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PubMed1992

Basement membrane components enhance isolated enterocyte growth.

Isolated enterocyte transplantation may have a potential role in increasing intestinal surface area. However, enterocytes are notoriously difficult to grow. Basement membrane components (BMC) promote adherence, migration, and differentiation of enterocytes. Our aim was to determine if BMC enhance enterocyte growth. Twenty-nine rabbits had 5-cm ileal segments resected and serosal pouches (n = 22) created from the serosal surface of the colon. Harvesting of enterocytes by warm trypsinization resulted in 92 +/- 6% cell viability and yielded 5.0 +/- 2.4 10(6) enterocytes/cm intestine. Enterocytes (10(5) were cultured in vitro (n = 7) in 10 ml growth media in plain flasks and flasks coated with laminin alone or Matrigel (an extract of mouse basement membrane containing laminin plus Type IV collagen and heparan sulfate). The cells were subcultured at 2 weeks and examined after Geimsa staining at 4 weeks. Epithelial growth was confirmed by light microscopy and staining for cytokeratin and quantitated by image analysis (JAVA). Epithelial coverage of the flasks was greater with Matrigel (80 +/- 15%) than laminin (66 +/- 15%) which was greater than control (44 +/- 20%) (P less than 0.01). For in vivo studies 10(5) harvested cells were infused into the serosal pouches either in growth media (n = 9) or media + Matrigel (n = 13). Epithelial growth in the pouch was evaluated by qualitative scoring of cytokeratin staining. Cytokeratin staining was similar on control colon serosa (n = 5) and serosa after infusion of cells in media alone. However, Matrigel significantly enhanced the area, depth, and intensity of staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Functional activity of isolated pig hepatocytes attached to different extracellular matrix substrates. Implication for application of pig hepatocytes in a bioartificial liver.

For the manufacture of a bioartificial liver for human application, large amounts of viable and active hepatocytes are needed. Pig hepatocytes are considered to be the best alternative to scarce human hepatocytes. In vitro hepatocyte functions have so far been tested under different circumstances, mainly with rat hepatocytes. Pig hepatocytes were isolated with a single two-step isolation procedure, resulting in a high yield of viable hepatocytes. The hepatocytes were tested for their ability to synthesise urea, to metabolise 7-ethoxycoumarin (cytochrome P450 activity), and to synthesise and secrete proteins. These activities of hepatocytes while attached to tissue culture plastic were compared to the activity of the cells attached to several extracellular matrix constituents: collagen I and IV, laminin, fibronectin, Engelbreth-Holm-Swarm Natrix and in the presence of Matrigel. With the exception of Matrigel, neither of the extracellular matrix substrates enhanced pig hepatocyte function compared to tissue culture plastic. However, relatively large amounts of murine proteins leak out of the Matrigel. The advisability of using Matrigel or other extracellular matrix proteins in a bioartificial liver loaded with pig hepatocytes is discussed.

Animals↗
PubMed1996

Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model.

The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model.

Cell Division↗
PubMed1995

Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma cell lines.

The influence of an artificial basement membrane (BM), Matrigel, and four individual extracellular matrix proteins, fibronectin, laminin, collagen I and vitronectin, on cell proliferation, morphology and migration was assessed in four glioma cell lines. Matrigel and individual BM proteins differentially inhibited cell proliferation of all cell lines studied. In addition, Matrigel was found to induce extensive morphological changes in glioma cells. Polycarbonate filters, of 8-microns porosity in modified Boyden chambers, were used to assess the chemoattraction activity of Matrigel and the individual proteins on glioma cells. All these components were found to stimulate cell migration, albeit to different extents but laminin proved to be the most effective chemoattractant for glioma cells in vitro. These data suggest that basement membrane proteins may inhibit proliferation and stimulate migration in order to facilitate invasion.

Cell Division↗
PubMed2004

Differential role of platelet granular mediators in angiogenesis.

