Non-steroidal anti-inflammatory drugs target the pro-tumorigenic extracellular matrix of the postpartum mammary gland

Breast cancer patients diagnosed postpartum have poor prognosis. The postpartum mammary gland undergoes tissue regression to return to the pre-pregnant state. This involution is characterized by wound healing programs known to be tumor promotional in other contexts. Previous studies have shown that mammary extracellular matrix (ECM) from nulliparous rats has tumor suppressive attributes, while mammary ECM from involuting mammary glands is promotional. In models of pregnancy-associated breast cancer, non-steroidal anti-inflammatory drug (NSAID) treatment targeted to postpartum involution inhibits tumor progression, in part by suppressing COX-2 dependent collagen deposition. Because mammary ECM proteins are coordinately regulated, NSAID treatment is anticipated to result in additional protective changes in the mammary extracellular matrix. Here, systemic NSAID treatment was utilized during postpartum involution to reduce mammary COX-2 activity. ECM was isolated from actively involuting glands of rats treated with NSAIDs and compared to ECM isolated from control-involution and nulliparous rats in 3D cell culture and xenograft assays. Compositional changes in ECM between groups were identified by proteomics. In four distinct 3D culture assays, normal and transformed mammary epithelial cells plated in NSAID-involution ECM, phenocopied cells plated in ECM from nulliparous rats rather than ECM from control-involution rats. Tumor cells mixed with NSAID-involution ECM and injected orthotopically in mice formed smaller tumors than cells mixed with control-involution ECM. Proteomic analyses identified and 3D culture assays implicated the ECM protein tenascin-C as a potential mediator of tumor progression during involution that is decreased by NSAID treatment. In summary, NSAID treatment decreases tumor-promotional attributes of postpartum involution mammary ECM.     

Effects of extracellular matrix on the expression of specific ovarian proteins

A unique ovarian follicle cell culture system has been established to analyze the effects of extracellular matrix (ECM) on early granulosa cell differentiation. Primary and early secondary follicles isolated from ovaries of sexually immature rabbits were grown on poly-D-lysine or Englebreth-Holm-Swarm basement membrane biomatrix substrata (EHS) in serum-free, hormonally defined medium. Granulosa cells from these follicles were examined for growth pattern characteristics and for secretory protein synthesis by two-dimensional (2D) PAGE. Whereas some proteins were synthesized by cells on either matrix, the expression of other secreted proteins was markedly affected by the ECM used. Secretion of zona pellucida (ZP) proteins was demonstrated by ELISA assays and immunoblots of one-dimensional (1D) and 2D-PAGE separations of secreted proteins probed with monoclonal and epitope-selected antibodies. Expression of two ZP proteins was altered by ECM: 55-kDa endo-beta-galactosidase (EBGD)-treated ZP glycoprotein (55-kDaEBGD) was secreted by cells grown on either ECM, but a greater amount of 75-kDaEBGD was secreted by cells grown on poly-D-lysine. These studies are the first to show that granulosa cells from early-stage follicles express ZP proteins in vitro in the absence of oocytes, although proper post-translational modification may not occur. They also demonstrate the dramatic effect of ECM on the expression of these and other secretory proteins.     

Isolation and characterization of proteoglycans synthesized by rat myoblasts L6J1 in culture

Proteoglycans synthesized by rat myoblasts L6J1 in culture were isolated using sorbent Q-Sepharose from culture medium, extracellular matrix (ECM), and cells. Elution of the sorbed material in a NaCl gradient separated proteoglycans from the bulk of proteins eluted at low concentration of the salt. Four fractions (fractions I-IV) were obtained for each component of the cell culture, including two proteoglycan fractions for the ECM and culture medium and one fraction for the myoblasts. Proteoglycans of the culture medium were virtually completely represented by proteoglycans of fetal calf serum. With enzymes chondroitinase ABC and heparinase III chondroitin/dermatan sulfate proteoglycans were shown to prevail in all components of the myoblast culture. The core proteins of proteoglycans were characterized by electrophoresis.     

The matrisome: in silico definition and in vivo characterization by proteomics of normal and tumor extracellular matrices

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, differentiation, and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico "matrisome" defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.     

