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1 - Article; Introduction: The poor efficacy of various anti-cancer treatments against metastatic cells has focused attention on the role of tumor microenvironment in cancer progression. To understand the contribution of the extracellular matrix (ECM) environment to this phenomenon, we isolated ECM surrogate invading cell populations from MDA-MB-231 breast cancer cells and studied their genotype and malignant phenotype. Methods: We isolated invasive subpopulations (INV) from non invasive populations (REF) using a 2D-Matrigel assay, a surrogate of basal membrane passage. INV and REF populations were investigated by microarray assay and for their capacities to adhere, invade and transmigrate in vitro, and to form metastases in nude mice. Results: REF and INV subpopulations were stable in culture and present different transcriptome profiles. INV cells were characterized by reduced expression of cell adhesion and cell-cell junction genes (44% of down regulated genes) and by a gain in expression of anti-apoptotic and pro-angiogenic gene sets. In line with this observation, in vitro INV cells showed reduced adhesion and increased motility through endothelial monolayers and fibronectin. When injected into the circulation, INV cells induced metastases formation, and reduced injected mice survival by up to 80% as compared to REF cells. In nude mice, INV xenografts grew rapidly inducing vessel formation and displaying resistance to apoptosis. Conclusion: Our findings reveal that the in vitro ECM microenvironment per se was sufficient to select for tumor cells with a stable metastatic phenotype in vivo characterized by loss of adhesion molecules expression and induction of proangiogenic and survival factors.

Type IV collagenases are secreted as latent 92 and 72 kDa proenzymes which are then activated extracellularly. The mechanisms by which they are activated in vivo are not clear. We have studied the activation of porcine endothelial cell type IV collagenases by tissue and plasma kallikrein, and found that tissue kallikrein was a very efficient activator of the 92 kDa type IV collagenase. Enzyme cleavage was observed at concentrations of tissue kallikrein as low as 0.1 μg/ml. Plasma kallikrein had no effect. By comparison, plasmin, which has been proposed to be the physiological activator of interstitial collagenase and stromelysin, and elastase were much less effective, and high concentrations (plasmin at 100–200 μg/ml and elastase at 20 μg/ml) were required to cause only a limited cleavage which was not associated with an increase in activity, as observed by the gelatin-gel lysis assay. In addition tissue kallikrein was found by immunohistochemistry to be present in the extracellular matrix of the intima of porcine aortic vessel wall. These findings suggest that tissue kallikrein can be a potential activator of the 92 kDa type IV collagenase in vivo. © 1993 Wiley-Liss, Inc.

Mutual interactions between human breast cancer cells and endothelial cells were studied in a model mimicking tumor cell intravasation. MDA-MB-231 tumor cells and human umbilical vein endothelial cells (HUVEC) were cocultured on opposite sides of a Transwell filter allowing tumor cell contacts with the basement membrane of the HUVEC forming endothelium and tumor cell transendothelial migration. Confocal microscopy analysis showed that transmigrating MDA-MB-231 cells lay under the HUVEC, thereby inducing HUVEC detachment and tumor cell-HUVEC contact-dependent apoptosis. GM6001 a matrix metalloproteinase (MMP) inhibitor inhibited almost completely, the MDA-MB-231 cell transendothelial migration and the anoikis process. In this intravasation model, a tumor cell invasive mechanism was demonstrated (i) induction of extensive endothelial anoikis induced by degradation of the extracellular matrix (ECM) components, (ii) activation of pro-matrix metalloproteinase (MMP)-2 into MMP-2 by the MT1-MMP-TIMP (tissue inhibitor metalloproteinase) 2-pro-MMP-2 membrane complex and (iii) attraction and migration of metastatic cell through apoptotic endothelium. These interactions could partly explain the necrosis-angiogenesis relationship in tumor angiogenesis.

The effects of sodium phenylacetate (NaPa), an antitumoral molecule, on cell death and matrix metalloproteinase (MMP) activities and synthesis were investigated in two metastatic breast tumour cell lines, MDA-MB-231 and MDA-MB-435, cultured on three-dimensional type I collagen gels (3-D cultures). In both cell lines, NaPa inhibited cell proliferation and induced apoptotic cell death as measured by TUNEL assay, with an IC(30) of 20 mM and 10 mM for MDA-MB-231 and MDA-MB-435 cells, respectively. In MDA-MB-231 cells, NaPa also induced (i) an autophagic process evidenced by the appearance of autophagic vacuoles and an increased phosphatase acid activity, (ii) the formation of pseudopodia and (iii) an increase in MMP-1 and MMP-9 secretion without affecting MT1-MMP. In NaPa-treated MDA-MB-435 cells, no autophagic vacuoles were formed but F-actin depolymerisation was observed. MMP-1, MMP-9 and MT1-MMP levels were strongly enhanced in these cells but MMPs were not secreted and accumulated intracellularly. When breast cancer cells were treated with NaPa in the presence of an MMP inhibitor (GM6001), apoptotic cell death decreased and the induction of autophagic vacuoles in MDA-MB-231 cells was inhibited. Taken together, these data suggest that MMPs are involved in the autophagic cell death and/or apoptosis of breast tumour cells.

