Effects Of Extracellular Matrix Stiffness Research Articles

Increased stiffness of extracellular matrix enhanced chemoresistance in 3D-bioprinted ovarian cancer model

Ovarian cancer is a gynecological malignancy with a high mortality rate. The ovarian cancer microenvironment is a crucial factor affecting the overall and progression-free survival rates of patients with ovarian cancer. The biophysical factors of the tumor microenvironment, such as stiffness, can affect the gene expression and behavior of tumor cells. In this study, we utilized 3D bioprinting technology to construct ovarian cancer tumor models with varying levels of stiffness in vitro to investigate the effect of extracellular matrix stiffness on drug resistance of tumor cells. Our findings indicate that increasing the stiffness of extracellular matrix can attenuate the sensitivity of tumor cells to chemotherapeutic agents. Additionally, the increased stiffness of 3D tumor model may promote malignant phenotypes, such as tumor stemness and tumor progression.

The role of matrix stiffness in breast cancer progression: a review.

The significance of matrix stiffness in cancer development has been investigated in recent years. The gradual elastic force the extracellular matrix imparts to cells, known as matrix stiffness, is one of the most important types of mechanical stimulation. Increased matrix stiffness alters the biological activity of cells, which promotes the growth of numerous malignancies, including breast cancer. Comprehensive studies have demonstrated that increasing matrix stiffness activates molecular signaling pathways that are closely linked to breast cancer progression. There are many articles exploring the relationship between mechanism hardness and breast cancer, so we wanted to provide a systematic summary of recent research advances. In this review, we briefly introduce the mechanism of matrix stiffness in breast cancer, elaborate on the effect of extracellular matrix stiffness on breast cancer biological behavior and signaling pathways, and finally, we will talk about breast cancer treatment that focuses on matrix stiffness.

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.

Abstract 5696: Effect of extracellular matrix stiffness on cancer cells radiation response

Abstract Over the past decade, it has become clear that the mechanical cues of extracellular matrix (ECM) are a major regulator of the cancer biology. In particular, ECM stiffness has shown an important role in chemoresistance modulation. However, despite the central role of radiotherapy in cancer management, little is known about the effect of ECM stiffness on cancer cells radiation response. In this study, we used polyacrylamide collagen-coated hydrogel model to reproduce a soft (0.7 kPa) and a stiff (70 kPa) matrix. The model was validated by atomic force microscopy force spectroscopy and biological analyses through cellular morphology, proliferation and YAP/TAZ mechanotransduction pathway that all confirmed the accuracy of our model. Then, lung cancer cells seeded on both soft and stiff hydrogels were irradiated, and the level of DNA damages were compared between the two conditions. First, alkaline comet assay revealed the total amount of DNA damages were higher in cells grown on a rigid ECM at 1 h post-irradiation than cells seeded on a soft matrix. Second, γH2AX/53BP1 foci assay showed that the number of DNA double strand breaks were also higher in cells grown on a rigid environment. However, the level of DNA damage was identical at 24 h post-irradiation for both stiffness, suggesting that a more efficient DNA damage repair response could take place for cells seeded in stiff matrix. In order to explain these observations, analyses on chromatin structure and nucleus shape were then performed. Data showed that cells cultured on stiff matrix displayed a larger nucleus, less condensed chromatin, and a decreased expression level of class I HDACs genes. Moreover, it was found that the level of total reactive oxygen species (ROS) and superoxide ion was also higher in cells grown on a rigid environment 24 h post-irradiation. Altogether these data suggest that a high stiffness may increase early DNA damages after irradiation, partially by remodeling chromatin that could expose DNA to radiation-induced genotoxic agents such as ROS. Additional investigations are required to understand better the mechanisms that could explain such differences, but these preliminary data highlighted the potential role of mechanical stress in cellular radiation response and paved the way to new therapeutic target for radiotherapy management. Citation Format: Jerome Lacombe, Melinda Muccio, Frederic Zenhausern. Effect of extracellular matrix stiffness on cancer cells radiation response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5696.

In situ monitoring of functional activity of extracellular matrix stiffness-dependent multidrug resistance protein 1 using scanning electrochemical microscopy.