Bioengineered Bruch's-like extracellular matrix promotes retinal pigment epithelial differentiation

In the eye, the retinal pigment epithelium (RPE) adheres to a complex protein matrix known as Bruch's membrane (BrM). The aim of this study was to provide enriched conditions for RPE cell culture through the production of a BrM-like matrix. Our hypothesis was that a human RPE cell line would deposit an extracellular matrix (ECM) resembling BrM. The composition and structure of ECM deposited by ARPE19 cells (ARPE19-ECM) was characterized. To produce ARPE19-ECM, ARPE19 cells were cultured in the presence dextran sulphate. ARPE19-ECM was decellularized using deoxycholate and characterized by immunostaining and western blot analysis. Primary human RPE and induced pluripotent stem cells were seeded onto ARPE19-ECM or geltrex coated surfaces and examined by microscopy or RT-PCR. Culture of ARPE19 cells with dextran sulphate promoted nuclear localization of SOX2, formation of tight junctions and deposition of ECM. ARPE19 cells deposited ECM proteins found in the inner layers of BrM, including fibronectin, vitronectin, collagens IV and V as well as laminin-alpha-5, but not those found in the middle elastic layer (elastin) or the outer layers (collagen VI). ARPE19-ECM promoted pigmentation in human RPE and pluripotent stem cell cultures. Expression of RPE65 was significantly increased on ARPE19-ECM compared with geltrex in differentiating pluripotent stem cell cultures. ARPE19 cells deposit ECM with a composition and structure similar to BrM in the retina. Molecular cues present in ARPE19-ECM promote the acquisition and maintenance of the RPE phenotype. Together, these results demonstrate a simple method for generating a BrM-like surface for enriched RPE cell cultures.     

Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos

Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.     

Simple and high yielding method for preparing tissue specific extracellular matrix coatings for cell culture

Background

The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu.

Methodology/Principal Findings

We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells.

Conclusions

This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell culture to more closely mimic the mature in vivo ECM microenvironment.     

Approaches in extracellular matrix engineering for determination of adhesion molecule mediated single cell function

The native extracellular matrix (ECM) and the cells that comprise human tissues are together engaged in a complex relationship; cells alter the composition and structure of the ECM to regulate the material and biologic properties of the surrounding environment while the composition and structure of the ECM modulates cellular processes that maintain healthy tissue and repair diseased tissue. This reciprocal relationship occurs via cell adhesion molecules (CAMs) such as integrins, selectins, cadherins and IgSF adhesion molecules. To study these cell-ECM interactions, researchers use two-dimensional substrates or three-dimensional matrices composed of native proteins or bioactive peptide sequences to study single cell function. While two-dimensional substrates provide valuable information about cell-ECM interactions, three-dimensional matrices more closely mimic the native ECM; cells cultured in three-dimensional matrices have demonstrated greater cell movement and increased integrin expression when compared to cells cultured on two-dimensional substrates. In this article we review a number of cellular processes (adhesion, motility, phagocytosis, differentiation and survival) and examine the cell adhesion molecules and ECM proteins (or bioactive peptide sequences) that mediate cell functionality.     

Chondrocyte-alginate constructs with or without TGF-β1 produces superior extracellular matrix expression than monolayer cultures

Tissue engineering approaches often require expansion of cell numbers in vitro to accelerate tissue regenerative processes. Although several studies have used this technique for therapeutic purposes, a major concern involving the use of isolated chondrocyte culture is the reduction of extracellular matrix (ECM) protein expressed due to the transfer of cells from the normal physiological milieu to the artificial 2D environment provided by the cell culture flasks. To overcome this issue, the use of alginate hydrogel beads as a substrate in chondrocyte cultures has been suggested. However, the resultant characteristics of cells embedded in this bead is elusive. To elucidate this, a study using chondrocytes isolated from rabbit knee articular cartilage expanded in vitro as monolayer and chondrocyte-alginate constructs was conducted. Immunohistochemical evaluation and ECM distribution was examined with or without transforming growth factor (TGF-β1) supplement to determine the ability of cells to express major chondrogenic proteins in these environments. Histological examination followed by transmission electron microscopy and scanning electron microscopy was performed to determine the morphology and the ultrastructural characteristics of these cells. Results demonstrated a significant increase in glycosaminoglycan/mg protein levels in chondrocyte cultures grown in alginate construct than in monolayer cultures. In addition, an abundance of ECM protein distribution surrounding chondrocytes cultured in alginate hydrogel was observed. In conclusion, the current study demonstrates that the use of alginate hydrogel beads in chondrocyte cultures with or without TGF-β1 supplement provided superior ECM expression than monolayer cultures.     

