice. Taken together, our study underscores that adipose tissue crosslinking and stiffening may orchestrate metabolic disorders resulting from adipocyte dysfunction in obesity.

Extracellular Matrix Stiffness and TGFβ2 Regulate YAP/TAZ Activity in Human Trabecular Meshwork Cells.

Primary open-angle glaucoma progression is associated with increased human trabecular meshwork (HTM) stiffness and elevated transforming growth factor beta 2 (TGFβ2) levels in the aqueous humor. Increased transcriptional activity of Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), central players in mechanotransduction, are implicated in glaucomatous HTM cell dysfunction. Yet, the detailed mechanisms underlying YAP/TAZ modulation in HTM cells in response to alterations in extracellular matrix (ECM) stiffness and TGFβ2 levels are not well understood. Using biomimetic ECM hydrogels with tunable stiffness, here we show that increased ECM stiffness elevates YAP/TAZ nuclear localization potentially through modulating focal adhesions and cytoskeletal rearrangement. Furthermore, TGFβ2 increased nuclear YAP/TAZ in both normal and glaucomatous HTM cells, which was prevented by inhibiting extracellular-signal-regulated kinase and Rho-associated kinase signaling pathways. Filamentous (F)-actin depolymerization reversed TGFβ2-induced YAP/TAZ nuclear localization. YAP/TAZ depletion using siRNA or verteporfin decreased focal adhesions, ECM remodeling and cell contractile properties. Similarly, YAP/TAZ inactivation with verteporfin partially blocked TGFβ2-induced hydrogel contraction and stiffening. Collectively, our data provide evidence for a pathologic role of aberrant YAP/TAZ signaling in glaucomatous HTM cell dysfunction, and may help inform strategies for the development of novel multifactorial approaches to prevent progressive ocular hypertension in glaucoma.

Abstract P5-12-13: Exploring the impact of the tumor microenvironment on nuclear morphometry: Lessons learned for sensitivity to cytotoxic treatment

Abstract Globally, breast cancer remains the leading cause of death in females. While systemic therapy for breast cancer may work effectively in the early phases, for more than 10% of primary and 50% of metastatic cases, the disease eventually progresses, resisting treatments. To overcome this issue, recognizing markers of resistance as early as possible is critical. Microenvironmental factors influence tumors, and increased stiffness of the extracellular matrix (ECM) is associated with cancer progression. Additionally, neighboring cells such as non-neoplastic epithelial cells(that may constitute an important portion of the tumor microenvironment -TME) are suspected to affect tumors, but details on their impact are lacking. Here, we asked whether the response to increased ECM stiffness differed depending on the degree of aggressiveness of triple negative breast cancers (TNBC) and the presence of the non-neoplastic epithelium. We investigated two morphometric parameters, nuclear circularity and area (used in cancer pathology), and tumors’ sensitivity to cisplatin to assess tumor aggressiveness- with TNBC cultured at normal (770 Pa) and increased (2000 and 3300 Pa) collagen I stiffness. Reduced nuclear circularity and increased heterogeneity for this parameter in the cancer cell population resulted from higher matrix stiffness and were associated with decreased sensitivity to the cytotoxic drug, cisplatin, notably in the more invasive cancer type. Interestingly, the impact of stiffness increase was reduced by the conditioned medium from the non-neoplastic cells. These findings warrant further investigations to identify whether changes in nuclear circularity that illustrate a global nuclear reorganization affect gene expression profiles linked to cell survival. Citation Format: Apekshya Chhetri. Exploring the impact of the tumor microenvironment on nuclear morphometry: Lessons learned for sensitivity to cytotoxic treatment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-13.

Stiff matrices enhance myoblast proliferation, reduce differentiation, and alter the response to fluid shear stress in vitro.

During myofiber regeneration, myoblasts are continuously subjected to shear stress. It is currently not known whether shear stress affects the regenerative capacity of myoblasts when extracellular matrix (ECM) stiffness increases (e.g. upon aging). Therefore, we aimed to assess (1) whether matrix stiffness and pulsating fluid shear stress affect myoblast proliferation and/or expression of differentiation-associated genes in myoblasts, and (2) whether matrix stiffness modulates the mechanoresponse of myoblasts to pulsating fluid shear stress. Myoblasts were seeded on matrigel-coated polyacrylamide gel matrices of varying stiffness, mimicking young ("soft", 0.5 kPa) and old ECM ("stiff", 20 kPa), as well as on matrigel-coated glass matrices with very high stiffness (40 ϺPa), and subjected to 1 h pulsating fluid shear stress (3 Pa/s or 4 Pa/s, 1 Hz). We found enhanced proliferation of myoblasts on stiff matrices, but reduced differentiation compared to myoblasts on soft matrices. Pulsating fluid shear stress significantly upregulated gene expression of proliferation-associated genes C-fos and Il-6, as well as expression of cytoskeletal α-actin in myoblasts seeded on glass. In contrast, pulsating fluid shear stress significantly downregulated gene expression of α-actin and Myogenin in myoblasts seeded on soft matrices. In conclusion, these results suggest that age and disease-associated increased ECM stiffness may contribute to declined regenerative capacity of myoblasts, by reducing their capacity to differentiate into new muscular tissue, at least in the absence of mechanical stimulation.

