Types Of Extracellular Matrix Research Articles

Reaching the Tumor: Mobility of Polymeric Micelles Inside an In Vitro Tumor-on-a-Chip Model with Dual ECM.

Degradable polymeric micelles are promising drug delivery systems due to their hydrophobic core and responsive design. When applying micellar nanocarriers for tumor delivery, one of the bottlenecks encountered in vivo is the tumor tissue barrier: crossing the dense mesh of cells and the extracellular matrix (ECM). Sometimes overlooked, the extracellular matrix can trap nanoformulations based on charge, size, and hydrophobicity. Here, we used a simple design of a microfluidic chip with two types of ECM and MCF7 spheroids to allow "high-throughput" screening of the interactions between biological interfaces and polymeric micelles. To demonstrate the applicability of the chip, a small library of fluorescently labeled polymeric micelles varying in their hydrophilic shell and hydrophobic core forming blocks was studied. Three widely used hydrophilic shells were tested and compared, namely, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and poly(acrylic acid), along with two enzymatically degradable dendritic hydrophobic cores (based on hexyl or nonyl end groups). Using ratiometric imaging of unimer:micelle fluorescence and FRAP inside the chip model, we obtained the local assembly state and dynamics inside the chip. Notably, we observed different micelle behaviors in the basal lamina ECM, from avoidance of the ECM structure to binding of the poly(acrylic acid) formulations. Binding to the basal lamina correlated with higher uptake into MCF7 spheroids. Overall, we proposed a simple microfluidic chip containing dual ECM and spheroids for the assessment of the interactions of polymeric nanocarriers with biological interfaces and evaluating nanoformulations' capacity to cross the tumor tissue barrier.

Extracellular Matrix Collagen I Differentially Regulates the Metabolic Plasticity of Pancreatic Ductal Adenocarcinoma Parenchymal Cell and Cancer Stem Cell.

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 10 percent largely due to the intense fibrotic desmoplastic reaction, characterized by high levels of extracellular matrix (ECM) collagen I that constitutes a niche for a subset of cancer cells, the cancer stem cells (CSCs). Cancer cells undergo a complex metabolic adaptation characterized by changes in metabolic pathways and biosynthetic processes. The use of the 3D organotypic model in this study allowed us to manipulate the ECM constituents and mimic the progression of PDAC from an early tumor to an ever more advanced tumor stage. To understand the role of desmoplasia on the metabolism of PDAC parenchymal (CPC) and CSC populations, we studied their basic metabolic parameters in organotypic cultures of increasing collagen content to mimic in vivo conditions. We further measured the ability of the bioenergetic modulators (BMs), 2-deoxyglucose, dichloroacetate and phenformin, to modify their metabolic dependence and the therapeutic activity of paclitaxel albumin nanoparticles (NAB-PTX). While all the BMs decreased cell viability and increased cell death in all ECM types, a distinct, collagen I-dependent profile was observed in CSCs. As ECM collagen I content increased (e.g., more aggressive conditions), the CSCs switched from glucose to mostly glutamine metabolism. All three BMs synergistically potentiated the cytotoxicity of NAB-PTX in both cell lines, which, in CSCs, was collagen I-dependent and the strongest when treated with phenformin + NAB-PTX. Metabolic disruption in PDAC can be useful both as monotherapy or combined with conventional drugs to more efficiently block tumor growth.

Targeting the YAP/TAZ mechanotransducers in solid tumour therapeutics.