OBJECTIVES: Platelets contain numerous substances regulating angiogenic response. However, the regulatory role of platelets in blood vessel development remains to be elucidated. We investigated the comprehensive effect of platelets as a cellular system on angiogenesis. METHODS: The following approaches were applied: (a) in vitro-aortic ring assay and chemotaxis assay; (b) in vivo-injection of platelet-containing matrigel plug subcutaneously into a mouse followed by immunohistochemical analysis of angiogenic response. RESULTS: Platelets stimulated formation of blood vessels in vitro in the rat aortic ring model via VEGF and bFGF, while blocking of platelet factor-4 promoted this effect. Addition of platelets to the matrigel followed by its subcutaneous injection into a mouse resulted in an intensive migration of fibroblasts into the matrigel as well as formation of blood capillaries de novo. This platelet effect was mediated through bFGF, VEGF, and heparanase. Furthermore, platelet releasate was found to induce endothelial cell chemotaxis. This effect was mediated by a concerted action of intraplatelet bFGF, PDGF, VEGF, and heparanase. CONCLUSION: Platelets affect different stages of the angiogenic response with a trend to a pro-angiogenic net effect despite the presence of angiogenesis inhibitors such as platelet factor 4. While a concomitant effect of bFGF and VEGF seemed to be essential for the entire process of vessel formation (aortic ring and matrigel models), PDGF and heparanase were effective only at the migration stage.

Animals↗
PubMed2006

A new model of experimental subretinal neovascularization in the rabbit.

Existing animal models of choroidal neovascularization (CNV) present several problems: they are hard to reproduce, they are inefficient, and the CNV created is not sustainable. The purpose of this study is to develop a highly efficient, reliable, sustainable rabbit model of CNV to facilitate the study of anti-angiogenic and anti-proliferative therapies for ocular diseases. Twenty-two pigmented rabbits were used in this study. Eleven rabbits received subretinal injections of either 10 microl of Matrigel with 500 ng of vascular endothelial growth factor (VEGF) or 20 microl of Matrigel with 750 ng of VEGF; eight rabbits received subretinal injections of either 10 or 20 microl of Matrigel only; three rabbits used as controls received subretinal injections of 20 microl phosphate-buffered saline (PBS) alone. Fundus photography, fluorescein angiography, optical coherence tomography, and histologic examinations were performed 1, 2, 4, and 9 weeks after injection. All experimental eyes showed angiographic leakage within this localized area 1 week after injection. The amount of leakage usually increased at weeks 2 and 4 and, in most cases, persisted at week 9. Control eyes demonstrated no leakage at any time point. Optical coherence tomography of treated eyes showed subretinal fluid and the presence of a lesion, possibly vascular or fibrotic, at the site of the leakage. Histologic analysis confirmed the presence of new subretinal blood vessels in the areas of Matrigel deposit. In conclusion, this novel method provides a highly reproducible, reliable, and sustainable rabbit model of experimental choroidal neovascularization. Such a model may prove useful for screening new anti-angiogenic therapies in a larger animal eye.

Animals↗
PubMed2001

Fractalkine: a novel angiogenic chemokine in rheumatoid arthritis.

Angiogenesis is an important aspect of the vasculoproliferation found in the rheumatoid arthritic (RA) pannus. We have previously implicated members of the CXC chemokine family as potent angiogenic mediators in RA. We investigated the possibility that the sole member of the CX(3)C chemokine family, fractalkine (fkn), induces angiogenesis and that fkn might mediate angiogenesis in RA. Recombinant human fkn significantly induced migration of human dermal microvascular endothelial cells (HMVECs), a facet of the angiogenic response, in the pmol/L range in a concentration-dependent manner (P < 0.05). Fkn also induced the formation of significantly more endothelial tubes on Matrigel than did a negative control (P < 0.05). Fkn significantly induced 2.3-fold more blood vessel growth than control in the in vivo Matrigel plug assays (P < 0.05). We identified HMVEC expression of the fkn receptor, CX(3)CR1. Next, we determined if RA synovial fluid (SF)-induced angiogenesis was fkn-dependent. SFs from six RA patients immunodepleted of soluble fkn induced 56% less migration of HMVECs than did sham-depleted RA SFs (P < 0.05). In vivo, immunodepletion of fkn from six RA SFs significantly inhibited their angiogenic activity in Matrigel plug assays (P < 0.05). Immunodepletion of fkn from five RA synovial tissue homogenates inhibited their ability to induce angiogenesis in in vivo Matrigel plug assays (P < 0.05). These results establish a new function for fkn as an angiogenic mediator and suggest that it may mediate angiogenesis in RA.

Arthritis, Rheumatoid↗
PubMed1996

Sulfotransferase gene expression in primary cultures of rat hepatocytes.

