Extracellular Matrix Stiffness Research Articles

Cancer-Associated Fibroblasts and Extracellular Matrix: Therapeutical Strategies for Modulating the Cholangiocarcinoma Microenvironment

During the last decade, immunotherapy has radically changed perspectives on anti-tumor treatments. However, solid tumor treatment by immunotherapy has not met expectations. Indeed, poor clinical response to treatment has highlighted the need to understand and avoid immunotherapy resistance. Cholangiocarcinoma (CCA) is the second cause of hepatic cancer-related deaths because of drug inefficacy and chemo-resistance in a majority of patients. Thus, intense research is ongoing to better understand the mechanisms involved in the chemo-resistance processes. The tumor microenvironment (TME) may be involved in tumor therapy resistance by limiting drug access. Indeed, cells such as cancer-associated fibroblasts (CAFs) alter TME by producing in excess an aberrant extracellular matrix (ECM). Interestingly, CAFs are the dominant stromal component in CCA that secrete large amounts of stiff ECM. Stiff ECM could contribute to immune exclusion by limiting anti-tumor T-cells drop-in. Herein, we summarize features, functions, and interactions among CAFs, tumor-associated ECM, and immune cells in TME. Moreover, we discuss the strategies targeting CAFs and the remodeling of the ECM to improve immunotherapy and drug therapies.

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.

Abstract 4561: 3D Reconstructed Pancreas: A model capturing the unique tumor microenvironment and stromal architecture of pancreatic cancer

Abstract As it is rarely discovered early, the prognosis for people with pancreatic cancer is poor, with an average 5-year survival rate of 10% and only 3% for those with metastatic illness. Pancreatic cancer patients have few therapeutic options and innovative therapies are sorely needed to improve treatment of the disease. This is mainly due to the late diagnosis and partially due to the biology of the disease. Capturing the components of the tumor microenvironment, which serve as both a physical barrier and a source of stromal-driven resistance to therapeutics, is one of the issues faced by drug developers searching for agents to combat pancreatic cancer. We have created a 3D model of the pancreatic tumor microenvironment, the Reconstructed Pancreas (r-Pancreas), using Mia PaCa-2, PANC-1, and BxPC-3 pancreatic tumor cell lines embedded in an extracellular matrix (ECM) that was formulated to recapitulate the typical ECM of pancreatic tumors. We have shown that gemcitabine was effective against pancreatic tumor cells cultured in r-Pancreas (IC50=0.4-0.8µM), while 5-fluorouracil was ineffective (IC50 not reached), mimicking clinical response. In nude mice, subcutaneous PANC-1 tumors exhibited similar responses to gemcitabine and 5-fluorocurcil. Treatment with gemcitabine resulted in a Day 63 median ΔT/ΔC of 37% while 5-fluorouracil resulted in a Day 63 median delta ΔT/ΔC of 102%. Incorporating a collagen-rich capsule increases the physiological relevance of the r-Pancreas model because it has been shown that human pancreatic tumors have an outer layer of stiff ECM that functions as a physical barrier and prevents drugs from penetrating the tumor. Recently, we showed that, at least in the Mia PaCa-2 and BxPC-3 tumor cell lines, introducing such a collagen capsule dramatically reduced sensitivity to gemcitabine. Disrupting the collagen capsule with MMP-9 restored the sensitivity of the tumor cells to gemcitabine demonstrating that the capsule provides a physical barrier to drug entry. Additionally, creating a co-culture of primary activated pancreatic stromal cells and pancreatic tumor cell lines produces a more complete 3D model that permits the testing of potential therapeutic agents inside the pancreatic tumor microenvironment. Hence, we have created a 3D model where pancreatic tumor cells were co-cultured with human pancreatic fibroblasts. According to our results, the IC50 values for pancreatic tumor cell lines treated with gemcitabine increased by twofold when co-cultured with activated pancreatic fibroblasts compared to control sets of tumor cells alone (IC50=1-3µM). Together, these data imply that, to be clinically relevant, in vitro models of pancreatic cancer must integrate tumor-specific components of the microenvironment, including the collagen capsule and activated fibroblasts. Citation Format: Arnat Balabyev, Justin D. Phillips, Michael Steffey, Eleanore J. Kirshner, Aayushi Ahlawat, Arlette H. Uihlein, Daniel Saims, Scott Wise, Julia Kirshner. 3D Reconstructed Pancreas: A model capturing the unique tumor microenvironment and stromal architecture of pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4561.