Extracellular matrix (ECM) interacts with solid tumour cells and cells from the tumour microenvironment (ΤΜΕ). Components of the ECM and various types of mechanical forces which are generated from ECM stiffening modulate properties of solid tumour cells, thereby affecting tumour proliferation, migration, angiogenesis, immune evasion, stemness and resistance to treatment. Clarification of the role of ECM-associated protein molecules during solid tumour progression offers new therapeutic strategies aiming at overcoming ECM-mediated oncogenic signalling and drug resistance.1, 2 A well-investigated signalling pathway linked to alterations of ECM rigidity is the Hippo signal transduction pathway. It is an evolutionary conserved kinase cassette that controls cell proliferation, apoptosis, self-renewal of stem cells and organ size, and is regulated by mechanical cues, the status of cellular energy and multiple receptor signalling.3 Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are key transcriptional regulators and the end nuclear effectors of the Hippo signal transduction cascade. YAP and TAZ are transcription coactivators and function by tethering to the transcriptional enhanced associate domain (TEAD) transcription factor (there are four TEAD proteins (TEAD1-4) in the human genome).3 Following activation of the Hippo pathway, the unphosphorylated Merlin protein phosphorylates/potentiates STE20-like protein kinase 1/2 (MST1/2)/large tumour suppressor 1/2 (LATS1/2) proteins to turn off YAP/TAZ by multiple-site phosphorylation. YAP/TAZ inactivation leads to YAP/TAZ cytoplasmic retention. Conversely, inactivation of the Hippo pathway leads to YAP/TAZ nuclear accumulation and target gene transcription.3 Changes in ECM stiffness elicit mechano-stimulated tumorigenic mechanisms through activation of YAP/TAZ (Figure 1). Activated YAP/TAZ induce the expression of genes that promote cell proliferation.3 For example, in oral squamous cell carcinoma, YAP potentiation triggers transcriptional activation of piezo type mechanosensitive ion channel component 1 (Piezo1), a mechano-induced Ca2+ channel, and this evokes tumour cell proliferation.4 YAP activation also promotes cell migration under stiffer ECM microenvironment, for example hepatocellular carcinoma (HCC) cells present increased migration in vitro when cultured under stiffer extracellular conditions. Mechanistically, stiffer ECM ignites c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPK) signalling leading to induction of YAP and its translocation to the nucleus. Subsequently, YAP transcriptionally mediates enhancement of aerobic glycolysis and migration of HCC cells.5 An alternative route involves upregulation of C-X-C motif chemokine receptor 4 (CXCR4), a known booster of HCC, by ECM stiffness. CXCR4 downregulates ubiquitin domain-containing protein 1 (UBTD1), thereby decreasing poly-ubiquitination and proteasome-dependent degradation of YAP. Sustainable YAP expression transcriptionally activates tumour-promoting genes.6 It has been documented that YAP/TAZ activation mediates resistance to pharmacological intervention as a result of changes in ECM stiffness.7 Biochemical and mechanical stimuli in the tumour stroma due to alterations of ECM stiffness participate in metastases and drug resistance.8 For instance, actin remodelling activates YAP/TAZ in melanoma cells and this event fosters resistance to serine/threonine-protein kinase B-Raf inhibitors (BRAFi).9 Furthermore, YAP/TAZ regulate programmed death-ligand 1 (PD-L1) expression and YAP specifically mediates downregulation of the antitumour immune response in BRAFi-resistant melanoma cells in a PD-L1-dependent manner.10 Alterations in ECM rigidity can also cause resistance to the human epidermal growth factor receptor 2 (HER2) inhibitors (e.g. lapatinib) through YAP/TAZ in breast cancer.11 Data from breast cancer cells reveal that intermediate-stiffness substrates are correlated with decreased uptake, increased efflux and resistance to doxorubicin, compared to high- and low-stiffness substrates. Doxorubicin resistance is associated with higher expression levels of the mechanosensory protein integrin-linked kinase (ILK) and activation of YAP. ILK can regulate YAP expression and activity in an intermediate ECM stiffness status and confer drug resistance to breast cancer cells.12 In a similar vein, chemoresistance in ovarian cancer is driven by chemotherapy-induced alterations in ECM stiffness and composition. ECM microenvironment evolves during platinum-based chemotherapy (cisplatin, carboplatin), as demonstrated by distinct expression profiles of core ECM-related genes in pre- and post-chemotherapy samples. Stiffer ECM and platinum treatment enhances spreading, focal adhesion formation and nuclear translocation of YAP/TAZ in high-grade serous carcinoma (HGSC) ovarian cells. Moreover, ECM stiffness functionally contributes to resistance of ovarian cancer cells to platinum-triggered apoptosis via focal adhesion kinase (FAK) and YAP signalling. Platinum-mediated resistance is attributed to activation of specific ECM proteins, such as vitronectin (VTN), fibronectin (FBN) and collagen VI (ColVI).8 On the contrary, chemotherapy resistance to cisplatin and doxorubicin in HCC is attributed to specific TAZ upregulation. TAZ can transcriptionally activate interleukin 8 (IL-8) to confer resistance; therefore, TAZ-IL-8 is a reasonable axis for therapeutic targeting.13 YAP/TAZ upregulation, in synergy with activating transcription factor 4 (ATF4), are also responsible for resistance to the multikinase inhibitor sorafenib in HCC.14 In experiments where hepatocellular tumour spheroids were employed, YAP/TAZ activation occurs in tumour and stromal cells of HCC.15 In a patient-derived hepatocellular tumour spheroid model, YAP inhibition can sensitize HCC cells with high YAP/TAZ expression to kinase inhibition by sorafenib.16 It should also be taken into consideration that although YAP functions as an oncoprotein that confers drug resistance, recent findings suggest that YAP may also act as a tumour suppressor. Androgen receptor (AR) interacts with TEAD to promote tumour growth in prostate cancer cells. YAP upregulation competes with AR for TEAD binding to suppress TEAD-mediated AR signalling and tumour progression in AR-positive prostate cancer. YAP can also impede anti-hormone therapy resistance caused by AR