Sensitive detection of cell-derived force and collagen matrix tension in microtissues undergoing large-scale densification

Mechanical forces generated by cells and the tension of the extracellular matrix (ECM) play a decisive role in establishment, homeostasis maintenance, and repair of tissue morphology. However, the dynamic change of cell-derived force during large-scale remodeling of soft tissue is still unknown, mainly because the current techniques of force detection usually produce a nonnegligible and interfering feedback force on the cells during measurement. Here, we developed a method to fabricate highly stretchable polymer-based microstrings on which a microtissue of fibroblasts in collagen was cultured and allowed to contract to mimic the densification of soft tissue. Taking advantage of the low-spring constant and large deflection range of the microstrings, we detected a strain-induced contraction force as low as 5.2 µN without disturbing the irreversible densification. Meanwhile, the microtissues displayed extreme sensitivity to the mechanical boundary within a narrow range of tensile stress. More importantly, results indicated that the cell-derived force did not solely increase with increased ECM stiffness as previous studies suggested. Indeed, the cell-derived force and collagen tension exchanged dramatically in dominating the microtissue strain during the densification, and the proportion of cell-derived force decreased linearly as the microtissue densified, with stiffness increasing to ∼500 Pa. Thus, this study provides insights into the biomechanical cross-talk between the cells and ECM of extremely soft tissue during large-extent densification, which may be important to guide the construction of life-like tissue by applying appropriate mechanical boundary conditions.

An Outside-In Switch in Integrin Signaling Caused by Chemical and Mechanical Signals in Reactive Astrocytes.

Astrocyte reactivity is associated with poor repair capacity after injury to the brain, where chemical and physical changes occur in the damaged zone. Astrocyte surface proteins, such as integrins, are upregulated, and the release of pro-inflammatory molecules and extracellular matrix (ECM) proteins upon damage generate a stiffer matrix. Integrins play an important role in triggering a reactive phenotype in astrocytes, and we have reported that αVβ3 Integrin binds to the Thy-1 (CD90) neuronal glycoprotein, increasing astrocyte contractility and motility. Alternatively, αVβ3 Integrin senses mechanical forces generated by the increased ECM stiffness. Until now, the association between the αVβ3 Integrin mechanoreceptor response in astrocytes and changes in their reactive phenotype is unclear. To study the response to combined chemical and mechanical stress, astrocytes were stimulated with Thy-1-Protein A-coated magnetic beads and exposed to a magnetic field to generate mechanical tension. We evaluated the effect of such stimulation on cell adhesion and contraction. We also assessed traction forces and their effect on cell morphology, and integrin surface expression. Mechanical stress accelerated the response of astrocytes to Thy-1 engagement of integrin receptors, resulting in cell adhesion and contraction. Astrocyte contraction then exerted traction forces onto the ECM, inducing faster cell contractility and higher traction forces than Thy-1 alone. Therefore, cell-extrinsic chemical and mechanical signals regulate in an outside-in manner, astrocyte reactivity by inducing integrin upregulation, ligation, and signaling events that promote cell contraction. These changes in turn generate cell-intrinsic signals that increase traction forces exerted onto the ECM (inside-out). This study reveals αVβ3 Integrin mechanoreceptor as a novel target to regulate the harmful effects of reactive astrocytes in neuronal healing.

Mechanosensitive expression of lamellipodin promotes intracellular stiffness, cyclin expression and cell proliferation.

Cell cycle control is a key aspect of numerous physiological and pathological processes. The contribution of biophysical cues, such as stiffness or elasticity of the underlying extracellular matrix (ECM), is critically important in regulating cell cycle progression and proliferation. Indeed, increased ECM stiffness causes aberrant cell cycle progression and proliferation. However, the molecular mechanisms that control these stiffness-mediated cellular responses remain unclear. Here, we address this gap and show good evidence that lamellipodin (symbol RAPH1), previously known as a critical regulator of cell migration, stimulates ECM stiffness-mediated cyclin expression and intracellular stiffening in mouse embryonic fibroblasts. We observed that increased ECM stiffness upregulates lamellipodin expression. This is mediated by an integrin-dependent FAK-Cas-Rac signaling module and supports stiffness-mediated lamellipodin induction. Mechanistically, we find that lamellipodin overexpression increased, and lamellipodin knockdown reduced, stiffness-induced cell cyclin expression and cell proliferation, and intracellular stiffness. Overall, these results suggest that lamellipodin levels may be critical for regulating cell proliferation. This article has an associated First Person interview with the first author of the paper.