Matrix Stiffness Influences Tubular Formation in Renal Tissue Engineering

Tubular structures contribute to essential organ functions. Therefore, controlling tubulogenesis is essential for bottom-up tissue engineering approaches. Tissue engineering strategies to form tubular structures utilize extracellular matrix (ECM) components and micropatterned molds. To improve the tubular formation rate, we studied the substrate stiffness’s influence on the tubulogenesis of murine inner medullary collecting duct (mIMCD) cells. mIMCD cells were seeded in micropatterned molds with different compositions of polydimethylsiloxane (PDMS) (1:5, 1:10, 1:15, 1:20, 1:30) and agarose (1%, 2%, 3% 5%). We established the Young’s modulus of the PDMS and agarose substrates and determined the ideal substrate stiffness for tube formation to be between 277 kPa and 2610 kPa. Within our parameters, optimal tube formation was observed at 439.9 kPa, a value similar to the Young’s Modulus found in the basement membrane of the murine renal tubular compartment. We also found that different substrate concentrations of agarose or PDMS are associated with different expression levels of the apical polarization marker Zonula occludens 1 (ZO-1) in the generated tubular structures. In addition to the substrate stiffness, we observed that the tube formation differed based on the substrate material, with agarose showing a generally greater tube formation rate. While previous research demonstrated that ECM stiffness influences cellular behavior towards tube formation, our results suggest that the stiffness of the substrate influences tubular formation independently of the ECM.

Preprint Highlight: Constriction forces imposed by basement membranes regulate developmental cell migration

Extracellular matrix (ECM) stiffness affects many processes of ECM-associated cells, such as migration and differentiation. Whether ECM mechanical properties also affect distant cell populations remains largely unexplored. This preprint studies migration of border cells within Drosophila ovarian follicles as a model system. Border cells migrate toward the center of the follicle by moving between nurse cells, which are surrounded by follicle cells that are themselves encased by the basement membrane ECM. Decreasing stiffness of the follicle ECM reduced cortical tension of follicle cells, and of nurse cells indirectly, accelerating border cell migration. Increasing follicle cell cortical tension slowed border cell migration. These results may inform how ECM stiffness affects cell migration within other complex tubular tissues, such as blood vessels. This preprint has been assigned the following badges: New Hypothesis, New Materials, Cross-Validation Read the preprint on bioRxiv ( Molina López et al., 2022 ): https://doi.org/10.1101/2022.12.19.520983 .

Matrix stiffness regulates tumor cell intravasation through expression and ESRP1-mediated alternative splicing of MENA

During intravasation, cancer cells cross the endothelial barrier and enter the circulation. Extracellular matrix stiffening has been correlated with tumor metastatic potential; however, little is known about the effects of matrix stiffness on intravasation. Here, we utilize invitro systems, a mouse model, specimens from patients with breast cancer, and RNA expression profiles from The Cancer Genome Atlas Program (TCGA) to investigate the molecular mechanism by which matrix stiffening promotes tumor cell intravasation. Our data show that heightened matrix stiffness increases MENA expression, which promotes contractility and intravasation through focal adhesion kinase activity. Further, matrix stiffening decreases epithelial splicing regulatory protein 1 (ESRP1) expression, which triggers alternative splicing of MENA, decreases the expression of MENA11a, and enhances contractility and intravasation. Altogether, our data indicate that matrix stiffness regulates tumor cell intravasation through enhanced expression and ESRP1-mediated alternative splicing of MENA, providing a mechanism by which matrix stiffness regulates tumor cell intravasation.