variants. This indicates that pharmacological targeting of the Hippo pathway may be effective to overcome resistance to anti-androgen treatment in prostate cancer.17 As most solid tumour cells depend on their mechanical properties to promote their malignant features, small-molecule compounds (derived from available banks or chemically synthesized) targeting YAP/TAZ, either directly or by disrupting vital interactions with transcription factors/complexes, have been developed and have already progressed to clinical trials with promising results.2, 7 A rational design of YAP/TAZ targeting involves hindering of the intermolecular interaction between TEAD and its coactivators YAP/TAZ. This seems an attractive therapeutic strategy to treat solid tumours with a dysregulated Hippo pathway.18 However, it has been difficult to locate suitable hotspot binding sites conducive to abrogate YAP/TAZ-TEAD interactions. YAP binds to the α-helix and Ω-loop distinct pockets of TEAD, which have been shown to be crucial drug-targeting inhibitory sites of the YAP-TEAD complex.18 The first small non-peptide molecules to disrupt the YAP-TEAD interaction that have been identified bind competitively to the specific Ω-loop pocket of TEAD, as they display the same level of affinity for TEAD as YAP does.18 A subsequent study offered further optimization of such Ω-loop-targeting class of low-molecular mass compounds and proved to be efficient in tumour-bearing mice following oral administration.19 Concerning peptide inhibitors, albeit more progress has been made in targeting the Ω-loop region, a peptide inhibitor with high affinity to the α-helix pocket was recently biochemically and structurally characterized.20 In gastrointestinal cancers, two peptide segments, which represent an α-helix and an Ω-loop, have been isolated from binding sites of YAP/TAZ with TEAD and are promising candidates for Hippo pathway targeting. Nevertheless, these self-inhibitory peptides are easily dissociated when they are not parts of the full-length proteins; hence, they are not able to competitively annul binding of TEAD to YAP/TAZ. To this end, a new rational design methodology was recently proposed that employs a chemical modification, termed hydrocarbon stapling technique, to retain the conformation of the α-helix peptide. This approach can improve peptide binding to TEAD and interfere effectively with coactivators-transcription factor complex formation.21 YAP is also implicated in the angiogenic process of diverse solid tumours and small-molecule inhibitors targeting that aspect of YAP function have been developed. Verteporfin (VP) is a low-molecular mass compound that suppresses YAP signalling by hampering YAP/TEAD interactions and can restrain proliferation, migration, tube formation and facilitate apoptosis of human umbilical vein endothelial cells (HUVECs).22 It causes reduction of massive vessel branches production, reduction of the number of blood vessels in vivo and downregulation of the expression of angiogenesis-engaged genes. Thus, VP has been shown to potently attenuate malignant progression of oesophageal squamous cell carcinoma (ESCC) cells via inhibition of tumour angiogenesis.22 Additionally, targeting angiogenesis can re-sensitize ESCC cells to treatment with paclitaxel.22 VP targeting of the YAP/TAZ pathway is also effective to overcome drug resistance in endometrial carcinoma (EC). YAP/TAZ mediates resistance to progestin through the phosphoinositide 3-kinase (PI3K)/Akt pathway, and VP sensitizes EC cells to progestin both in vitro and in vivo.23 It also demonstrates an additive antitumor effect when combined with the histone demethylase lysine-specific demethylase 1 (LSD1) inhibitor SP2509 in vitro and in vivo in oral squamous cell carcinoma cells.24 There are also selective inhibitors of TEAD. K-975 is a newly identified highly selective TEAD inhibitor that impairs YAP/TAZ-TEAD protein–protein interactions. It exerts antitumor effects by suppressing YAP/TAZ-TEAD signalling in pleural mesothelioma.25 Another (oral) TEAD inhibitor, IK-930, is currently in phase 1 clinical trial evaluation in patients with advanced solid tumours bearing or not gene alterations in the Hippo pathway (NCT05228015). Data regarding the TEAD palmitoylation inhibitor MGH-CP1 imply that TEAD inhibition alone is not enough to cause cancer-cell death, because it transcriptionally activates an alternative pathway of cell survival through the sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4)/PI3K/Akt axis. Dual targeting and inhibition of TEAD and Akt represents an efficient treatment option.26 Lastly, IAG933, a potent and direct small-molecule disruptor of the YAP-TEAD protein–protein interaction, is presently in clinical trial evaluation in patients with mesothelioma, neurofibromatosis 2 (NF2, also known as Merlin)/LATS1/2-mutated tumours and tumours with functional YAP/TAZ fusions (i.e. YAP/TAZ hybrids that hyperactivate a TEAD-based transcriptome) (NCT04857372), but its exact mechanism of action has not yet been disclosed.27, 28 Recent findings further implicate YAP/TAZ in signalling circuitries of the ΤΜΕ as candidate targets to improve chemotherapy efficacy. Nonetheless, as shown in breast tumours, stage-specific YAP/TAZ expression profiles and/or crosstalk with co-operating signalling molecules should also be taken into account in predicting response to combinatorial YAP/TAZ/TEAD-targeted treatment approaches.29, 30 Conclusively, accumulating evidence suggests that anomalous YAP/TAZ signalling contributes significantly to solid tumour development and progression and is a critical factor of intrinsic and acquired resistance to a variety of targeted regimens and chemotherapies. Further research is required to elucidate the underpinning mechanisms of YAP/TAZ operational aberrations in order to formulate more efficacious treatments for a broad spectrum of solid tumours and their clinical complications. Antonios N. Gargalionis: Conceptualization (equal); data curation (equal); writing – original draft (lead). Kostas A. Papavassiliou: Conceptualization (equal); data curation (equal); writing – original draft (equal). Athanasios G. Papavassiliou: Conceptualization (lead); data curation (lead); supervision (lead); writing – review and editing (lead). The authors declare no competing financial interests. Data sharing is not applicable—no new data generated.