The role of the arginine-glycine-aspartic acid-directed cellular binding to type I collagen and rat mesenchymal cells in colorectal tumour differentiation

The relationship between the adhesion of five human colorectal carcinoma cell lines to extracellular matrix (ECM) proteins, namely type I collagen, type IV collagen, fibronectin, laminin and basement membrane extract (Matrigel), and the ability of these cells to express morphological differentiation when grown in a basement membrane extract (Matrigel) or on normal rat mesenchymal cells has been examined. Two cell lines, SW1222 and HRA-19, organised into glandular structures, with well-defined polarity when cultured on both substrata as well as in three-dimensional (3D) collagen gel culture as previously shown. The remaining three cell lines (SW620, SW480 and HT29) grew as loose aggregates or as they would normally grow on tissue culture plastic. Addition to the culture medium of a hexapeptide, containing the cell-matrix recognition sequence arginine-glycine-aspartic acid (RGD), inhibited attachment and glandular formation of SW1222 and HRA-19 when these cells were grown on living mesenchymal cells, but not in Matrigel. The morphological differentiation of HRA-19 cells in 3D-collagen was also inhibited by the same RGD-containing peptide, as previously shown for SW1222 cells. Attachment of the remaining three cell lines was inhibited on mesenchyme but not in Matrigel, further supporting the specificity of the peptide effect on epithelial-mesenchymal binding. In conclusion we have shown that colorectal tumour cells are able to bind ECM proteins and that the cellular binding is an essential step in the induction of the morphological differentiation seen on living mesenchymal cells, in basement membrane extracts and in type I collagen gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell adhesion profiling using extracellular matrix protein microarrays

We have developed a microarray-based system for cell adhesion profiling of large panels of cell-adhesive proteins to increase the throughput of in vitro cell adhesion assays, which are currently primarily performed in multiwell plates. Miniaturizing cell adhesion assays to an array format required the development of protocols for the reproducible microspotting of extracellular matrix (ECM) protein solutions and for the handling of cell suspensions during the assay. We generated ECM protein microarrays with high reproducibility in microspot protein content using nitrocellulose-coated glass microslides, combined with piezoelectric microspotting of protein solutions. Protocols were developed that allowed us to use 5000 cells or fewer on an array of 4 x 4 mm consisting of 64 microspots. Using this microarray system, we identified differences of adhesive properties of three cell lines to 14 different ECM proteins. Furthermore, the sensitivity and accuracy of the assays were increased using microarrays with ranges of ECM protein amounts. This microarray system will be particularly useful for extensive comparative cell adhesion profiling studies when only low amounts of adhesive substrate and cells, such as stem cells or cells from biopsies, are available.     

Colocalization of laminin and fibronectin in bovinelens epithelial cells in vitro

Summary The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse clonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cellsurfaces and in between cells in the densely packed center of the colonies, and as “adhesive plaques” and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin. Supported by Public Health Service grant EY05570 from the National Eye Institute Bethesda, MD.     