Nondestructive ultrasound evaluation of microstructure-related material parameters of skeletal muscle: an in silico and in vitro study

Direct and nondestructive assessment of material properties of skeletal muscle in vivo shall advance our understanding of intact muscle mechanics and facilitate personalized interventions. However, this is challenged by intricate hierarchical microstructure of the skeletal muscle. We have previously regarded the skeletal muscle as a composite of myofibers and extracellular matrix (ECM), formulated shear wave propagation in the undeformed muscle using the acoustoelastic theory, and preliminarily demonstrated that ultrasound-based shear wave elastography (SWE) could estimate microstructure-related material parameters (MRMPs): myofiber stiffness μf, ECM stiffness μm, and myofiber volume ratio Vf. The proposed method warrants further validation but is hampered by the lack of ground truth values of MRMPs. In this study, we presented analytical and experimental validations of the proposed method using finite-element (FE) simulations and 3D-printed hydrogel phantoms, respectively. Three combinations of different physiologically relevant MRMPs were used in the FE simulations where shear wave propagations in the corresponding composite media were simulated. Two 3D-printed hydrogel phantoms with the MRMPs close to those of a real skeletal muscle (i.e., μf=2.02kPa, μm=52.42kPa, and Vf=0.675,0.832) for ultrasound imaging were fabricated by an alginate-based hydrogel printing protocol that we modified and optimized from the freeform reversible embedding of suspended hydrogels (FRESH) method in literature. Average percent errors of (μf,μm,Vf) estimates were found to be (2.7%,7.3%,2.4%)in silico and (3.0%,8.0%,9.9%)in vitro. This quantitative study corroborated the potential of our proposed theoretical model along with ultrasound SWE for uncovering microstructural characteristics of the skeletal muscle in an entirely nondestructive way.

Echinatin maintains glutathione homeostasis in vascular smooth muscle cells to protect against matrix remodeling and arterial stiffening

Decreased vascular compliance of the large arteries as indicated by increased pulse wave velocity is shown to be associated with atherosclerosis and the related cardiovascular events. The positive correlation between arterial stiffening and disease progression derives a hypothesis that softening the arterial wall may protect against atherosclerosis, despite that the mechanisms controlling the cellular pathological changes in disease progression remain unknown. Here, we established a mechanical-property-based screening to look for compounds alleviating the arterial wall stiffness through their actions on the interaction between vascular smooth muscle cells (VSMCs) and the wall extracellular matrix (ECM). We found that echinatin, a chalcone preferentially accumulated in roots and rhizomes of licorice (Glycyrrhiza inflata), reduced the stiffness of ECM surrounding cultured VSMCs. We examined the potential beneficial effects of echinatin on mitigating arterial stiffening and atherosclerosis, and explored the mechanistic basis by which the compound exert the effects. Administration of echinatin in mice fed on an adenine diet and in hyperlipidemia mice subjected to 5/6 nephrectomy mitigated arterial stiffening and atherosclerosis. Mechanistic insights were gained from the RNA-sequencing results showing that echinatin upregulated the expression of glutamate cysteine ligases (GCLs), both the catalytic (GCLC) and modulatory (GCLM) subunits. Further study indicated that upregulation of GCLC/GCLM in VSMCs by echinatin maintains the homeostasis of glutathione (GSH) metabolism; adequate availability of GSH is critical for counteracting arterial stiffening. As a consequence of regulating the GSH synthesis, echinatin inhibits ferroptosis and matrix remodeling that being considered two contributors of arterial stiffening and atherosclerosis. These data demonstrate a pivotal role of GSH dysregulation in damaging the proper VSMC-ECM interaction and uncover a beneficial activity of echinatin in preventing vascular diseases.

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.

Extracellular Matrix-Derived Biophysical Cues Mediate Interstitial Flow-Induced Sprouting Angiogenesis.