Abstract 1328: Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system

Abstract Despite the use of multi-agent conventional chemotherapy regimens, metastatic pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors with a 5-year survival of 9%. PDAC is characterized by a complex tumor microenvironment with multilayered interplay between neoplastic and stromal cells and the natural extracellular matrix (ECM). To date, models that recapitulate the complexity of these features are only partially reproduced ex vivo and lack the capacity to recreate PDAC tumor microenvironment physiology, fluid flow and the ECM architecture. To address these issues, we developed Curiochips MPS platform for metastatic PDAC on-a-chip. To replicate an accurate PDAC complex tumor microenvironment, we investigated human pancreatic cancer cell lines with/without SMAD4 mutation as a test model. SMAD4 is among the most frequently altered genes in PDAC and its mutation accelerates pancreatic cancer cell migration. To tailor cell-ECM interactions and recreate natural biochemical environment of tumors, we examined various ECM conditions. ECM substitutes such as type I collagen and fibrin gel offer mechanical properties to closely mimic the tumor ECM. We accessed the effect of ECM condition on the pancreatic stellate cells (PSC) activation and pancreatic cancer cell migration using PANC-1 (SMAD4-WT) and BxP3 (SMAD4-null) cell lines. Notably, type I collagen induced more PSC activation than fibrin gel. Interestingly BxP3 spheroids migrated toward to the endothelial compartment compared to PANC-1. Angiogenesis of endothelial cells was more prominent in co-culture with BxP3 than PANC-1. Furthermore, we are building the metastatic model using PDAC patient derived organoids and co-culture with primary PSC in different types of ECM. Preliminary analysis showed functional responses such as 3D organoid migration and aggregation, PSC activation and migration controlled by specific properties of EMC. Our data highlight the importance of the ECM composition on PDAC migration, PSC activation and angiogenesis in the context of genetic heterogeneity and presence/absence oncogenic mutation. The 3D co-culture platform, Curiochips offers suitable and tailored solution to investigate tumor-stroma interaction and potentially can be used as a platform for drug discovery and precision oncology to target PDAC desmoplastic tumor microenvironment. Citation Format: Sumin Kim, Yeongmin Choi, Jihye Baek, Bharti Garg, Jan Pencik, Andrew M. Lowy, Herve Tiriac, Sanghee Yoo. Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system [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 1328.

Discriminant Principal Component Analysis of ToF-SIMS Spectra for Deciphering Compositional Differences of MSC-Secreted Extracellular Matrices.

Identifying characteristic extracellular matrix (ECM) variants is a key challenge in mechanistic biology, bioengineering, and medical diagnostics. The reported study demonstrates the potential of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to detect subtle differences between human mesenchymal stromal cell (MSC)-secreted ECM types as induced by exogenous stimulation or emerging pathology. ToF-SIMS spectra of decellularized ECM samples are evaluated by discriminant principal component analysis (DPCA), an advanced multivariate analysis technique, to decipher characteristic compositional features. To establish the approach, signatures of major ECM proteins are determined from samples of pre-defined mixtures. Based on that, sets of ECM variants produced by MSCs in vitro are analyzed. Differences in the content of collagen, fibronectin, and laminin in the ECM resulting from the combined supplementation of MSC cultures with polymers that induce macromolecular crowding and with ascorbic acid are detected from the DPCA of ToF-SIMS spectra. The results are verified by immunostaining. Finally, the comparative ToF-SIMS analysis of ECM produced by MSCs of healthy donors and patients suffering from myelodysplastic syndrome display the potential of the novel methodology to reveal disease-associated alterations of the ECM composition.

Impact of hyaluronan size on localization and solubility of the extracellular matrix in the mouse brain.

Hyaluronan (HA) is a central component of the extracellular matrix (ECM) in the brain and plays a pivotal role in neural development and plasticity. Brain HA exists in 2 distinct forms of the ECM: the diffuse ECM, which is soluble in saline and detergents, and the condensed ECM, which forms aggregates, such as perineuronal nets (PNNs). Although the physiological functions of HA significantly differ depending on its size, size differences in HA have not yet been examined in the 2 ECM types, which is partly because of the lack of methods to rapidly and accurately measure the molecular weight (MW) of HA. In this study, we established a simple method to simultaneously assess the MW of HA in multiple crude biological samples. HA was purified through single-step precipitation from tissue extracts using biotinylated HA-binding protein and streptavidin-coupled magnetic beads, followed by separation on gel electrophoresis. By applying this method to HA in the mouse brain, we revealed that the condensed ECM contained higher MW HA than the diffuse ECM. Higher MW HA and lower MW HA exhibited different spatial distributions: the former was confined to PNNs, whereas the latter was widely present throughout the brain. Furthermore, the limited degradation of HA showed that only higher MW HA was required to form an insoluble HA-aggrecan complex. The present study demonstrated that the MW of HA in the brain strongly correlates with the localization and solubility of the ECM it forms.

Genetic Disorders of the Extracellular Matrix: From Cell and Gene Therapy to Future Applications in Regenerative Medicine.

