Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-based in vitro assays

In vitro analyses of basement membrane invasiveness employing Matrigel (a murine tumor extract rich in basement membrane components) have been performed on human breast cancer model systems. Constitutive invasiveness of different human breast cancer (HBC) cell lines has been examined as well as regulation by steroid hormones, growth factors, and oncogenes. Carcinoma cells exhibiting a mesenchymal-like phenotype (vimentin expression, lack of cell border associated uvomorulin) show dramatically increased motility, invasiveness, and metastatic potential in nude mice. These findings support the hypothesis that epithelial to mesenchymal transition (EMT)-like events may be instrumental in the metastatic progression of human breast cancer. The MCF-7 subline MCF-7ADR appears to have undergone such a transition. The importance of such a transition may be reflected in the emergence of vimentin expression as an indicator of poor prognosis in HBC. Matrix degradation and laminin recognition are highlighted as potential targets for antimetastatic therapy, and analyses of laminin attachment and the matrix metalloproteinase (MMP) family in HBC cell lines are summarized. Matrigel-based assays have proved useful in the study of the molecular mechanisms of basement membrane invasiveness, their regulation in HBC cells, and their potential as targets for antimetastatic therapy.

Hormonal carcinogenesis in breast cancer : cellular and molecular studies of malignant progression

We have established and characterized a series of variant cell lines in which to identify the critical factors associated with E2-induced malignant progression, and the acquisition to tamoxifen resistance in human breast cancer. Sublines of the hormone-dependent MCF-7 cell line (MCF7/MIII and MCF7/LCC1) form stable, invasive, estrogen independent tumors in the mammary fat pads of ovariectomized athymic nude mice. These cells retain expression of both estrogen (ER) and progesterone receptors (PGR), but retain sensitivity to each of the major structural classes of antiestrogens. The tamoxifen-resistant MCF7/LCC2 cells retain sensitivity to the inhibitory effects of the steroidal antiestrogen ICI 182780. By comparing the parental hormone-dependent and variant hormone-independent cells, we have demonstrated an altered expression of some estrogen regulated genes (PGR, pS2, cathepsin D) in the hormone-independent variants. Other genes remain normally estrogen regulated (ER, laminin receptor, EGF-receptor). These data strongly implicate the altered regulation of a specific subset or network of estrogen regulated genes in the malignant progression of human breast cancer. Some of the primary response genes in this network may exhibit dose-response and induction kinetics similar to pS2, which is constitutively upregulated in the MCF7/MIII, MCF7/LCC1 and MCF7/LCC2 cells.

Collagen induced MMP-2 activation in human breast cancer

Matrix metalloproteinase-2 (MMP-2), a zymogen requiring proteolytic activation for catalytic activity, has been implicated broadly in the invasion and metastasis of many cancer model systems, including human breast cancer (HBC). MMP-2 has been immunolocalized to carcinomatous human breast, where the degree of activation of MMP-2 correlates well with tumor grade and patient prognosis. Using Matrigel assays, we have stratified HBC cell lines for invasiveness in vitro, and compared this to their potential for metastatic spread in nude mice. HBC cell lines expressing the mesenchymal marker protein vimentin were found to be highly invasive in vitro, and tended to form metastases in nude mice. We have further discovered that culture on collagen-I gels (Vitrogen(TM): Vg) induces MMP-2-activator in highly invasive but not poorly invasive HBC cell lines. As seen for other MMP-2-activator inducing regimens, this induction requires protein synthesis and an intact MMP-2 hemopexin-like domain, appears to be mediated by a cell surface activity, and can be inhibited by metalloproteinase inhibitors. The induction is highly specific to collagen I, and is not seen with thin coatings of collagen I, collagen IV, laminin, or fibronectin, or with 3-dimensional gels of laminin, Matrigel, or gelatin. This review focuses on collagen I and MMP- 2, their localization and source in HBC, and their relationship(s) to MMP-2 activation and HBC metastasis. The relevance of collagen I in activation of MMP-2 in vivo is discussed in terms of stromal cell: tumor cell interaction for collagen I deposition, MMP-2 production and MMP-2-activation. Such cooperativity may exist in vivo for MMP-2 participation in HBC dissemination. A more complete understanding of the regulation of MMP-2-activator by type I collagen may provide new avenues for improved diagnosis and prognosis of human breast cancer.

