Cancer cells use different modes of migration, including integrindependent mesenchymal migration of elongated cells along elements of the 3D matrix as opposed to low-adhesion-, contractionbased amoeboid motility of rounded cells. We report that MDA-MB-231 human breast adenocarcinoma cells invade 3D Matrigel with a characteristic rounded morphology and with F-actin and myosin-IIa accumulating at the cell rear in a uropod-like structure. MDA-MB-231 cells display neither lamellipodia nor bleb extensions at the leading edge and do not require Arp2/3 complex activity for 3D invasion in Matrigel. Accumulation of phospho-MLC and blebbing activity were restricted to the uropod as reporters of actomyosin contractility, and velocimetric analysis of fluorescent beads embedded within the 3D matrix showed that pulling forces exerted to the matrix are restricted to the side and rear of cells. Inhibition of actomyosin contractility or β1 integrin function interferes with uropod formation, matrix deformation, and invasion through Matrigel. These findings support a model whereby actomyosin-based uropod contractility generates traction forces on the β1 integrin adhesion system to drive cell propulsion within the 3D matrix, with no contribution of lamellipodia extension or blebbing to movement.uring metastasis, tumor cells encounter various extracellular matrix (ECM) environments with distinct composition and architecture, including basement membrane (BM) and interstitial collagen networks (1-3). Although basic mechanisms of cell motility on 2D substrata are generally well understood, much less is known regarding the mechanics of cell migration in 3D matrix environments (1, 4-7). Recent evidence supports the conclusions that 2D and 3D migration can differ considerably and that different types of 3D motility exist depending on the biophysical properties of the 3D environment (viscoelasticity, confinement, porosity) (8-15). Understanding the mechanics of cancer cell migration in these different 3D environments is therefore of paramount importance.Schematically, two modes of 3D migration of invasive cells have been described; the models differ by the dependency on actomyosin contractility, requirement for integrin adhesion, and matrix remodeling. In the mesenchymal mode, which has some parallel with 2D migration of fibroblasts (1), invasive cells are elongated and require pericellular matrix proteolysis for extending Racdependent F-actin-based leading pseudopodia in an integrindependent manner (16)(17)(18)(19)(20). In contrast, some carcinoma cells invade with a low-adhesion amoeboid mode of migration, characterized by a round morphology, no stable intrinsic cell polarity, and a required high level of RhoA/ROCK-driven actomyosin contractility (17, 18). Amoeboid cell motility, which is typically associated with bleb formation, is independent of proteases, and it is generally thought that by forming blebs, tumor cells can squeeze themselves through preexisting voids in the matrix (5, 21). In vivo, bleb-based motility has been implicate...
One of the main difficulties with primary rat brain endothelial cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating cells would not. Treatment with either 4 lg/mL puromycin for the first 2 days of culture or 3 lg/mL puromycin for the first 3 days showed the best results without causing toxicity to the cells. Transendothelial electrical resistance was significantly increased in cell monolayers treated with puromycin compared with untreated cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 · 10 )6 cm/s) and a high electrical resistance (up to 500 W · cm 2 ). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for cellular and molecular biology studies as well as pharmacological investigations. Keywords: blood-brain barrier, in vitro model, P-glycoprotein, puromycin, rat brain microvessel endothelium. In the last decade, many efforts have been made to produce reliable in vitro models in order to study the blood-brain barrier (BBB). It is indeed important to better understand the complex cellular and molecular interactions at the interface between blood and brain. The BBB regulates the passage of physiological substances into and out of the CNS and protects it against potentially harmful substances present in the blood. It also prevents the passage of pharmacological substances into the CNS. In order to optimize drug delivery to the CNS, it is important to gain knowledge about the passage of drug candidates through the BBB, especially their effects on the CNS and their toxicity to this barrier (Begley 1996;Tsuji and Tamai 1997). The better we understand BBB regulation, the better we will be able to conceive treatments for CNS pathologies, including neurodegenerative diseases and brain tumours
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