Metazoans have evolved to produce various types of extracellular matrix (ECM) that provide structural support, cell adhesion, cell-cell communication, and regulated exposure to external cues. Epithelial cells produce and adhere to a specialized sheet-like ECM, the basement membrane, that is critical for cellular homeostasis and tissue integrity. Mesenchymal cells, such as chondrocytes in cartilaginous tissues and keratocytes in the corneal stroma, produce a pericellular matrix that presents optimal levels of growth factors, cytokines, chemokines, and nutrients to the cell and regulates mechanosensory signals through specific cytoskeletal and cell surface receptor interactions. Here, we discuss laminins, collagen types IV and VII, and perlecan, which are major components of these two types of ECM. We examinegenetic defects in these components that cause basement membrane pathologies such as epidermolysis bullosa, Alport syndrome, rare pericellular matrix-related chondrodysplasias, and corneal keratoconus and discuss recent advances in cell and gene therapies being developed for some of these disorders.

Investigating the Effects of IRF6 on Focal Adhesions in Keratinocytes

Focal adhesions are one of the two main types of adhesions between cells and the extra‐cellular matrix (ECM). Focal adhesions are dynamic complexes anchored to the actin cytoskeleton through intracellular adaptor proteins, and to the ECM through heterodimers of α and β integrins, forming a transmembrane link between the intra‐ and extra‐cellular space. β‐1 integrin is the primary β subunit found in keratinocytes, and its corresponding heterodimers show affinity for a wide range of ECM proteins including collagen, fibronectin, and laminin. Collagen is the main structural protein in cutaneous ECM (the dermis), while fibronectin is a glycoprotein found in the ECM that binds integrins and plays a crucial role in wound healing. Interferon regulatory factor 6 (IRF6) is a transcription factor expressed by epithelial cells that regulates keratinocyte differentiation, cell‐cell adhesions, and migration. It is well known that focal adhesions are required for cell migration, however the role of IRF6 in regulating focal adhesions is unknown. Therefore, we hypothesize that IRF6 regulates focal adhesion formation. To determine if IRF6 levels affect attachment to the ECM, we seeded wildtype and IRF6 knockdown keratinocytes on collagen IV and plastic and quantified the number of cells attached to the plate after thirty and sixty minutes. No differences were found between wild‐type and IRF6 knockdown keratinocytes on plastic. However, less IRF6 knockdown keratinocytes were attached on collagen IV compared to wildtypes. To explore how IRF6 affects focal adhesions, we plated IRF6 knockdown and wild‐type keratinocytes on different types of ECM that were representative of both normal and wounded conditions of the dermis. Immunofluorescent staining for β‐1 integrin and phalloidin was performed, followed by quantitative analysis of focal adhesion length and intensity. Initial observations indicate that IRF6 knockdown keratinocytes consistently have longer focal adhesions compared to the wildtype. Variations of β‐1 fluorescent intensity was also observed in wild‐type cells compared to IRF6 knockdown keratinocytes. These preliminary results suggest a role for IRF6 in regulating focal adhesions, providing a potential mechanism for the underlying defect observed in the migration of IRF6 deficient keratinocytes.

Extracellular Matrix Optimization for Enhanced Physiological Relevance in Hepatic Tissue-Chips.

The cellular microenvironment is influenced explicitly by the extracellular matrix (ECM), the main tissue support biomaterial, as a decisive factor for tissue growth patterns. The recent emergence of hepatic microphysiological systems (MPS) provide the basic physiological emulation of the human liver for drug screening. However, engineering microfluidic devices with standardized surface coatings of ECM may improve MPS-based organ-specific emulation for improved drug screening. The influence of surface coatings of different ECM types on tissue development needs to be optimized. Additionally, an intensity-based image processing tool and transepithelial electrical resistance (TEER) sensor may assist in the analysis of tissue formation capacity under the influence of different ECM types. The current study highlights the role of ECM coatings for improved tissue formation, implying the additional role of image processing and TEER sensors. We studied hepatic tissue formation under the influence of multiple concentrations of Matrigel, collagen, fibronectin, and poly-L-lysine. Based on experimental data, a mathematical model was developed, and ECM concentrations were validated for better tissue development. TEER sensor and image processing data were used to evaluate the development of a hepatic MPS for human liver physiology modeling. Image analysis data for tissue formation was further strengthened by metabolic quantification of albumin, urea, and cytochrome P450. Standardized ECM type for MPS may improve clinical relevance for modeling hepatic tissue microenvironment, and image processing possibly enhance the tissue analysis of the MPS.