Supernatants of acquired immunodeficiency syndrome-related Kaposi's sarcoma cells induce endothelial cell chemotaxis and invasiveness

Kaposi's sarcoma (KS) in general, and acquired immunodeficiency syndrome-related KS (AIDS-KS) in particular, is a highly invasive and intensely angiogenic neoplasm of unknown cellular origin. We have recently established AIDS-KS cells in long term culture and reported the development of KS-like lesions in nude mice inoculated with these cells. Here, we have examined the in vitro invasiveness of basement membrane by AIDS-KS cells, as well as the effect(s) of their supernatants on the migration and invasiveness of human vascular endothelial cells. AIDS-KS cells were highly invasive in the Boyden chamber invasion assay and formed invasive, branching colonies in a 3-dimensional gel (Matrigel). Normal endothelial cells form tube-like structures on Matrigel. AIDS-KS cell-conditioned media induced endothelial cells to form invasive clusters in addition to tubes. KS-cell-conditioned media, when placed in the lower compartment of the Boyden chamber, stimulated the migration of human and bovine vascular endothelial cells across filters coated with either small amounts of collagen IV (chemotaxis) or a Matrigel barrier (invasion). Basic fibroblast growth factor could also induce endothelial cell chemotaxis and invasion in these assays. However, when antibodies to basic fibroblast growth factor were used the invasive activity induced by the AIDS-KS-cell-conditioned media was only marginally inhibited, suggesting that the large quantities of basic fibroblast growth factor-like material released by the AIDS-KS cells are not the main mediators of this effect. Specific inhibitors of laminin and collagenase IV action, which represent critical determinants of basement membrane invasion, blocked the invasiveness of the AIDS-KS cell-activated endothelial cells in these assays. These data indicate that KS cells appear to be of smooth muscle origin but secrete a potent inducer of endothelial cell chemotaxis and invasiveness which could be responsible for angiogenesis and the resulting highly vascularized lesions. These assays appear to be a model to study the invasive spread and angiogenic capacity of human AIDS-related KS and should prove useful in the identification of molecular mediators and potential inhibitors of neoplastic neovascularization.

The invasive and metastatic properties of hormone-independent but hormone-responsive variants of MCF-7 human breast cancer cells

We have previously isolated a series of MCF-7 human breast cancer cell variants which no longer require estrogen-supplementation for tumor growth in nude mice (Clarke et al. Proc Natl Acad Sci USA 86: 3649-3653, 1989). We now report that these hormone-independent and hormone-responsive variants (MIII, MCF7/LCC1) can invade locally from solid mammary fat pad tumors, and produce primary extensions on the surface of intraperitoneal structures including liver, pancreas, and diaphragm. Both lymphatic and hematogenous dissemination are observed, resulting in the establishing of pulmonary, bone, and renal metastases. The pattern of metastasis by MIII and MCF7/LCC1 cells closely resembles that frequently observed in breast cancer patients, and provides the first evidence of metastasis from MCF-7 cells growing in vivo without supplementary estrogen. The interexperimental incidence of metastases, and the time from cell inoculation to the appearance of metastatic disease are variable. The increased metastatic potential is not associated with an increase in either the level of laminin attachment, laminin receptor mRNA expression, or secreted type IV collagenolytic activity. We also did not detect a significant decrease in the steady-state mRNA levels of the metastasis inhibitor nm23 gene. However, when growing without estrogen in vitro, MCF7/LCC1 cells produce elevated levels of the estrogen-inducible cathepsin D enzyme.

Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell lines in vitro and in vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-rasH, v-rasKi, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determined in vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection with ras, despite similar ras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-rasH + SV40T and v -rasH + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.

Myogel, a novel, basement membrane-rich, extracellular matrix derived from skeletal muscle, is highly adipogenic in vivo and in vitro