Sprouting angiogenesis is orchestrated by an intricate balance of biochemical and mechanical cues in the local tissue microenvironment. Interstitial flow has been established as a potent regulator of angiogenesis. Similarly, extracellular matrix (ECM) physical properties, such as stiffness and microarchitecture, have also emerged as important mediators of angiogenesis. However, the interplay between interstitial flow and ECM physical properties in the initiation and control of angiogenesis is poorly understood. Using a three-dimensional (3D) microfluidic tissue analogue of angiogenic sprouting with defined interstitial flow superimposed over ECM with well-characterized physical properties, we found that the addition of hyaluronan (HA) to collagen-based matrices significantly enhances sprouting induced by interstitial flow compared to responses in collagen-only hydrogels. We confirmed that both the stiffness and matrix pore size of collagen-only hydrogels were increased by the addition of HA. Interestingly, interstitial flow-potentiated sprouting responses in collagen/HA matrices were not affected when functionally blocking the HA receptor CD44. In contrast, enzymatic depletion of HA in collagen/HA matrices with hyaluronidase (HAdase) resulted in decreased stiffness, pore size, and interstitial flow-mediated sprouting to the levels observed in collagen-only matrices. Taken together, these results suggest that HA enhances interstitial flow-mediated angiogenic sprouting through its alterations to collagen ECM stiffness and pore size.

The extracellular matrix of dystrophic mouse diaphragm accounts for the majority of its passive stiffness and is resistant to collagenase digestion

The healthy skeletal muscle extracellular matrix (ECM) has several functions including providing structural integrity to myofibers, enabling lateral force transmission, and contributing to overall passive mechanical properties. In diseases such as Duchenne Muscular dystrophy, there is accumulation of ECM materials, primarily collagen, which results in fibrosis. Previous studies have shown that fibrotic muscle is often stiffer than healthy muscle, in part due to the increased number and altered architecture of collagen fibers within the ECM. This would imply that the fibrotic matrix is stiffer than the healthy matrix. However, while previous studies have attempted to quantify the extracellular contribution to passive stiffness in muscle, the outcomes are dependent on the type of method used. Thus, the goals of this study were to compare the stiffness of healthy and fibrotic muscle ECM and to demonstrate the efficacy of two methods for quantifying extracellular-based stiffness in muscle, namely decellularization and collagenase digestion. These methods have been demonstrated to remove the muscle fibers or ablate collagen fiber integrity, respectively, while maintaining the contents of the extracellular matrix. Using these methods in conjunction with mechanical testing on wildtype and D2.mdx mice, we found that a majority of passive stiffness in the diaphragm is dependent on the ECM, and the D2.mdx diaphragm ECM is resistant to digestion by bacterial collagenase. We propose that this resistance is due to the increased collagen cross-links and collagen packing density in the ECM of the D2.mdx diaphragm. Taken altogether, while we did not find increased stiffness of the fibrotic ECM, we did observe that the D2.mdx diaphragm conveyed resistance against collagenase digestion. These findings demonstrate how different methods for measuring ECM-based stiffness each have their own limitations and can produce different results.

Effect of Extracellular Matrix Stiffness on Candesartan Efficacy in Anti-Fibrosis and Antioxidation.

Myocardial fibrosis progression and imbalanced redox state are closely associated with increased extracellular matrix (ECM) stiffness. Candesartan (CAN), an angiotensin II (Ang II) receptor inhibitor, has shown promising anti-fibrosis and antioxidant efficacy in previous cardiovascular disease studies. However, the effect of ECM stiffness on CAN efficacy remains elusive. In this study, we constructed rat models with three different degrees of myocardial fibrosis and treated them with CAN, and then characterized the stiffness, cardiac function, and NADPH oxidase-2 (NOX2) expression of the myocardial tissues. Based on the obtained stiffness of myocardial tissues, we used polyacrylamide (PA) gels with three different stiffness to mimic the ECM stiffness of cardiac fibroblasts (CFs) at the early, middle, and late stages of myocardial fibrosis as the cell culture substrates and then constructed CFs mechanical microenvironment models. We studied the effects of PA gel stiffness on the migration, proliferation, and activation of CFs without and with CAN treatment, and characterized the reactive oxygen species (ROS) and glutathione (GSH) levels of CFs using fluorometry and scanning electrochemical microscopy (SECM). We found that CAN has the best amelioration efficacy in the cardiac function and NOX2 levels in rats with medium-stiffness myocardial tissue, and the most obvious anti-fibrosis and antioxidant efficacy in CFs on the medium-stiffness PA gels. Our work proves the effect of ECM stiffness on CAN efficacy in myocardial anti-fibrosis and antioxidants for the first time, and the results demonstrate that the effect of ECM stiffness on drug efficacy should also be considered in the treatment of cardiovascular diseases.