Hepatocyte cultures have been used in pharmacotoxicological studies, and sulfotransferases (ST) are important drug-metabolizing enzymes in liver. The expression of sulfotransferases in hepatocyte cultures has not been examined systematically. In the present study, the mRNA levels of different sulfotransferases in male and female rat hepatocytes were examined by northern-blot analyses. Various culture conditions such as different matrices (collagen, matrigel, collagen sandwich, or co-culture with epithelial cells), medium (Way-mouth's MB 752/1 and Modified Chee's Medium) and glucocorticoid supplementation (dexamethasone, 0.1 microM) were compared. Phenol ST (ST1A1) mRNA levels decreased to about 50% of initial mRNA levels within 10 hr of culture. At 96 hr, ST1A1 mRNA levels were approximately 20% of initial values when cultured on collagen, matrigel or co-culture. The two media did not differ in ability to maintain ST1A1 mRNA levels in the absence of dexamethasone (DEX); however, DEX addition to either medium resulted in ST1A1 mRNA levels greater than 100% of the initial mRNA levels at 96 hr, with the greatest increase observed using the matrigel substratum and Chee's medium. In the absence of DEX, the mRNA levels of N-hydroxy-2-acetylaminoflurene sulfortransferase (ST1C1), estrogen sulfotransferase (ST1E2) and hydroxysteroid sulfotransferase (ST-20/21, ST-40/41, ST-60) fell to approximately 20% of their initial levels within 24 hr, and to less than 5% at 96 hr. The loss of expression of these sulfotransferases was observed with all culture conditions. Addition of DEX to the media resulted in ST-40/41 and ST-60 mRNA expression at 20 and 35% of their initial values, respectively, in cultures maintained on matrigel and Chee's medium at 96 hr. These data suggest that sulfotransferases lose their constitutive expression in hepatocyte culture, but retain their inducibility.

Albumins↗
PubMed2001

In vitro tubulogenesis of endothelial cells by relaxation of the coupling extracellular matrix-cytoskeleton.

OBJECTIVE: This investigation aimed at determining the importance of the rigidity of the adhesive support and the participation of the cytoskeleton in tubulogenesis of endothelial cells in vitro. METHODS: The morphotype, biosynthetic phenotype and cytoskeleton organization of human umbilical vein endothelial cells (HUVEC) were analyzed on supports of variable mechanical resistance. RESULTS: Western blot analysis revealed a strong reduction of the expression of actin and focal-adhesion plaque (FAP) proteins in HUVEC organized in tube-like structures (TLS) on soft matrigel or on matrigel co-polymerized with heat-denatured collagen as compared to HUVEC remaining in a monolayer pattern on rigid matrigel-coat or on matrigel co-polymerized with type I collagen. Human skin fibroblasts morphotype was not altered in these culture conditions and the pattern of FAP proteins and actin was not modulated. By using polyacrylamide gels polymerized with various concentrations of bis-acrylamide to modulate the mechanical resistance of the support and cross-linked to a constant amount of gelatin to provide an equal density of attachment sites, it was shown that the less rigid the support, the more endothelial cells switched to a tube-like pattern. Collagen type I-induced tubulogenesis was accompanied by a profound and reversible remodeling of the actin-FAP complex suggesting a weakening of the bridging between extracellular matrix (ECM) and the cytoskeleton. Human skin fibroblasts and smooth muscle cells, used as control cells, adhered strongly to the collagen, did not form TLS and their network of actin stress fibers was not remodeled. The inhibition of collagen type I-induced tubulogenesis by agents altering the actin cytoskeleton-FAP complex including calpain type I inhibitor, orthovanadate, KT5720 and jasplakinolide, further supports the determinant role of mechanical coupling between the cells and the matrix in tubulogenesis. CONCLUSIONS: A reduced tension between the endothelial cells and the extracellular matrix, originating in the support or within the cells is sufficient to trigger an intracellular signaling cascade leading to tubulogenesis, an event mimicking one of the last steps of angiogenesis.

Blotting, Western↗
PubMed2002

Determination of bone marrow-derived endothelial progenitor cell significance in angiogenic growth factor-induced neovascularization in vivo.