3D Cell Culture for the Study of Microenvironment-Mediated Mechanostimuli to the Cell Nucleus: An Important Step for Cancer Research.

The discovery that the stiffness of the tumor microenvironment (TME) changes during cancer progression motivated the development of cell culture involving extracellular mechanostimuli, with the intent of identifying mechanotransduction mechanisms that influence cell phenotypes. Collagen I is a main extracellular matrix (ECM) component used to study mechanotransduction in three-dimensional (3D) cell culture. There are also models with interstitial fluid stress that have been mostly focusing on the migration of invasive cells. We argue that a major step for the culture of tumors is to integrate increased ECM stiffness and fluid movement characteristic of the TME. Mechanotransduction is based on the principles of tensegrity and dynamic reciprocity, which requires measuring not only biochemical changes, but also physical changes in cytoplasmic and nuclear compartments. Most techniques available for cellular rheology were developed for a 2D, flat cell culture world, hence hampering studies requiring proper cellular architecture that, itself, depends on 3D tissue organization. New and adapted measuring techniques for 3D cell culture will be worthwhile to study the apparent increase in physical plasticity of cancer cells with disease progression. Finally, evidence of the physical heterogeneity of the TME, in terms of ECM composition and stiffness and of fluid flow, calls for the investigation of its impact on the cellular heterogeneity proposed to control tumor phenotypes. Reproducing, measuring and controlling TME heterogeneity should stimulate collaborative efforts between biologists and engineers. Studying cancers in well-tuned 3D cell culture platforms is paramount to bring mechanomedicine into the realm of oncology.

Matrix Rigidity Controls Epithelial-Mesenchymal Plasticity and Tumor Metastasis via a Mechanoresponsive EPHA2/LYN Complex.

Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis invivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.

Biomimetic Matrix Stiffness Modulates Hepatocellular Carcinoma Malignant Phenotypes and Macrophage Polarization through Multiple Modes of Mechanical Feedbacks.

The extracellular matrix (ECM) stiffening is an important sign of local microenvironment change, which is considered as a hallmark of many diseases including hepatocellular carcinoma (HCC). The fates of both cancer cells and immune cells can be regulated by mechanical feedbacks acquired from ECM, but there is a lack of a precise study of mechanical feedback modes in different cell phenotypes following with the progressively increasing ECM stiffness. Herein, we used a biopolymeric film without further modification of adhesive molecules, as a natural local niche to mimic a gradually stiffening manner from HCC onset in liver cirrhosis to its metastasis in the spinal cord. Three distinct manners of mechanical feedbacks were found: the gradual manner in HCC cell spreading, migration and early apoptosis to oxaliplatin, the stepwise manner in HCC cell adhesion, proliferation, focal adhesion (FA) formation, drug resistance, and macrophage M1 polarization; the specific manner in the stages of the progression of epithelial-mesenchymal transition at different stiffness ranges. Further investigation of molecular mechanisms confirmed those mechanical feedback manners by signaling activation of FA kinase, phosphatidylinositol 3-kinase, and expression of pro-/antiapoptotic and pro-/anti-inflammatory genes. Our results pave a novel avenue to know about mechanical feedbacks from ECM, which could be used for future cancer studies and in vitro drug screening applications.

Glycation alters the mechanical behavior of kidney extracellular matrix

The mechanical properties of the extracellular matrix (ECM) are important in maintaining normal physiological function, and changes in ECM mechanics drive disease. The biochemical structure of the ECM is modified with aging and in diseases such as diabetes. One mechanism of ECM modification is the non-enzymatic reaction between sugars and ECM proteins resulting in formation of advanced glycation end products (AGEs). Some AGE reactions result in formation of molecular crosslinks within or between matrix proteins, but it is not clear how sugar-mediated biochemical modification of the ECM translates to changes in kidney ECM mechanical properties. AGE-mediated changes in ECM mechanics may have pathological consequences in diabetic kidney disease. To determine how sugars alter the mechanical properties of the kidney ECM, we employ custom methodologies to evaluate the mechanical properties of isolated tubular basement membrane (TBM) and glomerular ECM. Results show that the mechanical properties of TBM and glomerular ECM stiffness were altered by incubation in glucose and ribose. Mechanical behavior of TBM and glomerular ECM were further evaluated using mechanical models for hyperelastic materials in tension and compression. Increased ECM stiffness following sugar modification corresponded to increased crosslinking as determined by ECM fluorescence and reduced pepsin extractability of sugar modified ECM. These results show that sugar-induced modifications significantly affect the mechanical properties of kidney ECM. AGE-mediated changes in ECM mechanics may be important in progression of chronic diseases including diabetic kidney disease.