Interplay Between Extracellular Matrix Remodeling and Angiogenesis in Tumor Ecosystem.

Studying the complex mechanisms of tumorigenesis and examining the interactions of neoplastic cells within tumor ecosystem are critical to explore the possibility of effective cancer treatment modalities. Dynamic tumor ecosystem is constantly evolving and is composed of tumor cells, extracellular matrix (ECM), secreted factors, and stromal cancer-associated fibroblasts (CAF), pericytes, endothelial cells (EC), adipocytes, and immune cells. ECM remodeling by synthesis, contraction, and/or proteolytic degradation of ECM components and release of matrix-sequestered growth factors create a microenvironment that promotes EC proliferation, migration, and angiogenesis. Stromal CAFs release multiple angiogenic cues (angiogenic growth factors, cytokines, and proteolytic enzymes) which interact with ECM proteins, thus contribute to enhance proangiogenic/promigratory properties and support aggressive tumor growth. Targeting angiogenesis brings about vascular changes including reduced adherence junction proteins, basement membrane and pericyte coverage, and increased leakiness. This facilitates ECM remodeling, metastatic colonization and chemoresistance. Owing to significant role of denser and stiffer ECM in inducing chemoresistance, direct or indirect targeting of ECM components is being reported as major axis of anticancer treatment. Exploring the agents targeting angiogenesis and ECM in a context specific manner may lead to reduced tumor burden by promoting conventional therapeutic effectiveness and overcoming the hurdles of therapy resistance.

Abstract P5-11-01: Epithelial-Mesenchymal Plasticity is Regulated by Inflammatory Signaling Networks Coupled to Cell Morphology

Abstract Purpose: During development and homeostatic processes such as wound repair, certain cells undergo a remarkable process where they radically transform in cell shape and state, from epithelial to mesenchymal cells. This ability is referred to as ‘Epithelial-Mesenchymal Plasticity’ (EMP), triggered by both mechanical (i.e. loss of cell-cell contact) and soluble cues (i.e. TGFβ), and is absolutely essential in both embryonic and adult organisms. Dysregulation of EMP also occurs in cancer; where tumor cells undergo EMP to become metastatic, stem-like, and drug resistant. Critically, increased EMP correlates with increased cancer severity. However, it is largely a black box as to how EMP is regulated and how epithelial cells sense physical, geometrical, and soluble cues in their environment to assume a mesenchymal fate. This work inferred that cell shape is a determinant of not only fates – but of long-term outcomes. That is, we provide mechanistic explanations between cell context, environment, and cancer severity. In this work, we investigated how mechanical and soluble cues are coupled to the dynamics of signaling pathways that regulate transcriptional and post-transcriptional events that underpin EMP. Especially with regards to mechanical cues, we attempt to unlock the ‘black box’ as to how changes in adhesion, ECM (Extracellular Matrix) stiffness, and environment geometry are coupled to the transcriptional events that drive EMP. Results: We show that changes in cell and nuclear shape result from the actions of the cytoskeleton and important drivers of EMP in upregulating ‘interlocking’ networks that promote EMP-driving inflammation and suppressing insulin signaling. Using a combination of cell biology, proteomics, and new statistical methods, we provide a systems biology model demonstrating: Cell shape → MT bound Kinesin-1 activity and nuclear shape → inflammation (IKK, JNK), insulin signaling (IRS), and YAP/TAZ → EMP Our work connects observable changes in phenotype to causal changes in signaling network architecture and cell fates. We used an integrative -omic approach to analyze tumors from breast cancer patients. We identified a novel tumor suppressor – JAM3 – whose loss is associated with altered nuclear shape in vivo, inhibiting JAM3 in cells, or stimulation with canonical EMP inducer TGFβ to promote EMP, and thus changes the organization of microtubules and alters nuclear shape. During EMP we observe there is upregulation of a pro-inflammatory, insulin resistant, signaling network that is predictive of mesenchymal states across cancer. EMP following JAM3 depletion and/or TGFβ stimulation is rescued by inhibition of Kinesin-1 motors. This rescue is explained by changes in inflammatory and insulin signaling. We show that while Kinesin-1 activity is responsible for upregulation in canonical signalling and network ‘hubs’, changes in nuclear shape upregulate ‘effectors’ of these hubs. Thus, microtubules and nuclei differentially regulate different parts of ‘interlocking’ networks. Conclusions: This work has integrated image-omics, comprehensive global proteomics, and quantitative cell biology to provide a mechanistic ¬and systems-level understanding of how epithelial cells differentiate into mesenchymal forms during disease development and progression. This work is of major significance for three reasons. First, it shows how cell shape can mechanistically regulate cell fates on an unprecedented systems-level. Second, we identify an EMP network that is conserved across cancers and may indeed be conserved across both normal and diseased mesenchymal cells. Indeed, we speculate that different types of diseased cells may all share the same network. Finally, we introduce the concept of interlocking networks – where hubs and effectors are regulated by different cellular components. Our work has been extensively validated, using chemical and genetic approaches and in vivo model of in human breast cancer. Citation Format: Zheng Yin. Epithelial-Mesenchymal Plasticity is Regulated by Inflammatory Signaling Networks Coupled to Cell Morphology [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-11-01.