A Unified Mechanism for Aminopeptidase N-based Tumor Cell Motility and Tumor-homing Therapy

Tumor cell surface aminopeptidase N (APN or CD13) has two puzzling functions unrelated to its enzymatic activity: mediating tumor cell motility and serving as a receptor for tumor-homing peptides (peptides that bring anti-cancer drugs to tumor cells). To investigate APN-based tumor-homing therapy, we determined the crystal structure of APN complexed with a tumor-homing peptide containing a representative Asn-Gly-Arg (NGR) motif. The tumor-homing peptide binds to the APN enzymatic active site, but it resists APN degradation due to a distorted scissile peptide bond. To explore APN-based tumor cell motility, we examined the interactions between APN and extracellular matrix (ECM) proteins. APN binds to, but does not degrade, NGR motifs in ECM proteins that share similar conformations with the NGR motif in the APN-bound tumor-homing peptide. Therefore, APN-based tumor cell motility and tumor-homing therapy rely on a unified mechanism in which both functions are driven by the specific and stable interactions between APN and the NGR motifs in ECM proteins and tumor-homing peptides. This study further implicates APN as an integrin-like molecule that functions broadly in cell motility and adhesion by interacting with its signature NGR motifs in the extracellular environment.     

Pectin-based injectable biomaterials for bone tissue engineering

A variety of natural polymers and proteins are considered to be 3D cell culture structures able to mimic the extracellular matrix (ECM) to promote bone tissue regeneration. Pectin, a natural polysaccharide extracted from the plant cell walls and having a chemical structure similar to alginate, provides interesting properties as artificial ECM. In this work, for the first time, pectin, modified with an RGD-containing oligopeptide or not, is used as an ECM alternative to immobilize cells for bone tissue regeneration. The viability, metabolic activity, morphology, and osteogenic differentiation of immobilized MC3T3-E1 preosteoblats demonstrate the potential of this polysaccharide to keep immobilized cells viable and differentiating. Preosteoblasts immobilized in both types of pectin microspheres maintained a constant viability up to 29 days and were able to differentiate. The grafting of the RGD peptide on pectin backbone induced improved cell adhesion and proliferation within the microspheres. Furthermore, not only did cells grow inside but also they were able to spread out from the microspheres and to organize themselves in 3D structures producing a mineralized extracellular matrix. These promising results suggest that pectin can be proposed as an injectable cell vehicle for bone tissue regeneration.     

Vasculogenic and angiogenic potential of adipose stromal vascular fraction cell populations in vitro

Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell source that contains endothelial cells, pericytes, smooth muscle cells, stem cells, and other accessory immune and stromal cells. The SVF cell population has been shown to support vasculogenesis in vitro as well vascular maturation in vivo. Matrigel, an extracellular matrix (ECM) mixture has been utilized in vitro to evaluate tube formation of purified endothelial cell systems. We have developed an in vitro system that utilizes freshly isolated SVF and ECM molecules both in pure form (fibrin, laminin, collagen) as well as premixed form (Matrigel) to evaluate endothelial tip cell formation, endothelial stalk elongation, and early stages of branching and inosculation. Freshly isolated SVF rat demonstrate cell aggregation and clustering (presumptive vasculogenesis) on Matrigel ECM within the first 36 h of seeding followed by tip cell formation, stalk cell formation, branching, and inosculation (presumptive angiogenesis) during the subsequent 4 days of culture. Purified ECM molecules (laminin, fibrin, and collagen) promote cell proliferation but do not recapitulate events seen on Matrigel. We have created an in vitro system that provides a functional assay to study the mechanisms of vasculogenesis and angiogenesis in freshly isolated SVF to characterize SVF’s blood vessel forming potential prior to clinical implantation.     

Odontoblasts induced from mesenchymal cells of murine dental papillae in three-dimensional cell culture