Extracellular matrix (ECM) stiffness affects the drug resistance behavior of cancer cells, while multidrug resistance protein 1 (MRP1) on the cell membrane confers treatment resistance via actively transporting drugs out of cancer cells. However, the relationship between ECM stiffness and MRP1 functional activity in cancer cells remains elusive, mainly due to the technical challenge of in situ monitoring. Herein, we engineered in vitro cancer cell models using breast cancer cells (MCF-7 and MDA-MB-231 cells) as the reprehensive cells on polyacrylamide (PA) gels with three stiffness, mimicking different developmental stages of cancer. We in situ characterized the functional activity of MRP1 and investigated the effect of ECM stiffness on MRP1 of cancer cells before and after vincristine treatment using scanning electrochemical microscopy (SECM) with ferrocenecarboxylic acid (FcCOOH) as the redox mediator and endogenous glutathione (GSH) as the indicator. The SECM results show that the functional activity of MRP1 is enhanced with increasing ECM stiffness, and the MRP1-mediated vincristine efflux activity of MCF-7 cells is more affected by ECM stiffness than that of MDA-MB-231 cells. This work, for the first time, applied SECM to in situ and quantitatively monitor the functional activity of MRP1 in cancer cells in different tumor mechanical microenvironments, which could help to elucidate the mechanism of matrix stiffness-dependent drug resistance behavior in cancer cells.

Influence of Substrate Stiffness on Barrier Function in an iPSC-Derived In Vitro Blood-Brain Barrier Model.

Vascular endothelial cells respond to a variety of biophysical cues such as shear stress and substrate stiffness. In peripheral vasculature, extracellular matrix (ECM) stiffening alters barrier function, leading to increased vascular permeability in atherosclerosis and pulmonary edema. The effect of ECM stiffness on blood-brain barrier (BBB) endothelial cells, however, has not been explored. To investigate this topic, we incorporated hydrogel substrates into an in vitro model of the human BBB. Induced pluripotent stem cells were differentiated to brain microvascular endothelial-like (BMEC-like) cells and cultured on hydrogel substrates of varying stiffness. Cellular changes were measured by imaging, functional assays such as transendothelial electrical resistance (TEER) and p-glycoprotein efflux activity, and bulk transcriptome readouts. The magnitude and longevity of TEER in iPSC-derived BMEC-like cells is enhanced on compliant substrates. Quantitative imaging shows that BMEC-like cells form fewer intracellular actin stress fibers on substrates of intermediate stiffness (20 kPa relative to 1 and 150 kPa). Chemical induction of actin polymerization leads to a rapid decline in TEER, agreeing with imaging readouts. P-glycoprotein activity is unaffected by substrate stiffness. Modest differences in RNA expression corresponding to specific signaling pathways were observed as a function of substrate stiffness. iPSC-derived BMEC-like cells exhibit differences in passive but not active barrier function in response to substrate stiffness. These findings may provide insight into BBB dysfunction during neurodegeneration, as well as aid in the optimization of more complex three-dimensional neurovascular models utilizing compliant hydrogels. The online version contains supplementary material available at 10.1007/s12195-021-00706-8.

Extracellular Matrix Stiffness Regulates DNA Methylation by PKCα-Dependent Nuclear Transport of DNMT3L.

Extracellular matrix (ECM) stiffness has profound effects on the regulation of cell functions. DNA methylation is an important epigenetic modification governing gene expression. However, the effects of ECM stiffness on DNA methylation remain elusive. Here, it is reported that DNA methylation is sensitive to ECM stiffness, with a global hypermethylation under stiff ECM condition in mouse embryonic stem cells (mESCs) and embryonic fibroblasts compared with soft ECM. Stiff ECM enhances DNA methylation of both promoters and gene bodies, especially the 5' promoter regions of pluripotent genes. The enhanced DNA methylation is functionally required for the loss of pluripotent gene expression in mESCs grown on stiff ECM. Further experiments reveal that the nuclear transport of DNA methyltransferase 3-like (DNMT3L) is promoted by stiff ECM in a protein kinase C α (PKCα)-dependent manner and DNMT3L can be binding to Nanog promoter regions during cell-ECM interactions. These findings unveil DNA methylation as a novel target for the mechanical sensing mechanism of ECM stiffness, which provides a conserved mechanism for gene expression regulation during cell-ECM interactions.

Matrix Stiffness and Colorectal Cancer.