The hippo pathway orchestrates cytoskeletal organisation during intervertebral disc degeneration

Yes-associated protein (YAP) activity responded to physical and mechanical cues such as extracellular matrix (ECM), cell density and the mechanical regulation of YAP controlled cellular proliferation and inhibition of apoptotic signals. The intervertebral disc (IVD) comprises a heterogeneous population of cells, including those of the nucleus pulposus (NP) and annulus fibrosus (AF), which are diverse in phenotype, partly due to the different ECM and mechanical loads they experience. How do IVD cells sense microenvironment and what is the relationship between YAP and cytoskeleton in the process of intervertebral disc degeneration (IDD) are not well understood. First, Hippo pathway and cytoskeleton organisation were assessed in the NP and AF of immature (4 weeks), mature (14 weeks), aged (50 weeks), and degenerated (14 weeks, 4 weeks after annulus puncture) IVDs. Second, to assess the effect of ECM composition and cell density on cytoskeleton and YAP levels, we seeded cells at different densities on three types of ECM. In this study, YAP and F-actin activity decreased gradually with age in natural IDD. Hippo signalling was suppressed in the early stages of disc injury, demonstrating the potential for endogenous repair, but this repair did not prevent further disc degeneration. β-tubulin and vimentin filaments provide the cell with its shape and its elastic properties in resisting mechanical forces. The Hippo pathway and cytoskeleton were shown to be regulated by cell density and the ECM composition.

Evaluating the Effect of Tissue Selection on the Characteristics of Extracellular Matrix Hydrogels from Decellularized Porcine Bladders

Porcine urinary bladder is one of the most used organs to fabricate extracellular matrix (ECM) hydrogel. Although there are two different ECM types inside a bladder, i.e., urinary bladder matrix (UBM) and a subtype ECM (sECM), most studies have only employed UBM for hydrogel fabrication, and overlooked the potential use of sECM. In another aspect, the delamination of UBM from bladders is a time-consuming process; consequently, the use of the whole bladder (WB) will likely increase production yield. Therefore, the objective of this study was to fabricate hydrogels from sECM and WB and compare them to UBM. The results indicated that different layers of the bladder shared almost the same biochemical composition. In terms of gelation kinetics, rheology and morphology, although hydrogels from UBM and sECM exhibited some discrepancies, those from UBM and WB interestingly possessed almost the same characteristics. In in vitro studies, all the hydrogels possessed nearly the same biochemical effects towards L929 viability and C2C12 differentiation. These results could preliminarily indicate that the use of sECM should no longer be ignored, and WB could be a promising substitution for UBM hydrogels, eliminating the need for time-consuming delamination processes, as well as increasing the possibility of mass production.

Extracellular Matrix in Neural Plasticity and Regeneration.

The extracellular matrix (ECM) is a fundamental component of biological tissues. The ECM in the central nervous system (CNS) is unique in both composition and function. Functions such as learning, memory, synaptogenesis, and plasticity are regulated by numerous ECM molecules. The neural ECM acts as a non-specific physical barrier that modulates neuronal plasticity and axon regeneration. There are two specialized types of ECM in the CNS, diffuse perisynaptic ECM and condensed ECM, which selectively surround the perikaryon and initial part of dendritic trees in subtypes of neurons, forming perineuronal nets. This review presents the current knowledge about the role of important neuronal ECM molecules in maintaining the basic functions of a neuron, including electrogenesis and the ability to form neural circuits. The review mainly focuses on the role of ECM components that participate in the control of key events such as cell survival, axonal growth, and synaptic remodeling. Particular attention is drawn to the numerous molecular partners of the main ECM components. These regulatory molecules are integrated into the cell membrane or disposed into the matrix itself in solid or soluble form. The interaction of the main matrix components with molecular partners seems essential in molecular mechanisms controlling neuronal functions. Special attention is paid to the chondroitin sulfate proteoglycan 4, type 1 transmembrane protein, neural-glial antigen 2 (NG2/CSPG4), whose cleaved extracellular domain is such a molecular partner that it not only acts directly on neural and vascular cells, but also exerts its influence indirectly by binding to resident ECM molecules.

The Bone Extracellular Matrix in Bone Formation and Regeneration.

Bone regeneration repairs bone tissue lost due to trauma, fractures, and tumors, or absent due to congenital disorders. The extracellular matrix (ECM) is an intricate dynamic bio-environment with precisely regulated mechanical and biochemical properties. In bone, ECMs are involved in regulating cell adhesion, proliferation, and responses to growth factors, differentiation, and ultimately, the functional characteristics of the mature bone. Bone ECM can induce the production of new bone by osteoblast-lineage cells, such as MSCs, osteoblasts, and osteocytes and the absorption of bone by osteoclasts. With the rapid development of bone regenerative medicine, the osteoinductive, osteoconductive, and osteogenic potential of ECM-based scaffolds has attracted increasing attention. ECM-based scaffolds for bone tissue engineering can be divided into two types, that is, ECM-modified biomaterial scaffold and decellularized ECM scaffold. Tissue engineering strategies that utilize the functional ECM are superior at guiding the formation of specific tissues at the implantation site. In this review, we provide an overview of the function of various types of bone ECMs in bone tissue and their regulation roles in the behaviors of osteoblast-lineage cells and osteoclasts. We also summarize the application of bone ECM in bone repair and regeneration. A better understanding of the role of bone ECM in guiding cellular behavior and tissue function is essential for its future applications in bone repair and regenerative medicine.

The extracellular matrix in development.

