Cell-matrix Crosstalk Research Articles

Structure-Mechanics Principles and Mechanobiology of Fibrocartilage Pericellular Matrix: A Pivotal Role of Type V Collagen.

The pericellular matrix (PCM) is the immediate microniche surrounding resident cells in various tissue types, regulating matrix turnover, cell-matrix cross-talk and disease initiation. This study elucidated the structure-mechanical properties and mechanobiological functions of the PCM in fibrocartilage, a family of connective tissues that sustain complex tensile and compressive loads in vivo. Studying the murine meniscus as the model tissue, we showed that fibrocartilage PCM contains thinner, random collagen fibrillar networks that entrap proteoglycans, a structure distinct from the densely packed, highly aligned collagen fibers in the bulk extracellular matrix (ECM). In comparison to the ECM, the PCM has a lower modulus and greater isotropy, but similar relative viscoelastic properties. In Col5a1 +/- menisci, the reduction of collagen V, a minor collagen localized in the PCM, resulted in aberrant fibril thickening with increased heterogeneity. Consequently, the PCM exhibited a reduced modulus, loss of isotropy and faster viscoelastic relaxation. This disrupted PCM contributes to perturbed mechanotransduction of resident meniscal cells, as illustrated by reduced intracellular calcium signaling, as well as upregulated biosynthesis of lysyl oxidase and tenascin C. When cultured in vitro, Col5a1 +/- meniscal cells synthesized a weakened nascent PCM, which had inferior properties towards protecting resident cells against applied tensile stretch. These findings underscore the PCM as a distinctive microstructure that governs fibrocartilage mechanobiology, and highlight the pivotal role of collagen V in PCM function. Targeting the PCM or its molecular constituents holds promise for enhancing not only meniscus regeneration and osteoarthritis intervention, but also addressing diseases across various fibrocartilaginous tissues.

An osteosarcoma-on-a-chip model for studying osteosarcoma matrix-cell interactions and drug responses

Marrow niches in osteosarcoma (OS) are a specialized microenvironment that is essential for the maintenance and regulation of OS cells. However, existing animal xenograft models are plagued by variability, complexity, and high cost. Herein, we used a decellularized osteosarcoma extracellular matrix (dOsEM) loaded with extracellular vesicles from human bone marrow-derived stem cells (hBMSC-EVs) and OS cells as a bioink to construct a micro-osteosarcoma (micro-OS) through 3D printing. The micro-OS was further combined with a microfluidic system to develop into an OS-on-a-chip (OOC) with a built-in recirculating perfusion system. The OOC system successfully integrated bone marrow niches, cell‒cell and cell–matrix crosstalk, and circulation, allowing a more accurate representation of OS characteristics in vivo. Moreover, the OOC system may serve as a valuable research platform for studying OS biological mechanisms compared with traditional xenograft models and is expected to enable precise and rapid evaluation and consequently more effective and comprehensive treatments for OS.

Characterization of Bioinks Prepared via Gelifying Extracellular Matrix from Decellularized Porcine Myocardia.

Since the emergence of 3D bioprinting technology, both synthetic and natural materials have been used to develop bioinks for producing cell-laden cardiac grafts. To this end, extracellular-matrix (ECM)-derived hydrogels can be used to develop scaffolds that closely mimic the complex 3D environments for cell culture. This study presents a novel cardiac bioink based on hydrogels exclusively derived from decellularized porcine myocardium loaded with human-bone-marrow-derived mesenchymal stromal cells. Hence, the hydrogel can be used to develop cell-laden cardiac patches without the need to add other biomaterials or use additional crosslinkers. The scaffold ultrastructure and mechanical properties of the bioink were characterized to optimize its production, specifically focusing on the matrix enzymatic digestion time. The cells were cultured in 3D within the developed hydrogels to assess their response. The results indicate that the hydrogels fostered inter-cell and cell-matrix crosstalk after 1 week of culture. In conclusion, the bioink developed and presented in this study holds great potential for developing cell-laden customized patches for cardiac repair.