In recent years, a growing consensus is emerging that the mechanical microenvironment of tumors is far more critical in the onset of tumor, tumor progression, invasion, and metastasis. Matrix stiffness, one of the sources of mechanical stimulation, affects tumor cells as well as non-tumor cells in multiple different molecular signaling pathways in solid tumors such as colorectal tumors, which lead to tumor invasion and metastasis, immune evasion and drug resistance. This review will illustrate the relationship between matrix stiffness and colorectal cancer from the following aspects. First, briefly introduce the mechanical microenvironment and colorectal cancer, then explain the origin of colorectal cancer extracellular matrix stiffness, and then synthesize the study of matrix stiffness of colorectal cancer in recent years to elaborate the effects of extracellular matrix stiffness in colorectal cancer’s biological behavior and signaling pathways, and finally we will discuss the transformation treatment for the matrix stiffness of colorectal cancer. An in-depth understanding of matrix stiffness and colorectal cancer can help researchers conduct further experiments to find new targets for the treatment of colorectal cancer.

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells.

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types' responses to matrix stiffness have been characterized, how adherent cells' susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

A Multi-well Format Polyacrylamide-based Assay for Studying the Effect of Extracellular Matrix Stiffness on the Bacterial Infection of Adherent Cells

Extracellular matrix stiffness comprises one of the multiple environmental mechanical stimuli that are well known to influence cellular behavior, function, and fate in general. Although increasingly more adherent cell types' responses to matrix stiffness have been characterized, how adherent cells' susceptibility to bacterial infection depends on matrix stiffness is largely unknown, as is the effect of bacterial infection on the biomechanics of host cells. We hypothesize that the susceptibility of host endothelial cells to a bacterial infection depends on the stiffness of the matrix on which these cells reside, and that the infection of the host cells with bacteria will change their biomechanics. To test these two hypotheses, endothelial cells were used as model hosts and Listeria monocytogenes as a model pathogen. By developing a novel multi-well format assay, we show that the effect of matrix stiffness on infection of endothelial cells by L. monocytogenes can be quantitatively assessed through flow cytometry and immunostaining followed by microscopy. In addition, using traction force microscopy, the effect of L. monocytogenes infection on host endothelial cell biomechanics can be studied. The proposed method allows for the analysis of the effect of tissue-relevant mechanics on bacterial infection of adherent cells, which is a critical step towards understanding the biomechanical interactions between cells, their extracellular matrix, and pathogenic bacteria. This method is also applicable to a wide variety of other types of studies on cell biomechanics and response to substrate stiffness where it is important to be able to perform many replicates in parallel in each experiment.

Single-cell study of the extracellular matrix effect on cell growth by in situ imaging of gene expression.

Cell behaviors are known to be regulated by the cellular microenvironment. Traditional cell-population based analysis methods need to separate cells from their extracellular matrix (ECM) and cannot resolve the heterogeneity of cell behaviors. Herein, an in situ single-cell analysis method based on rolling circle amplification was exploited to image gene expression in single cells for investigating the effect of ECM stiffness on cell growth. This method enables the simultaneous quantifying of the cell phenotype and gene expression at the single-cell level, which can help in understanding the underlying molecular mechanism of cell growth. It is found that ECM stiffness could affect cell growth via regulating the expression level of the cytoskeleton-assembly associated genes PFN1 and CFL1 and their co-expression pattern. Therefore, this single-cell analysis platform may facilitate us to tap into the study of "single-cell phenotypes" and elucidate the disease association of ECMs.

Abstract 12: The Effect of Extracellular Matrix Stiffness on Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation

Differentiation of human mesenchymal stem cells (hMSCs) has been shown to be influenced by the surrounding microenvironment. It is important to understand the physiological implications of the hMSC microenvironment regarding differentiation within the body, especially in patients with cardiovascular disease. We are interested in understanding the influence of differing extracellular matrix (ECM) stiffness found in the body that the MSC encounters during its journey from the bone marrow to the infarct scar in patients recovering from a myocardial infarction. As hMSCs must respond rapidly to their environment, we also investigated the influence of microRNA at different surface tensions. HMSCs were isolated from the bone marrow of patients undergoing open heart surgery and cultured in standard DMEM/F12 with 20% FBS. We plated these cells on fibronectin-coated plates with surface tensions of 2kPa, simulating bone marrow; 15kPa, simulating left ventricle; and 100kPa, simulating a fibrotic environment. Protein and mRNA were collected for further analysis. Our data have revealed that softer surface tensions, representing a bone marrow-undifferentiating environment, cause a decrease in the protein expression of EDA-fibronectin and alpha-smooth muscle actin. There were also increases in mRNA of myosin heavy chain-9 and 10, and collagen-1. Softer surface tensions also show a slight increase in miR-301a, although at 15kPa, miR-301a expression is increased even further. Our lab has previously shown that miR-301a is involved in maintaining a proliferative phenotype of hMSCs. Interestingly, Dicer1, responsible for processing microRNAs, is upregulated at softer surface tensions and attenuated at 15kPa. Dicer1 mRNA expression is attenuated at 15kPa. These results indicate that ECM stiffness influences hMSC differentiation and the increase in Dicer1 found with softer matrices could represent an umbrella miRNA inhibition effect to effectively suppress hMSC differentiation.

Abstract 220: Role of ECM Stiffness in Microvascular Inflammation

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is a critical regulator of inflammation as it inhibits leukocyte adhesion to vessel endothelium. Notably, reduced eNOS activity/NO bioavailability and chronic inflammation are key features, and often major determinants, of atherosclerosis and diabetic cardiovascular complications. Past approaches aimed at normalizing eNOS activity have focused primarily on the role of soluble, genetic or shear stress-mediated physical factors. However, extracellular matrix (ECM) mechanics also plays a crucial role in endothelial cell (EC) activation and function. Importantly, tissue mechanics is altered in both atherosclerosis and diabetes that are marked by chronic inflammation and impaired eNOS activity. Thus, we investigated whether there exists a causal relationship between ECM stiffness and eNOS/NO-dependent endothelial inflammation. To test this hypothesis, we cultured ECs on fibronectin-coated elastic substrates of varying stiffness (220 Pa/soft, 1000 Pa/intermediate and 4000 Pa/stiff). EC mechanosensitivity was first confirmed by quantifying the density of actin cytoskeletal filaments, which increased with increasing ECM stiffness. EC-leukocyte adhesion (inflammation) on these substrates, however, followed a biphasic trend where ECs on both 220 Pa (soft) and 4000 Pa (stiff) substrates exhibited greater leukocyte adhesion than those cultured on intermediate 1000 Pa substrates. This biphasic effect of ECM stiffness on endothelial inflammation correlated inversely with levels of eNOS-activation and NO production on these substrates. Consistent with these data, NF-kB activation was enhanced on 220 Pa and 4000 Pa while being suppressed on 1000 Pa substrates. Preliminary data implicate differential levels of calcium influx via mechanosensitive calcium ion channels in this ECM stiffness-dependent eNOS activation and endothelial inflammation. These new findings can potentially lead to superior cardiovascular disease management as they identify ECM stiffness both as a novel therapeutic target for immunomodulatory strategies as well as a key physical design criterion for the development of instructive biomaterials for cardiovascular tissue engineering.

Regulation of Dendrite Arborization by Substrate Stiffness is Mediated by Glutamate Receptors

Brain injury or disease can initiate changes in local or global stiffness of brain tissue. While stiffness of the extracellular environment is known to affect the morphology and function of many cell types, little is known about how the dendrites of neurons respond to changes in brain stiffness. To assess how extracellular stiffness affects dendrite morphology, we took biomaterial and biomedical engineering approaches. We cultured mixed and pure hippocampal neurons on hydrogels composed of polyacrylamide (PA) of varying stiffnesses to mimic the effects of extracellular matrix stiffness on dendrite morphology. The majority of investigations of cortical and spinal cord neurons on soft hydrogels examined branching at early time points (days in vitro (DIV) 2-7), an important distinction from our study, where we include later time points that encompass the peak of branching (DIV 10-12). At DIV 12, dendrite branching was altered by stiffness for both pure and mixed neuronal cultures. Furthermore, we treated hippocampal cultures with glutamate receptor antagonists and with astrocyte-conditioned media. Blocking AMPA and NMDA receptors affected the changes in dendrite branching seen at varying rigidities. Moreover, extracellular factors secreted by astrocytes also change dendrite branching seen at varying rigidities. Thus, astrocytes and ionotropic glutamate receptors contribute to mechanosensing.