In an organ culture system under a three-dimensional microenvironment that provides the conditions needed for odontoblast differentiation, a row of odontoblasts can be induced (Kikuchi et al. 1996, 2001). Therefore, in a newly designed three-dimensional cell culture system that fulfils the conditions necessary for odontoblast differentiation (Kikuchi et al. 2002), we examined whether dental papilla cells in rat mandibular incisors could differentiate into tubular dentine-forming cells. In our previously established organ culture system, CM-Dil-labeled cells that were microinjected into isolated dental papillae were replaced by a row of odontoblasts. In a three-dimensional cell culture system, which consists of two kinds of type I collagen in the upper layer over multi-layered cells seeded onto collagen containing Matrigel in the lower layer and which acts as a structural meshwork, dental papilla cells were incubated as multi-layered cells in an artificial extracellular matrix (ECM). The cells aggregated to form a cell mass and invaginated as a cell mass into the ECM. The cells also extended fine fibrillar processes into the ECM. With regard to invagination, the proteolytic activities of matrix metalloproteinase-2 (MMP-2)/membrane type 1-matrix metalloproteinase (MT 1-MMP) were observed on the outer multi-layers of cells within a cell mass adjacent to the ECM. The cell mass progressively shrank to about one-half to one-third of its original diameter and was organized as a tissue surrounded by a newly secreted ECM, like dental pulp-dentine. The cells adjacent to the secreted ECM were constructed as a row of polarized columnar cells. They extended slender processes into the new ECM, which is characteristic of tubular matrix. Dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP 1) genes, which are specific for odontoblast differentiation, were expressed in an aggregated cell mass where tubular matrix-forming cells were induced. Furthermore, the tubular matrix became mineralized under prolonged culture. These results imply that the putative progenitor cells/stem cells residing in dental papillae can differentiate into odontoblasts under appropriate conditions in vitro.     

Effect of extracellular matrix on prolactin secretion and mRNA accumulation in GH3 cells

The matrix upon which cells grow affects their morphology, growth rate, response to external stimuli, and protein synthesis. GH3 cells, a well-characterized rat pituitary tumor cell line, synthesize and secrete growth hormone and prolactin (Prl). These cells are rounded, attach loosely, and form clumps when plated on plastic. GH3 cells plated on an extracellular matrix (ECM) from bovine corneal endothelial cells become flattened and strongly adherent to the culture dish, and have an initial increased rate of proliferation. Cells cultured on plastic have a 48-hr lag period before the start of cell division; this can be shortened by increasing the concentration of serum in the medium. Since GH3 cells store little Prl, hormone release is a good index of Prl synthesis. Prl secretion from cells cultured on extracellular matrix is twice as great as from cells cultured on plastic. The increase in Prl secretion from cells grown on extracellular matrix paralleled by a concomitant increase in the accumulation of prolactin mRNA. Cells cultured on plastic secrete more Prl in response to TRH stimulation than do cells cultured on ECM. Cells grown on either surface were unresponsive to dopamine. Thus, culturing cells on ECM may change their morphology and affect the synthesis and regulation of specific cellular proteins and their mRNAs.     

Secreted protein acidic and rich in cysteine is a matrix scavenger chaperone

Secreted Protein Acidic and Rich in Cysteine (SPARC) is one of the major non-structural proteins of the extracellular matrix (ECM) in remodeling tissues. The functional significance of SPARC is emphasized by its origin in the first multicellular organisms and its high degree of evolutionary conservation. Although SPARC has been shown to act as a critical modulator of ECM remodeling with profound effects on tissue physiology and architecture, no plausible molecular mechanism of its action has been proposed. In the present study, we demonstrate that SPARC mediates the disassembly and degradation of ECM networks by functioning as a matricellular chaperone. While it has low affinity to its targets inside the cells where the Ca(2+) concentrations are low, high extracellular concentrations of Ca(2+) activate binding to multiple ECM proteins, including collagens. We demonstrated that in vitro, this leads to the inhibition of collagen I fibrillogenesis and disassembly of pre-formed collagen I fibrils by SPARC at high Ca(2+) concentrations. In cell culture, exogenous SPARC was internalized by the fibroblast cells in a time- and concentration-dependent manner. Pulse-chase assay further revealed that internalized SPARC is quickly released outside the cell, demonstrating that SPARC shuttles between the cell and ECM. Fluorescently labeled collagen I, fibronectin, vitronectin, and laminin were co-internalized with SPARC by fibroblasts, and semi-quantitative Western blot showed that SPARC mediates internalization of collagen I. Using a novel 3-dimensional model of fluorescent ECM networks pre-deposited by live fibroblasts, we demonstrated that degradation of ECM depends on the chaperone activity of SPARC. These results indicate that SPARC may represent a new class of scavenger chaperones, which mediate ECM degradation, remodeling and repair by disassembling ECM networks and shuttling ECM proteins into the cell. Further understanding of this mechanism may provide insight into the pathogenesis of matrix-associated disorders and lead to the novel treatment strategies.