The Maastricht Acquisition Platform for Studying Mechanisms of Cell-Matrix Crosstalk (MAPEX): An Interdisciplinary and Systems Approach towards Understanding Thoracic Aortic Disease.

Current management guidelines for ascending thoracic aortic aneurysms (aTAA) recommend intervention once ascending or sinus diameter reaches 5-5.5 cm or shows a growth rate of >0.5 cm/year estimated from echo/CT/MRI. However, many aTAA dissections (aTAAD) occur in vessels with diameters below the surgical intervention threshold of <55 mm. Moreover, during aTAA repair surgeons observe and experience considerable variations in tissue strength, thickness, and stiffness that appear not fully explained by patient risk factors. To improve the understanding of aTAA pathophysiology, we established a multi-disciplinary research infrastructure: The Maastricht acquisition platform for studying mechanisms of tissue-cell crosstalk (MAPEX). The explicit scientific focus of the platform is on the dynamic interactions between vascular smooth muscle cells and extracellular matrix (i.e., cell-matrix crosstalk), which play an essential role in aortic wall mechanical homeostasis. Accordingly, we consider pathophysiological influences of wall shear stress, wall stress, and smooth muscle cell phenotypic diversity and modulation. Co-registrations of hemodynamics and deep phenotyping at the histological and cell biology level are key innovations of our platform and are critical for understanding aneurysm formation and dissection at a fundamental level. The MAPEX platform enables the interpretation of the data in a well-defined clinical context and therefore has real potential for narrowing existing knowledge gaps. A better understanding of aortic mechanical homeostasis and its derangement may ultimately improve diagnostic and prognostic possibilities to identify and treat symptomatic and asymptomatic patients with existing and developing aneurysms.

Smooth Muscle-Alpha Actin R149C Pathogenic Variant Downregulates Integrin Recruitment at Cell-Matrix Adhesions and Decreases Cellular Contractility.

Thoracic aortic aneurysm is found in patients with ACTA2 pathogenic variants. ACTA2 missense variants are associated with impaired aortic smooth muscle cell (SMC) contraction. This study tested the hypothesis that the Acta2R149C/+ variant alters actin isoform expression and decreases integrin recruitment, thus, reducing aortic contractility. Stress relaxation measurements in thoracic aortic rings showed two functional regimes with a reduction of stress relaxation in the aorta from Acta2R149C/+ mice at low tension, but not at high tension values. Contractile responses to phenylephrine and potassium chloride were 50% lower in Acta2R149C/+ mice than in wild-type (WT) mice. Additionally, SMC were immunofluorescently labeled for specific proteins and imaged by confocal or total internal reflection fluorescence microscopy. The quantification of protein fluorescence of Acta2R149C/+ SMC showed a downregulation in smooth muscle α-actin (SMα-actin) and a compensatory upregulation of smooth muscle γ-actin (SMγ-actin) compared to WT cells. These results suggest that downregulation of SMα-actin leads to reduced SMC contractility, while upregulation of SMγ-actin may lead to increased SMC stiffness. Decreased α5β1 and α2β1 integrin recruitment at cell-matrix adhesions further reduce the ability of mutant cells to participate in cell-matrix crosstalk. Collectively, the results suggest that mutant Acta2R149C/+ aortic SMC have reduced contractility and interaction with the matrix, which are potential long-term contributing factors to thoracic aortic aneurysms.