Background/Aims Biological and synthetic scaffolds play important roles in tissue engineering and are being developed towards human clinical applications. Based on previous work from our laboratory, we propose that extracellular matrices from skeletal muscle could be developed for adipose tissue engineering. Methods Extracellular matrices (Myogels) extracted from skeletal muscle of various species were assessed using biochemical assays including ELISA and Western blotting. Biofunctionality was assessed using an in vitro differentiation assay and a tissue engineering construct model in the rat. Results Myogels were successfully extracted from mice, rats, pigs and humans. Myogels contained significant levels of laminin α4- and α2-subunits and collagen I compared to Matrigel™, which contains laminin 1 (α1β1γ1) and collagen IV. Levels of growth factors such as fibroblast growth factor 2 were significantly higher than Matrigel, vascular endothelial growth factor-A levels were significantly lower and all other growth factors were comparable. Myogels reproducibly stimulated adipogenic differentiation of preadipocytes in vitro and the growth of adipose tissue in the rat. Conclusions We found Myogel induces adipocyte differentiation in vitroand shows strong adipogenic potential in vivo, inducing the growth of well-vascularised adipose tissue. Myogel offers an alternative for current support scaffolds in adipose tissue engineering, allowing the scaling up of animal models towards clinical adipose tissue engineering applications.

Intrinsics and dynamics of fat grafts : an in vitro study

Background Despite a revived interest in fat grafting procedures, clinicians still fail to demonstrate clearly the in vivo behavior of fat grafts as a dynamic tissue substitute. However, the basic principles in cellular biology teach us that cells can survive and develop, provided that a structural matrix exists that directs their behavior. The purpose of this in vitro study was to analyze that behavior of crude fat grafts, cultured on a three-dimensional laminin-rich matrix. Methods Nonprocessed, human fat biopsy specimens (approximately 1 mm) were inoculated on Matrigel-coated wells to which culture medium was added. The control group consisted of fat biopsy specimens embedded in medium alone. The cellular proliferation pattern was followed over 6 weeks. Additional cultures of primary generated cellular spheroids were performed and eventually subjected to adipogenic differentiation media. Results A progressive outgrowth of fibroblast-like cells from the core fat biopsy specimen was observed in both groups. Within the Matrigel group, an interconnecting three-dimensional network of spindle-shaped cells was established. This new cell colony reproduced spheroids that functioned again as solitary sources of cellular proliferation. Addition of differentiation media resulted in lipid droplet deposition in the majority of generated cells, indicating the initial steps of adipogenic differentiation. Conclusions The authors noticed that crude, nonprocessed fat biopsy specimens do have considerable potential for future tissue engineering-based applications, provided that the basic principles of developmental, cellular biology are respected. Spontaneous in vitro expansion of the stromal cells present in fat grafts within autologous and injectable matrices could create "off-the-shelf" therapies for reconstructive procedures.

The role of biological extracellular matrix scaffolds in vascularized three-dimensional tissue growth in vivo

An in vivo murine vascularized chamber model has been shown to generate spontaneous angiogenesis and new tissue formation. This experiment aimed to assess the effects of common biological scaffolds on tissue growth in this model. Either laminin-1, type I collagen, fibrin glue, hyaluronan, or sea sponge was inserted into silicone chambers containing the epigastric artery and vein, one end was sealed with adipose tissue and the other with bone wax, then incubated subcutaneously. After 2, 4, or 6 weeks, tissue from chambers containing collagen I, fibrin glue, hyaluronan, or no added scaffold (control) had small amounts of vascularized connective tissue. Chambers containing sea sponge had moderate connective tissue growth together with a mild "foreign body" inflammatory response. Chambers containing laminin-1, at a concentration 10-fold lower than its concentration in Matrigel™, resulted in a moderate adipogenic response. In summary, (1) biological hydrogels are resorbed and gradually replaced by vascularized connective tissue; (2) sponge-like matrices with large pores support connective tissue growth within the pores and become encapsulated with granulation tissue; (3) laminin-containing scaffolds facilitate adipogenesis. It is concluded that the nature and chemical composition of the scaffold exerts a significant influence on the amount and type of tissue generated in this in vivo chamber model.

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and β-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, β-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.

Optimising the cell source and biomaterials for the generation of vascularized adipose tissue in vivo