OBJECTIVE: Our laboratory and others recently provided evidence indicating that endothelial progenitor cells (EPCs) participate in postnatal neovascularization. However, the extent to which EPCs contribute to adult neovascularization remains unclear. To address this issue, we investigated the quantitative contribution of EPCs to newly formed vascular structures in an in vivo Matrigel plug assay and corneal micropocket assay. MATERIALS AND METHODS: Lethally irradiated FVB mice were transplanted with bone marrow (BM) mononuclear cells from transgenic mice constitutively expressing beta-galactosidase (beta-gal) encoded by the lacZ gene regulated by an endothelial-specific tie-2 promoter. Reconstitution of the transplanted BM leads to the expression of lacZ in mice, which is restricted to BM cells expressing tie-2. RESULTS: Four weeks after BM transplantation (BMT), tie-2/lacZ/BMT mice were implanted with either Matrigel containing fibroblast growth factor-2 subcutaneously or with a vascular endothelial growth factor pellet into the cornea. After 7 days, the Matrigel plug or the cornea was removed and analyzed by X-gal staining or immunostaining for beta-gal. X-gal staining of the Matrigel plug identified 5.7% +/- 1.2% of endothelial cells (ECs) as cells originated from BM-derived EPCs, whereas the more sensitive technique of immunofluorescence identified 26.5% +/- 0.9% of ECs. Similarly, EPC-derived cells comprised 5.0% +/- 2.4% and 17.7% +/- 3.6% of the ECs in corneal neovascularization identified by X-gal staining and immunohistochemistry, respectively. Ki67 staining of the corneal tissue documented that the majority of EPC-derived cells were actively proliferating in situ. CONCLUSION: These findings suggest that BM-derived EPCs make a significant contribution to angiogenic growth factor-induced neovascularization that may account for up to 26% of all ECs.

Animals↗
PubMed1998

Humatrix, a novel myoepithelial matrical gel with unique biochemical and biological properties.

Myoepithelial cells in situ and in vitro exert important paracrine effects on carcinoma cells which are mediated by high expression of extracellular matrix molecules, proteinase inhibitors and angiogenic inhibitors. Myoepithelial xenografts (human matrix secreting (HMS)-X, HMS-3X and HMS-4X) established from benign human salivary gland and breast myoepithelial tumors accumulate an abundant extracellular matrix which can be extracted with 6 M urea and 2 M guanidinium hydrochloride to form a gel at 25-37 degrees C. This gel, termed Humatrix, exhibits different biochemical and biological properties than the conventional non-human matrical gels in existence, i.e. Matrigel and Vitrogen 100. Whereas Matrigel consists mainly of basement membrane molecules, e.g. laminin, type IV collagen and heparan sulfate proteoglycan, and Vitrogen 100 consists mainly of non-basement membrane molecules, e.g. type I and type III collagen, Humatrix contains significant amounts of both basement membrane and non-basement membrane molecules, including large amounts of chondroitin sulfate proteoglycan. Like Matrigel, Humatrix contains bound growth factors, including epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I); unlike Matrigel, which contains predominantly significant quantities of bound proteinases, including tissue-type plasminogen activator (tPA), matrix metalloproteinase (MMP)-2 and MMP-9, and angiogenic factors, including basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta, Humatrix contains predominantly bound proteinase inhibitors such as protease nexin II (PN-II) and alpha1-antitrypsin and angiogenic inhibitors such as thrombospondin-1. Humatrix selectively stimulates the growth and tumorigenicity of human myoepithelial cell lines but inhibits invasion, angiogenesis and metastasis of other non-myoepithelial malignant cell lines. Because of its myoepithelial origin Humatrix represents a more natural source of extracellular matrix molecules and bound factors that carcinoma cells encounter in vivo.

Animals↗
PubMed1990

Reconstituted basement membrane matrix stimulates interstitial procollagenase synthesis by human fibroblasts in culture.

Synthesis of interstitial collagenase by human fibroblasts was compared when cultured on plastic, in the presence or absence of soluble laminin, on a type I collagen gel and on a gel of basement membrane components (matrigel). Fibroblasts cultured on matrigel or on type I collagen gel displayed an increase in the steady-state levels of mRNA for interstitial procollagenase that was proportional to its enzymatic activity. Laminin, the main component of matrigel, had no effect on the interstitial collagenase synthesis by fibroblasts. We suggest that matrigel, which stimulates the interstitial collagenase production at a transcriptional step, could regulate the catabolic potential of fibroblasts.

Basement Membrane↗
PubMed2002

Invasiveness of human pleural mesothelioma cells is influenced in vitro by the three-dimensional structure of the ECM and its composition.