Abstract P6-14-09: Non-canonical Wnt/Ror2 signaling regulates tumor cell invasion and dissemination in breast cancer through cell-matrix crosstalk

Abstract BACKGROUND: Metastasis is the main cause of mortality for breast cancer patients. The early steps of cancer metastasis require that tumor cells actively invade and disseminate from the primary tumor to distant organs. Cell-extracellular matrix (ECM) interactions represent fundamental interactions during tumor invasion and metastasis, yet how particular signal-transduction factors prompt the conversion of tumor cells into migratory populations capable of systemic dissemination remains elusive. OBJECTIVES: Wnt signaling is a known regulator of cell fate, migration, and polarity during various key biological processes. We previously discovered an inverse correlation between the canonical Wnt signature and a non-canonical Wnt receptor, Ror2, across breast cancers in TCGA. The objective of this study is to investigate how canonical and non-canonical Wnt signaling orchestrate tumor cell behavior during cancer invasion and metastasis. METHODS: We used clinically relevant syngeneic TP53-null Genetically Engineered Mouse Model (GEMM) tumors that molecularly reflect the different breast cancer intrinsic subtypes. In vivo transplantation of GEMM models and in vitro 3D tumor organoids were employed to elucidate the molecular mechanism underlying the migration, invasion, and metastasis of tumor cells upon genetic perturbation of Wnt/Ror2 signaling. RESULTS/DISCUSSION: From 3-dimensional (3D) tumor organoid models, we identified novel transcriptional alterations encompassing cell-cell adhesion, cytoskeletal remodeling, and ECM organization upon Ror2 loss. At protein levels, we discovered a significant increase in collagen fibril organization and integrin-α5 and integrin-β3 expression following Ror2 depletion. In addition, the matrisomal protein fibronectin (FN) was concomitantly upregulated and assembled in Ror2-deficient tumor cells at sites of invasion. Consequently, we observed FAK activation and actin cytoskeleton alterations in Ror2-deficient tumor cells, leading to a promigratory tumor cell behavior. The altered cytoskeleton and cell-ECM interaction enhanced the rigidity of the Ror2-depleted cells. Furthermore, Inhibition of either integrin or FAK activation abrogated the increased invasion driven by Ror2-loss. Unexpectedly, these changes were distinct from processes regulated by Wnt/ß-catenin activation. From both spontaneous metastasis and experimental metastasis models, we discovered a shift from canonical to non-canonical Wnt signaling throughout the progression of lung metastasis, indicating that the balance of Wnt signaling may serve as a switch to dictate cell functions at different stages of metastatic development. Consistently, we found enhanced initial colonization in the lungs when Ror2 is depleted in these breast cancer cells. Together, these studies provide new insight into how canonical and alternative Wnt pathways coordinate cell-cell and cell-ECM exchanges during breast cancer progression and metastasis. Supported by grant NIH-CA016303 Citation Format: Hongjiang Si, Na Zhao, Andrea Pedroza, Chad Creighton, Jeffrey Rosen, Kevin Roarty. Non-canonical Wnt/Ror2 signaling regulates tumor cell invasion and dissemination in breast cancer through cell-matrix crosstalk [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 P6-14-09.

Reshaping in vitro Models of Breast Tissue: Integration of Stromal and Parenchymal Compartments in 3D Printed Hydrogels.

Breast tissue consists of an epithelial parenchyma embedded in stroma, of heterogeneous and complex composition, undergoing several morphological and functional alterations throughout females' lifespan. Improved knowledge on the crosstalk between parenchymal and stromal mammary cells should provide important insights on breast tissue dynamics, both under healthy and diseased states. Here, we describe an advanced 3D in vitro model of breast tissue, combining multiple components, namely stromal cells and their extracellular matrix (ECM), as well as parenchymal epithelial cells, in a hybrid system. To build the model, porous scaffolds were produced by extrusion 3D printing of peptide-modified alginate hydrogels, and then populated with human mammary fibroblasts. Seeded fibroblasts were able to adhere, spread and produce endogenous ECM, providing adequate coverage of the scaffold surface, without obstructing the pores. On a second stage, a peptide-modified alginate pre-gel laden with mammary gland epithelial cells was used to fill the scaffold's pores, forming a hydrogel in situ by ionic crosslinking. Throughout time, epithelial cells formed prototypical mammary acini-like structures, in close proximity with fibroblasts and their ECM. This generated a heterotypic 3D model that partially recreates both stromal and parenchymal compartments of breast tissue, promoting cell-cell and cell-matrix crosstalk. Furthermore, the hybrid system could be easily dissolved for cell recovery and subsequent analysis by standard cellular/molecular assays. In particular, we show that retrieved cell populations could be discriminated by flow cytometry using cell-type specific markers. This integrative 3D model stands out as a promising in vitro platform for studying breast stroma-parenchyma interactions, both under physiological and pathological settings.