Breast cancer treatments are limited by secondary effects and chemoresistances or hormone resistances. Sodium phenylacetate (NaPa), a nontoxic metabolite, has been shown to induce in vivo and in vitro antiproliferative effects on various cell types in our laboratory. We have previously shown that NaPa treatment induced breast tumor cell apoptosis without acquired drug resistance. On the other hand, we have demonstrated that a dextran derivative (CMDB) was not only antitumoral but also antiangiogenic in a MCF-7ras tumor model in animals. Recently, we have synthesized a novel hybrid molecule, CMDB-NaPa ester, called NaPaC, and tested its effect on the proliferation of MDA-MB-231 and MCF-7 breast tumor cells. NaPaC inhibits, dose dependently, the proliferation of MDA-MB-231 cell (IC50 0.5 mmol/l) and of MCF-7 cell (IC50 1.5 mmol/l). Primary cultured endothelial cells (HUVEC) are weakly affected by NaPaC treatment. Cytostatic effect of NaPaC was evidenced from the accumulation of tumor cells in G0/G1 phase after 96 h of treatment. As compared with the NaPa parent molecule, this new molecule was 10-fold more efficient on these two tumor cell lines. Moreover, NaPaC induces a strong apoptotic effect, as measured with Annexin V-positive cells, on these tumor cells. In order to understand the interactions between tumor cells and endothelial cells, the conditioned media were prepared and added to HUVEC cells. Our results showed a clear killing effect on HUVEC cells. However, this killing effect can be rescued by adding NaPaC. Taken together, our results showed that NaPaC is a powerful anti-tumoral molecule, with cytostatic and proapoptotic effects on MDA-MB-231 and MCF-7 tumor cells. Further studies should be conducted to better understand the mechanism of these mutual interactions between tumor cells and endothelial cells, especially the killing effect on HUVEC cells.

Thrombomodulin is synthesized by mesangial cells. Thrombomodulin (TM), an endothelial receptor for thrombin, endowed with a powerful anticoagulant activity, plays an important role in the antithrombogenicity of the vascular endothelium. Its presence within the human renal glomerulus is already known but was thought to be only endothelial. We looked for TM expression in human mesangial cells (MC), both in situ , in freshly prepared glomeruli, and in primary culture. Both fresh and cultured MC were strongly reactive for TM by immunocytochemical methods. Total TM antigen measured on MC lysates and surface TM activity on MC were 0.292 ± 0.075 ng/mg of cellular proteins and 1.20 ± 0.02 pmole of activated protein C/min/mg of cellular proteins, respectively. As shown by the presence of numerous transcripts detected by in situ hybridization, TM was shown to be synthesized by MC in vivo and in culture. The synthesis of active TM by both endothelial and mesangial cells within the renal glomerulus stresses the importance of its role in maintaining renal hemostatic equilibrium, and sheds some light on the conflicting reports of TM over- and underexpression in glomerulopathies to open a new field for investigation.

Thrombomodulin, a major anticoagulant proteoglycan of the endothelial cell membrane, is a thrombin receptor that acts as a cofactor for protein C activation. It has previously been shown that thrombomodulin, present in human epidermis and in lysates of cultured keratinocytes, is implicated in cellular differentiation during mouse fetal development. The role of retinoic acid in keratinocyte differentiation prompted us to study retinoic acid regulation of thrombomodulin expression in primary cultures of keratinocytes isolated from adult human skin, grown at low (undifferentiated keratinocytes) and normal calcium levels (differentiated keratinocytes). Thrombomodulin antigen levels and total and surface activities were measured in cultures without and with retinoic acid. Thrombomodulin mRNA visualized by in situ hybridization was quantified by computer-based image analysis. Functional thrombomodulin was expressed on the surface and in the cytoplasm of cultured human keratinocytes regardless of the calcium concentration. In contrast, retinoic acid induced significant increases in the total antigen level and in surface and intracellular thrombomodulin activities only in keratinocytes grown in a low-calcium medium. In these undifferentiated keratinocytes, quantification of mRNA transcripts showed a threefold increase after retinoic acid stimulation. Thus, functional thrombomodulin is a human keratinocyte surface protein whose expression is controlled through the keratinocyte differentiation program and is modulated in vitro by retinoic acid.

The histological, ultrastructural and permeability aspects of the intima in 60 70-week-old spontaneously hypertensive (SHR) and Wistar Kyoto normotensive (WK) rats were studied and compared. The intima of aorta, coronary and renal arteries was unequally thickened owing to the smooth muscle cell (SMC) migration and proliferation, blood cell immigration and endothelial cell activation. The present work describes intimal changes that may act as potential atherogenic factors, i.e. hyper-reactivity of endothelial cells; decreased thinness of endothelial cell periphery; reduced intercellular junction pathways; increased quantity of basement lamina and glycosaminoglycan subendothelial material; platelet and monocyte-macrophage infiltration; widened fenestrae in the internal elastic lamina, and smooth muscle cell migration and proliferation. These changes might possibly be able to reduce the atheroresistance of this species by reducing the barrier function, increasing the trapping effect and stimulating smooth muscle cell proliferation and fibrogenesis. They are believed to promote the development of arterial lipidosis when hyperlipemia is an added risk factor.