The grade of malignancy of a neoplasm is influenced by the invasive and metastatic potential of the tumor cells. The extracellular matrix of tissues is known to interact with many aspects of the biological behavior of tumor cells, such as differentiation and invasiveness. Therefore we studied the influence of the extracellular matrix on the morphology and invasiveness of the human biphasic pleural mesothelioma cell line MSTO-211H in vitro. The major components of the two strata encountered by a pleural mesothelioma cell leaving the epithelial community were mimicked by plating cells either on collagen I, the major component of the underlying stratum fibrosum, being encountered by cells under pathological conditions or on reconstituted basement membrane (Matrigel) in order to simulate the basement membrane of the stratum serosum of the mesothelium, which is the matrix cells have contact to under physiological conditions. Growth on collagen I leads to cell separation and invasion into the matrix, whereas growth of MSTO-211H cells on Matrigel results in the formation of a huge and dense network of cells extending throughout the whole plating area. The morphology of cell contacts between the two populations varies impressively. While cells on collagen I hardly find to each other in groups, and if so, with a broad intercellular cleft, Matrigel induces the tight approach of membranes of neighbouring cells with formation of syncytium-like structures. Administration of the main ECM components laminin and collagen IV alone and together in equimolar concentrations as present in Matrigel, does not result in any morphological changes when compared to cells growing on plastic substrates or on collagen I. Therefore, collagen I increases cell separation and invasiveness whereas an intact basement membrane seems to prevent the cells from separating and spreading, thus lowering their invasive potential.

Collagen↗
PubMed2003

Basement membrane-like matrix inhibits proliferation and collagen synthesis by activated rat hepatic stellate cells: evidence for matrix-dependent deactivation of stellate cells.

During liver fibrosis hepatic stellate cells become activated, transforming into proliferative myofibroblastic cells expressing type I collagen and alpha-smooth muscle actin. They become the major producers of the fibrotic neomatrix in injured liver. This study examines if activated stellate cells are a committed phenotype, or whether they can become deactivated by extracellular matrix. Stellate cells isolated from normal rat liver proliferated and expressed mRNA for activation markers, alpha-smooth muscle actin, type I procollagen and tissue inhibitor of metalloproteinases-1 following 5-7 day culture on plastic, but culture on Matrigel suppressed proliferation and mRNA expression. Activated stellate cells were recovered from plastic by trypsinisation and replated onto plastic, type I collagen films or Matrigel. Cells replated on plastic and type I collagen films proliferated and remained morphologically myofibroblastic, expressing alpha-smooth muscle actin and type I procollagen. However, activated cells replated on Matrigel showed <30% of the proliferative rate of these cells, and this was associated with reduced cellular expression of proliferating cell nuclear antigen and phosphorylation of mitogen-activated protein kinase in response to serum. Activated HSC replated on Matrigel for 3-7 days progressively reduced their expression of mRNA for type I procollagen and alpha-smooth muscle actin and both became undetectable after 7 days. We conclude that basement membrane-like matrix induces deactivation of stellate cells. Deactivation represents an important potential mechanism mediating recovery from liver fibrosis in vivo where type I collagen is removed from the liver and stellate cells might re-acquire contact with their normal basement membrane-like pericellular matrix.

Actins↗
PubMed1997

Human neutrophils do not degrade major basement membrane components during chemotactic migration.

At sites of inflammation, circulating neutrophils (PMNs) migrate through microvessel walls into the subendothelial interstitium. While endothelial passage is mediated by adhesion proteins, including those of the integrin, selectin and immunoglobulin superfamily classes, the mechanisms used to cross the subendothelial basement membrane (BM) are unclear. Studies examining tumour cell invasion and lymphocyte extravasation suggest several possible mechanisms, including proteolysis. Different cells, however, may use different mechanisms to effect passage. To examine neutrophil-basement membrane interactions in more detail, human PMNs were embedded within reconstituted BM (Matrigel) and used in migration assays. The integrity of the gel following migration was assessed by assaying for the release of incorporated radiolabelled products and by-immunoblotting for specific matrix molecule epitopes. PMNs migrated through Matrigel in response to the chemotactic peptide FMLP. Degradation products of laminin, heparan sulphate proteoglycan or of gelatin, however, were not detected. In contrast, phorbol ester, which triggers activation without migration, released approximately 40% of incorporated HSPG, 30% of gelatin and 20% of laminin as intact molecules or degraded fragments. Electron microscopy of migrating cells demonstrated pseudopodia associated with channels within the Matrigel. Although the serine proteinase inhibitor DFP, plasma and a specific anti-neutrophil elastase IgG blocked degradation, these agents failed to inhibit migration. Migration was inhibited, however, when the Matrigel concentration was increased to 10 mg/ml. Thus, although PMNs will degrade matrix components they do not do so during migration, and proteolytic remodelling of the BM is not a pre-requisite for neutrophil passage.

Basement Membrane↗
PubMed2004

Human ovarian tissue cultures: extracellular matrix composition, coating density and tissue dimensions.