We initially described a rat chamber model with an inserted arteriovenous pedicle which spontaneously generates 3-dimensional vascularized connective tissue (Tanaka Y et al., Br J Plast Surg 2000; 53: 51-7). More recently we have developed a murine chamber model containing reconstituted basement membrane (Matrigel®) and FGF-2 that generates vascularized adipose tissue in vivo (Cronin K et al., Plast Reconstr Surg 2004; in press). We have extended this work to assess the cellular and matrix requirements for the Matrigel®- induced neo-adipogenesis. We found that chambers sealed to host fat were unable to grow new adipose tissue. In these chambers the Matrigel® became vascularized with maximal outgrowth of vessels extending to the periphery at 6 weeks. A small amount of adipose tissue was found adjacent to the vessels, most likely arising from periadventitial adipose tissue. In contrast, chambers open to interaction with endogenous adipose tissue showed abundant new fat, and partial exposure to adjacent adipose tissue clearly showed neo-adipogenesis only in this area. Addition of small amounts of free fat to the closed chamber containing Matrigel® was able to induce neo-adipogenesis. Addition of small pieces of human fat also caused neo-adipogenesis in immunocompromised (SCID) mice. Also, we found Matrigel® to induce adipogenesis of Lac-Z-tagged (Rosa-26) murine bone marrow-derived mesenchymal stem cells, and cells similar to these have been isolated from human adipose tissue. Given that Matrigel® is a mouse product and cannot be used in humans, we have started investigating alternative matrix scaffolds for adipogenesis such as the PDA-approved PLGA, collagen and purified components derived from Matrigel®, such as laminin-1. The optimal conditions for adipogenesis with these matrices are still being elucidated. In conclusion, we have demonstrated that a precursor cell source inside the chamber is essential for the generation of vascularized adipose tissue in vivo. This technique offers unique potential for the reconstruction of soft tissue defects and may enable the generation of site-specific tissue using the correct microenvironment.

UpaG, a new member of the Trimeric Autotransporter family of Adhesins in Uropathogenic Escherichia coli

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

Pancreatic islet basement membrane loss and remodeling after mouse islet isolation and transplantation: Impact for allograft rejection

The isolation of islets by collagenase digestion can cause damage and impact the efficiency of islet engraftment and function. In this study, we assessed the basement membranes (BMs) of mouse pancreatic islets as a molecular biomarker for islet integrity, damage after isolation, and islet repair in vitro as well as in the absence or presence of an immune response after transplantation. Immunofluorescence staining of BM matrix proteins and the endothelial cell marker platelet endothelial cell adhesion molecule-1 (PECAM-1) was performed on pancreatic islets in situ, isolated islets, islets cultured for 4 days, and islet grafts at 3-10 days posttransplantation. Flow cytometry was used to investigate the expression of BM matrix proteins in isolated islet β-cells. The islet BM, consisting of collagen type IV and components of Engelbreth-Holm-Swarm (EHS) tumor laminin 111, laminin α2, nidogen-2, and perlecan in pancreatic islets in situ, was completely lost during islet isolation. It was not reestablished during culture for 4 days. Peri- and intraislet BM restoration was identified after islet isotransplantation and coincided with the migration pattern of PECAM-1(+) vascular endothelial cells (VECs). After islet allotransplantation, the restoration of VEC-derived peri-islet BMs was initiated but did not lead to the formation of the intraislet vasculature. Instead, an abnormally enlarged peri-islet vasculature developed, coinciding with islet allograft rejection. The islet BM is a sensitive biomarker of islet damage resulting from enzymatic isolation and of islet repair after transplantation. After transplantation, remodeling of both peri- and intraislet BMs restores β-cell-matrix attachment, a recognized requirement for β-cell survival, for isografts but not for allografts. Preventing isolation-induced islet BM damage would be expected to preserve the intrinsic barrier function of islet BMs, thereby influencing both the effector mechanisms required for allograft rejection and the antirejection strategies needed for allograft survival.

Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region

Ulcerative colitis is a common form of inflammatory bowel disease with a complex etiology. As part of the Wellcome Trust Case Control Consortium 2, we performed a genome-wide association scan for ulcerative colitis in 2,361 cases and 5,417 controls. Loci showing evidence of association at P 1 × 10 5 were followed up by genotyping in an independent set of 2,321 cases and 4,818 controls. We find genome-wide significant evidence of association at three new loci, each containing at least one biologically relevant candidate gene, on chromosomes 20q13 (HNF4A; P = 3.2 × 10 17), 16q22 (CDH1 and CDH3; P = 2.8 × 10 8) and 7q31 (LAMB1; P = 3.0 × 10 8). Of note, CDH1 has recently been associated with susceptibility to colorectal cancer, an established complication of longstanding ulcerative colitis. The new associations suggest that changes in the integrity of the intestinal epithelial barrier may contribute to the pathogenesis of ulcerative colitis. © 2009 Nature America, Inc. All rights reserved.