Periostin in cardiovascular disease and development: a tale of two distinct roles.

Tissue development and homeostasis are dependent upon the concerted synthesis, maintenance, and degradation of extracellular matrix (ECM) molecules. Cardiac fibrosis is now recognized as a primary contributor to incidence of heart failure, particularly heart failure with preserved ejection fraction, wherein cardiac filling in diastole is compromised. Periostin is a cell-associated protein involved in cell fate determination, proliferation, tumorigenesis, and inflammatory responses. As a non-structural component of the ECM, secreted 90kDa periostin is emerging as an important matricellular factor in cardiac mesenchymal tissue development. In addition, periostin's role as a mediator in cell-matrix crosstalk has also garnered attention for its association with fibroproliferative diseases in the myocardium, and for its association with TGF-β/BMP signaling. This review summarizes the phylogenetic history of periostin, its role in cardiac development, and the major signaling pathways influencing its expression in cardiovascular pathology. Further, we provide a synthesis of the current literature to distinguish the multiple roles of periostin in cardiac health, development and disease. As periostin may be targeted for therapeutic treatment of cardiac fibrosis, these insights may shed light on the putative timing for application of periostin-specific therapies.

Dynamic interplay between adhesion surfaces in carcinomas: Cell-cell and cell-matrix crosstalk.

Cell-cell and cell-matrix signaling and communication between adhesion sites involve mechanisms which are required for cellular functions during normal development and homeostasis; however these cellular functions and mechanisms are often deregulated in cancer. Aberrant signaling at cell-cell and cell-matrix adhesion sites often involves downstream mediators including Rho GTPases and tyrosine kinases. This review discusses these molecules as putative mediators of cellular crosstalk between cell-cell and cell-matrix adhesion sites, in addition to their attractiveness as therapeutic targets in cancer. Interestingly, inter-junctional crosstalk mechanisms are frequently typified by the way in which bacterial and viral pathogens opportunistically infect or intoxicate mammalian cells. This review therefore also discusses the concept of learning from pathogen-host interaction studies to better understand coordinated communication between cell-cell and cell-matrix adhesion sites, in addition to highlighting the potential therapeutic usefulness of exploiting pathogens or their products to tap into inter-junctional crosstalk. Taken together, we feel that increased knowledge around mechanisms of cell-cell and cell-matrix adhesion site crosstalk and consequently a greater understanding of their therapeutic targeting offers a unique opportunity to contribute to the emerging molecular revolution in cancer biology.

Fabrication and characterization of nano-fibrous bilayer composite for skin regeneration application

Full thickness wound healing with minimal scarring and complete restoration of normal skin properties still remains as a clinical challenge. In this study, a bilayer skin substitute has been fabricated to biomimic the microstructure of natural extracellular matrix of the skin. Human amniotic membrane (HAM) and silk fibroin nano-fibers were combined to produce bilayer construct, which was further treated and characterized. HAM was obtained from healthy mothers and de-epithelized by means of fine enzymatic method to preserve the extracellular structure. Fibroin protein was extracted from fresh Bombyx mori cocoons and transformed to uniform nano-fiberous structure, which was used as a coating layer on the de-epithelized membrane. Surface modification through oxygen plasma treatment was attempted to further induce hydrophilicity. Subsequently, scaffolds were fully characterized in terms of morphology, mechanical properties, hydrophilicity and cell culture response. Histological and immunohistological staining demonstrated localization of fibronectin, cell denudation and structural integrity of HAM after de-epithelization. Scanning electron microscopy images showed bead-free silk fibroin nano-fibers with the average diameter of 250nm. Water contact angle of bilayer scaffolds reduced dramatically to 26.34° after oxygen plasma treatment, which is correlated with more hydrophilic surface. Due to fibroin nano-fiber coating, mechanical properties of HAM improved significantly. Tensile Young’s modulus and tensile strength increased from 16.14MPa and 68.46MPa to 25.69MPa and 108.03MPa, respectively. 14days in vitro cultivation of mouse embryonic fibroblasts on the scaffolds revealed that bilayer scaffolds are able to support cell attachment and proliferation. Plasma-etched scaffolds provided the best niche for cell-matrix crosstalk by allowing cells to penetrate beneath the pores and to integrate in fibers direction. The obtained results suggest that the presented nano-fibrous bilayer composite based on HAM is a potential substitute for skin regeneration application.

Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

BackgroundHeparan sulfate (HS) is present on the surface of virtually all mammalian cells and is a major component of the extracellular matrix (ECM), where it plays a pivotal role in cell-cell and cell-matrix cross-talk through its large interactome. Disruption of HS biosynthesis in mice results in neonatal death as a consequence of malformed lungs, indicating that HS is crucial for airway morphogenesis. Neonatal mortality (~50%) in newborns with congenital diaphragmatic hernia (CDH) is principally associated with lung hypoplasia and pulmonary hypertension. Given the importance of HS for lung morphogenesis, we investigated developmental changes in HS structure in normal and hypoplastic lungs using the nitrofen rat model of CDH and semi-synthetic bacteriophage ('phage) display antibodies, which identify distinct HS structures.ResultsThe pulmonary pattern of elaborated HS structures is developmentally regulated. For example, the HS4E4V epitope is highly expressed in sub-epithelial mesenchyme of E15.5 - E17.5 lungs and at a lower level in more distal mesenchyme. However, by E19.5, this epitope is expressed similarly throughout the lung mesenchyme.We also reveal abnormalities in HS fine structure and spatiotemporal distribution of HS epitopes in hypoplastic CDH lungs. These changes involve structures recognised by key growth factors, FGF2 and FGF9. For example, the EV3C3V epitope, which was abnormally distributed in the mesenchyme of hypoplastic lungs, is recognised by FGF2.ConclusionsThe observed spatiotemporal changes in HS structure during normal lung development will likely reflect altered activities of many HS-binding proteins regulating lung morphogenesis. Abnormalities in HS structure and distribution in hypoplastic lungs can be expected to perturb HS:protein interactions, ECM microenvironments and crucial epithelial-mesenchyme communication, which may contribute to lung dysmorphogenesis. Indeed, a number of epitopes correlate with structures recognised by FGFs, suggesting a functional consequence of the observed changes in HS in these lungs. These results identify a novel, significant molecular defect in hypoplastic lungs and reveals HS as a potential contributor to hypoplastic lung development in CDH. Finally, these results afford the prospect that HS-mimetic therapeutics could repair defective signalling in hypoplastic lungs, improve lung growth, and reduce CDH mortality.

The matricellular functions of small leucine-rich proteoglycans (SLRPs)

The small leucine-rich proteoglycans (SLRPs) are biologically active components of the extracellular matrix (ECM), consisting of a protein core with leucine rich-repeat (LRR) motifs covalently linked to glycosaminoglycan (GAG) side chains. The diversity in composition resulting from the various combinations of protein cores substituted with one or more GAG chains along with their pericellular localization enables SLRPs to interact with a host of different cell surface receptors, cytokines, growth factors, and other ECM components, leading to modulation of cellular functions. SLRPs are capable of binding to: (i) different types of collagens, thereby regulating fibril assembly, organization, and degradation; (ii) Toll-like receptors (TLRs), complement C1q, and tumor necrosis factor-alpha (TNFα), regulating innate immunity and inflammation; (iii) epidermal growth factor receptor (EGF-R), insulin-like growth factor receptor (IGF-IR), and c-Met, influencing cellular proliferation, survival, adhesion, migration, tumor growth and metastasis as well as synthesis of other ECM components; (iv) low-density lipoprotein receptor-related protein (LRP-1) and TGF-β, modulating cytokine activity and fibrogenesis; and (v) growth factors such as bone morphogenic protein (BMP-4) and Wnt-I-induced secreted protein-1 (WISP-1), controlling cell proliferation and differentiation. Thus, the ability of SLRPs, as ECM components, to directly or indirectly regulate cell-matrix crosstalk, resulting in the modulation of various biological processes, aptly qualifies these compounds as matricellular proteins.