Research Progress of Hippo-Yap/Taz Signaling in Skeletal Muscle Hypertrophy

The Hippo pathway is an important pathway to control cell proliferation and organ size. By sensing extracellular matrix stiffness and intercellular mechanical force and converting them into molecular signal transduction, through a series of cascade amplification effects of kinases, the final the phosphorylation of YAP/TAZ, the main downstream target of the Hippo pathway, plays a series of regulatory roles. Although many studies have shown that Hippo signaling changes during skeletal muscle hypertrophy, the complex mechanism of Hippo signaling in skeletal muscle hypertrophy remains to be further studied. In this review, we summarize the relationship between Hippo-YAP/TAZ signaling and skeletal muscle hypertrophy and sort out the contribution of the main factors in the process of skeletal muscle hypertrophy.

Enhanced Expression of a Novel Lamin A/C Splice Variant in Idiopathic Pulmonary Fibrosis Lung.

In idiopathic pulmonary fibrosis (IPF), the normal delicate lung architecture is replaced with rigid extracellular matrix (ECM) as a result of the accumulation of activated myofibroblasts and excessive deposition of ECM. Lamins have a role in fostering mechanosignaling from the ECM to the nucleus. Although there is a growing number of studies on lamins and associated diseases, there are no prior reports linking aberrations in lamins with pulmonary fibrosis. Here, we discovered, through analysis of RNA sequencing data, a novel isoform of lamin A/C that is more highly expressed in IPF compared with control lung. This novel LMNA (lamin A/C) splice variant includes retained introns 10 and 11 and exons 11 and 12 as documented by rapid amplification of cDNA ends. We found that this novel isoform is induced by stiff ECM. To better clarify the specific effects of this novel isoform of lamin A/C and how it may contribute to the pathogenesis of IPF, we transduced the lamin transcript into primary lung fibroblasts and alveolar epithelial cells and found that it impacts several biological effects, including cell proliferation, senescence, cell contraction, and the transition of fibroblasts to myofibroblasts. We also observed that type II epithelial cells and myofibroblasts in the IPF lung exhibited wrinkled nuclei, and this is notable because this has not been previously described and is consistent with laminopathy-mediated cellular effects.

Matrix stiffness induces epithelial-to-mesenchymal transition via Piezo1-regulated calcium flux in prostate cancer cells

Cells utilize calcium channels as one of the main signaling mechanisms to sense changes in the extracellular space and convert these changes to intracellular signals. Calcium regulates several key signaling networks, such as the induction of EMT. The current study expands on the understanding of how EMT is controlled via the mechanosensitive calcium channel Piezo1 in cancerous cells, which senses changes in the extracellular matrix stiffness. We model the biophysical environment of healthy and cancerous prostate tissue using polyacrylamide gels of different stiffnesses. Significant increases in calcium steady-state concentration, vimentin expression, and aspect ratio, and decreases in E-cadherin expression were observed by increasing matrix stiffness and also after treatment with Yoda1, a chemical agonist of Piezo1. Overall, this study concludes that Piezo1-regulated calcium flux plays a role in prostate cancer cell metastatic potential by sensing changes in ECM stiffness and modulating EMT markers.