ABSTRACTAs the crucial non-cellular component of tissues, the extracellular matrix (ECM) provides both physical support and signaling regulation to cells. Some ECM molecules provide a fibrillar environment around cells, while others provide a sheet-like basement membrane scaffold beneath epithelial cells. In this Review, we focus on recent studies investigating the mechanical, biophysical and signaling cues provided to developing tissues by different types of ECM in a variety of developing organisms. In addition, we discuss how the ECM helps to regulate tissue morphology during embryonic development by governing key elements of cell shape, adhesion, migration and differentiation.

Extracellular matrix type modulates mechanotransduction of stem cells

Extracellular matrix (ECM) is comprised of different types of proteins, which change in composition and ratios during morphogenesis and disease progression. ECM proteins provide cell adhesion and impart mechanical cues to the cells. Increasing substrate stiffness has been shown to induce Yes-associated protein (YAP) translocation from the cytoplasm to the nucleus, yet these mechanistic studies used fibronectin only as the biochemical cue. How varying the types of ECM modulates mechanotransduction of stem cells remains largely unknown. Using polyacrylamide hydrogels with tunable stiffness as substrates, here we conjugated four major ECM proteins commonly used for cell adhesion: fibronectin, collagen I, collagen IV and laminin, and assessed the effects of varying ECM type and density on YAP translocation in human mesenchymal stem cells (hMSCs). For all four ECM types, increasing ECM ligand density alone can induce YAP nuclear translocation without changing substrate stiffness. The ligand threshold for such biochemical ligand-induced YAP translocation differs across ECM types. While stiffness-dependent YAP translocation can be induced by all four ECM types, each ECM requires a different optimized ligand density for this to occur. Using antibody blocking, we further identified integrin subunits specifically involved in mechanotransduction of different ECM types. Finally, we demonstrated that altering ECM type further modulates hMSC osteogenesis without changing substrate stiffness. These findings highlight the important role of ECM type in modulating mechanotransduction and differentiation of stem cells, and call for future mechanistic studies to further elucidate the role of changes in ECM compositions in mediating mechanotransduction during morphogenesis and disease progression. Statement of SignificanceOur study addresses a critical gap of knowledge in mechanobiology. Increasing substrate stiffness has been shown to induce nuclear YAP translocation, yet only on fibronectin-coated substrates. However, extracellular matrix (ECM) is comprised of different protein types. How varying the type of ECM modulates stem cell mechanotransduction remains largely unknown.We here reveal that the choice of ECM type can directly modulate stem cell mechanotransduction, filling this critical gap.This work has broad impacts in mechanobiology and biomaterials, as it provides the first evidence that varying ECM type can impact YAP translocation independent of substrate stiffness, opening doors for a more rational biomaterials design tuning ECM properties to control cell fate for promoting normal development and for preventing disease progression.

Extracellular matrix derived by human umbilical cord-deposited mesenchymal stem cells accelerates chondrocyte proliferation and differentiation potential in vitro.

The extracellular matrix (ECM) is a dynamic and intricate three-dimensional (3D) microenvironment with excellent biophysical, biomechanical, and biochemical properties that may directly or indirectly regulate cell behavior, including proliferation, adhesion, migration, and differentiation. Compared with tissue-derived ECM, cell-derived ECM potentially has more advantages, including less potential for pathogen transfer, fewer inflammatory or anti-host immune responses, and a closer resemblance to the native ECM microenvironment. Different types of cell-derived ECM, such as adipose stem cells, synovium-derived stem cells and bone marrow stromal cells, their effects on articular chondrocytes which have been researched. In this study, we aimed to develop a 3D cell culture substrate using decellularized ECM derived from human umbilical cord-derived mesenchymal stem cells (hUCMSCs), and evaluated the effects on articular chondrocytes. We evaluated the morphology and components of hUCMSC-derived ECM using physical and chemical methods. Morphological, histological, immunohistochemical, biochemical, and real-time PCR analyses demonstrated that proliferation and differentiation capacity of chondrocytes using the 3D hUCMSC-derived ECM culture substrate was superior to that using non-coated two-dimensional plastic culture plates. In conclusion, 3D decellularized ECM derived from hUCMSCs offers a tissue-specific microenvironment for in vitro culture of chondrocytes, which not only markedly promoted chondrocyte proliferation but also preserved the differentiation capacity of chondrocytes. Therefore, our findings suggest that a 3D cell-derived ECM microenvironment represents a promising prospect for autologous chondrocyte-based cartilage tissue engineering and regeneration. The hUCMSC-derived ECM as a biomaterial is used for the preparation of scaffold or hybrid scaffold products which need to further study in the future.

Polydopamine-Based Interfacial Engineering of Extracellular Matrix Hydrogels for the Construction and Long-Term Maintenance of Living Three-Dimensional Tissues.