Human ovarian tissue can be successfully cryopreserved for fertility preservation. Optimal use of this approach requires the development of reliable restoration methods, including in-vitro culture of follicles. A culture system has been established, but improvement of the basic handling and techniques is necessary. Ovarian biopsies were collected from 33 women, cut into small pieces and cultured for 7-14 days on an extracellular matrix. Three separate studies investigated tissue dimensions (slices and cubes), coating density of extracellular matrix (diluted, thin and thick), and different extracellular matrix compositions (regular Matrigel, growth factor reduced Matrigel and laminin). Initial recruitment of primordial follicles and reduction in follicle viability was observed in all cultures compared with uncultured tissue. After 7 days of culture, more viable follicles were present in the cubed tissue, which also showed significant activation of growth, observed in tissue slices only after 14 days of culture. A diluted coating of Matrigel supported a greater proportion of viable follicles in 7-day cultures, whereas composition of the extracellular matrix had no effect. Human ovarian follicles can grow and develop in vitro within cortical tissue, and may benefit from culture as cubes on diluted Matrigel. This technique may provide a solution to the successful recovery and growth of follicles from frozen human ovarian tissue even though it will take time and much more optimization before it can be used in clinical practice.

Adult↗
PubMed2000

Specific expression of matrix metalloproteinases 1, 3, 9 and 13 associated with invasiveness of breast cancer cells in vitro.

Several matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) were studied in highly invasive (MDA-MB-231) and slightly invasive (MCF-7, T47D, BT-20) breast cancer cell lines. Investigations were carried out at the protein level and/or at the mRNA level, either in cells cultured as monolayers on plastic, or in cells seeded on a thin layer of Matrigel basement membrane matrix. Analysis of MMP expression by RT-PCR showed expression of MMP-1. MMP-3, and MMP-13 in highly invasive MDA-MB-231 cells, but not in slightly invasive cell lines. The extracellular secretion of MMP-1 and MMP-3 by MDA-MB 231 cells could be also shown by ELISA. TIMP-1 and TIMP-2 mRNAs were found in all cell lines, however, the extracellular secretion of both TIMPs was much higher in MDA-MB-231 cells than in the other cell lines. When the cells were cultured on Matrigel matrix, MMP-9 expression was induced in MDA-MB-231 cells only, as assessed by RT-PCR and zymography experiments. The invasive potential of MDA-MB-231 cells evaluated in vitro through Matrigel was significantly inhibited by the MMP inhibitor BB-2516, by 25% and 50% at the concentrations of 2 x 10(-6) M and 10(-5) M, respectively. In conclusion, our data show that highly invasive MDA-MB-231 cells but not slightly invasive T47D, MCF-7 and BT-20 cells express MMP-1, MMP-3, MMP-9 and MMP-13. MMP-9 which is specifically up-regulated by cell contact to Matrigel, may play a key role in the invasiveness of MDA-MB-231 cells through basement membranes.

Base Sequence↗
PubMed1997

Assessment of the invasive potential of human gynecological tumor cell lines with the in vitro Boyden chamber assay: influences of the ability of cells to migrate through the filter membrane.

The Boyden chamber assay is widely used for in vitro measurement of the invasive capacity of cells. However, results can be affected significantly if certain precautions are not taken. Using the Boyden chamber assay we investigated in vitro the invasive potential of a variety of human gynecological tumor cell lines to degrade and migrate through the artificial basement membrane matrix Matrigel. However, in the absence of this Matrigel layer large differences were observed in the ability of cells to adhere to, migrate through and attach to the lower side of the filter membranes. These differences were influenced by cell density, degree of directional locomotion, and the size of the filter pores. To adjust for these influences (which are not directly correlated to the capacity of cells to traverse the Matrigel layer), invasion results were corrected for the ability of cells to migrate through the filter membrane. In addition, the invasion of MDA-MB-231 cells was used as an internal standard to compensate for variations in the Matrigel layer between different experiments. Overall, in our experimental set up, the five human breast cancer cell lines were the most invasive (mean invasion +/- SEM relative to MDA-MB-231 invasion: 104.7 +/- 6.1%), the five human ovarian cancer cell lines the least invasive (60.2 +/- 2.2%) and the six human endometrial cancer cell lines showed an intermediate capacity (79.1 +/- 3.5%). In conclusion, the Boyden chamber assay can be used reliably for studying the invasive potential of cells in vitro, if the ability of the cells to migrate through the filter is taken into account, and a reference cell line is included to enable comparison of the data obtained from independently performed experiments on different cell lines.

3T3 Cells↗