CD44 Variant Isoforms Promote Metastasis Formation by a Tumor Cell-Matrix Cross-talk That Supports Adhesion and Apoptosis Resistance

CD44 designates a large family of proteins with a considerable structural and functional diversity, which are generated from one gene by alternative splicing. As such, the overexpression of CD44 variant isoform (CD44v) has been causally related to the metastatic spread of cancer cells. To study the underlying mechanism, stable knockdown clones with deletion of exon v7 containing CD44 isoforms (CD44v(kd)) of the highly metastatic rat adenocarcinoma line BSp73ASML (ASML(wt)) were established. ASML-CD44v(kd) clones hardly form lung metastases after intrafootpad application and the metastatic load in lymph nodes is significantly reduced. Rescuing, albeit at a reduced level, CD44v expression in ASML-CD44v(kd) cells (ASML-CD44v(rsc)) restores the metastatic potential. The following major differences in ASML(wt), ASML-CD44v(kd), and ASML-CD44v(rsc) clones were observed: (a) ASML(wt) cells produce and assemble a matrix in a CD44v-dependent manner, which supports integrin-mediated adhesion and favors survival. This feature is lost in the ASML-CD44v(kd) cells. (b) CD44v cross-linking initiates phosphatidylinositol 3-kinase/Akt activation in ASML(wt) cells. Accordingly, apoptosis resistance is strikingly reduced in ASML-CD44v(kd) cells. The capacity to generate an adhesive matrix but not apoptosis resistance is restored in ASML-CD44v(rsc) cells. These data argue for a 2-fold effect of CD44v on metastasis formation: CD44v-mediated matrix formation is crucial for the settlement and growth at a secondary site, whereas apoptosis resistance supports the efficacy of metastasis formation.

Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

Tumorigenicity of IL-1α– and IL-1β–Deficient Fibrosarcoma Cells

Analyzing the growth of fibrosarcoma lines derived from IL-1α–, IL-1β–, or IL-1αβ–knockout (−/−) mice in the immunocompetent host revealed that tumor-derived IL-1α and IL-1β exert strong and opposing effects on immune response induction, which prohibited the evaluation of a potential impact on tumorigenicity. Therefore, in vivo growth of IL-1–deficient tumor lines was evaluated in nu/nu mice and was compared with in vitro growth characteristics. All IL-1–deficient fibrosarcoma lines grow in immunocompromised mice. However, IL-1α−/−β–competent (comp) lines grow more aggressively, efficiently induce angiogenesis, and recruit inflammatory cells. Despite stronger tumorigenicity of IL-1βcomp lines, IL-1α strengthens anchorage-independent growth, but both IL-1α and IL-1β support drug resistance. Corresponding to the aggressive growth, IL-1βcomp cells display increased matrix adhesion, motility, and cable formation on matrigel, likely supported by elevated αv/β3 and matrix metalloproteinase expression. Recruitment of myeloid cells requires IL-1β but is regulated by IL-1α, because inflammatory chemokine and cytokine expression is stronger in IL-1α−/−βcomp than in IL-1wt lines. This regulatory effect of tumorderived IL-1α is restricted to the tumor environment and does not affect systemic inflammatory response induction by tumor-derived IL-1β. Both sarcoma cell–derived IL-1α and IL-1β promote tumor growth. However, IL-1α exerts regulatory activity on the tumor cell–matrix cross-talk, and only IL-1β initiates systemic inflammation.