Extended preconditioning on soft matrices directs human mesenchymal stem cell fate via YAP transcriptional activity and chromatin organization.

Dynamic extracellular matrix (ECM) mechanics plays a crucial role in tissue development and disease progression through regulation of stem cell behavior, differentiation, and fate determination. Periodontitis is a typical case characterized by decreased ECM stiffness within diseased periodontal tissues as well as with irreversible loss of osteogenesis capacity of periodontal tissue-derived human periodontal tissue-derived MSCs (hMSCs) even returning back to a physiological mechanical microenvironment. We hypothesized that the hMSCs extendedly residing in the soft ECM of diseased periodontal tissues may memorize the mechanical information and have further effect on ultimate cell fate besides the current mechanical microenvironment. Using a soft priming and subsequent stiff culture system based on collagen-modified polydimethylsiloxane substrates, we were able to discover that extended preconditioning on soft matrices (e.g., 7 days of exposure) led to approximately one-third decrease in cell spreading, two-third decrease in osteogenic markers (e.g., RUNX2 and OPN) of hMSCs, and one-thirteenth decrease in the production of mineralized nodules. The significant loss of osteogenic ability may attribute to the long-term residing of hMSCs in diseased periodontal tissue featured with reduced stiffness. This is associated with the regulation of transcriptional activity through alterations of subcellular localization of yes-associated protein and nuclear feature-mediated chromatin organization. Collectively, we reconstructed phenomena of irreversible loss of hMSC osteogenesis capacity in diseased periodontal tissues in our system and revealed the critical effect of preconditioning duration on soft matrices as well as the potential mechanisms in determining ultimate hMSC fate.

Stiff matrix induces exosome secretion to promote tumour growth.

Tissue fibrosis and extracellular matrix (ECM) stiffening promote tumour progression. The mechanisms by which ECM regulates its contacting cells have been extensively studied. However, how stiffness influences intercellular communications in the microenvironment for tumour progression remains unknown. Here we report that stiff ECM stimulates the release of exosomes from cancer cells. We delineate a molecular pathway that links stiff ECM to activation of Akt, which in turn promotes GTP loading to Rab8 that drives exosome secretion. We further show that exosomes generated from cells grown on stiff ECM effectively promote tumour growth. Proteomic analysis revealed that the Notch signalling pathway is activated in cells treated with exosomes derived from tumour cells grown on stiff ECM, consistent with our gene expression analysis of liver tissues from patients. Our study reveals a molecular mechanism that regulates exosome secretion and provides insight into how mechanical properties of the ECM control the tumour microenvironment for tumour growth.

Effects of biomechanical and biochemical stimuli on angio- and vasculogenesis in a complex microvasculature-on-chip

The endothelium of blood vessels is a vital organ that reacts differently to subtle changes in stiffness and mechanical forces exerted on its environment (extracellular matrix (ECM)). Upon alteration of these biomechanical cues, endothelial cells initiate signaling pathways that govern vascular remodeling. The emerging organs-on-chip technologies allow the mimicking of complex microvasculature networks, identifying the combined or singular effects of these biomechanical or biochemical stimuli. Here, we present a microvasculature-on-chip model to investigate the singular effect of ECM stiffness and mechanical cyclic stretch on vascular development. Following two different approaches for vascular growth, the effect of ECM stiffness on sprouting angiogenesis and the effect of cyclic stretch on endothelial vasculogenesis are studied. Our results indicate that ECM hydrogel stiffness controls the size of the patterned vasculature and the density of sprouting angiogenesis. RNA sequencing shows that the cellular response to stretching is characterized by the upregulation of certain genes such as ANGPTL4+5, PDE1A, and PLEC.