Diverse biological processes in the body rely on the ability of cells to exert contractile forces on their extracellular matrix (ECM). In three-dimensional (3D) cell culture, however, this intrinsic cellular property can cause unregulated contraction of ECM hydrogel scaffolds, leading to a loss of surface anchorage and the resultant structural failure of in vitro tissue constructs. Despite advances in the 3D culture technology, this issue remains a significant challenge in the development and long-term maintenance of physiological 3D in vitro models. Here, we present a simple yet highly effective and accessible solution to this problem. We leveraged a single-step surface functionalization technique based on polydopamine to drastically increase the strength of adhesion between hydrogel scaffolds and cell culture substrates. Our method is compatible with different types of ECM and polymeric surfaces and also permits prolonged shelf storage of functionalized culture substrates. The proof-of-principle of this technique was demonstrated by the stable long-term (1 month) 3D culture of human lung fibroblasts. Furthermore, we showed the robustness and advanced application of the method by constructing a dynamic cell stretching system and performing over 100 000 cycles of mechanical loading on 3D multicellular constructs for visualization and quantitative analysis of stretch-induced tissue alignment. Finally, we demonstrated the potential of our technique for the development of microphysiological in vitro models by establishing microfluidic 3D co-culture of vascular endothelial cells and fibroblasts to engineer self-assembled, perfusable 3D microvascular beds.

Investigation of the Extracellular Matrix Effect for the QCM/CCD Cell Activity Monitoring System.

A real-time quartz crystal microbalance (QCM) cell activity monitoring system coupled with micro CCD cameras was developed to investigate the cultured cell activity, which could measure the viscoelastic characteristics of the cell with the QCM and observe the cell morphology changes with CCD camera simultaneously. Both the viscoelastic characteristics and the shape of the cultured cell are important factors to estimate the cell activity and the cell adhesion. The extracellular matrix (ECM) on the surface of the QCM is essential to culture the cell stably in the QCM monitoring system. To find the ECM optimization condition, the adhesive strength of cultured cells on the ECM modified glass surface was measured by using rotating water stream and CCD camera. After culturing HepG2 cells for 24 hours on the ECM modified glass plates, the glass plates were dipped in the PBS solution and rotated with 1,000, 1,300, and 1,500 rpm for 30 seconds. The adhesiveness of ECMs was investigated by calculating the remained cells after rotating. Four types of ECM, such as amino group, carboxyl group, collagen monomer, and collagen polymer, were used and tested. The current paper improves the sensing system of previous report so that measurements of four ECMs can be simultaneously conducted under the same conditions in order to enhance reliability. A collagen polymer exposed ECM was the most stable on an adhesiveness point of view, but not suitable for the QCM cell activity monitoring due to the decrease of the QCM sensitivity. The sensitivity of the QCM cell activity monitoring system using collagen monomer as ECM is about 2.6 times better than that using collagen polymer. A collagen monomer exposed ECM was more stable than amino group and carboxyl group exposed ECMs based on an adhesiveness point of view. Therefore, a collagen monomer exposed ECM was the most stable and suitable for the QCM cell activity monitoring system among the four ECMs. The changes of the resonance frequency and the resonance resistance of the ECM film with the cultured cells were investigated and compared the results of CCD camera images. From these results, we showed the QCM cell activity monitoring system coupled with the micro CCD camera could be applied to the evaluation of the cell activities.

The endoplasmic reticulum–resident collagen chaperone Hsp47 interacts with and promotes the secretion of decorin, fibromodulin, and lumican

The build-up of diversified and tissue-specific assemblies of extracellular matrix (ECM) proteins depends on secreted and cell surface-located molecular arrays that coordinate ECM proteins into discrete designs. The family of small leucine-rich proteins (SLRPs) associates with and dictates the structure of fibrillar collagens, which form the backbone of most ECM types. However, whether SLRPs form complexes with proteins other than collagens is unclear. Here, we demonstrate that heat shock protein 47 (Hsp47), a well-established endoplasmic reticulum-resident collagen chaperone, also binds the SLRPs decorin, lumican, and fibromodulin with affinities comparable with that in the Hsp47-type I collagen interaction. Furthermore, we show that a lack of Hsp47 inhibits the cellular secretion of decorin and lumican. Our results expand the understanding of the concerted molecular interactions that control the secretion and organization of a functional collagenous ECM.

Erasure of striatal chondroitin sulfate proteoglycan-associated extracellular matrix rescues aging-dependent decline of motor learning.

Cognitive decline is a feature of aging. Accumulating evidence suggests that the brain extracellular matrix (ECM) is involved in the process of aging-dependent cognitive impairment and neurodegeneration by regulating synaptic neurotransmission and affecting neuroplasticity. Age-related changes in brain structure and cognition are not uniform across the whole brain. Being one of the most vulnerable brain regions to aging-dependent alterations, striatum is integral to several central nervous system functions, such as motor, cognition, and affective control. However, the striatal ECM is largely understudied. We first describe 2 major types of chondroitin sulfate proteoglycan (CSPG)-associated ECM in striatum: perineuronal nets and diffusive ECM. Both types of ECM accumulate in an aging-dependent manner. The accumulation of CSPG-associated ECM correlates with aging-dependent decline in striatum-related cognitive functions, including motor learning and working memory. Enzymatic depletion of CSPG-associated ECM in aged mice via chondroitinase ABC significantly improves motor learning, suggesting that changes in neural ECM CSPGs regulate striatal plasticity. Our study provides a greater understanding of the role of neural ECM underlying striatal plasticity, which is an important precursor to design appropriate therapeutic strategies for normal and pathologic aging.