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after 3 days in culture. In the case of type III collagen, this was more obvious at later stages of culture, thus reflecting the delay in expression of scattering activity observed with this subtype (Table 1). On fibronectin and laminin, NBT-II cells formed a monolayer of cells with desmosomes (Fig. 5, A and B). Cell scattering on collagens therefore correlates with a de crease in L-CAM expression at the cell surface and the disso ciation of desmosomes. Time-lapse videomicroscopy and scan ning electron microscopy (not shown) revealed the existence of small membranous processes between the cells detaching from the peripheral halo on collagens. These probably coincide with the desmosome reactivity remaining in the interconnections mentioned. Quantitation of NBT-II Cell Motility during Kpithelium-to- Mesenchyme Transition on Collagens. In the initial phases of cell-to-substratum interaction analyzed, NBT-II cellular aggre gates remained spherical, and the first cells were seen to detach from the cohesive mass after various periods of time, depending on the substrate (Table 1). Time-lapse video analyses of the formation of the halo around the aggregates revealed that it arose from the progressive emi gration of cells in contact with the substrate. In standard medium and on substrates preventing cell scattering (fibronec tin, laminin, uncoated substrate), the peripheral cells remained adherent to one another at the periphery, and the growth of the halo was mainly the consequence of both active proliferation and epithelial migration as a sheet of cells (as inferred from video observations). However, this epithelial sheet migration did not last more than 3 to 4 days, as evidenced by the absence of total flattening of the aggregate and the presence of numerous bulging multilayered foci; these cells were not found to migrate after 5 to 6 days in culture and remained strongly anchored to the substrate. The tracks of isolated cells scattered on collagens were re corded, and this analysis revealed additional differences be tween the collagen subtypes (Fig. 6). The average speeds of migration differed from 25 to almost 100 fitn/h, and the effec tive translocation from the site of emergence in the peripheral halo to the final position of the cell was more efficient for cells 133
Source publication
During metastatic spread, locomotion mediated by extracellular matrix components of basement membranes and connective tissues has been invoked as a prerequisite to invasion. We studied the interactions of the rat bladder carcinoma cell line NBT-II with fibronectin, laminin, and collagens (types I, III, IV, and V). They all promoted cell attachment...
Citations
... E-cadherin is one of the most commonly used markers for epithelial traits, and Snail and EGFR are the common markers for mesenchyma [34]. Matrix metalloproteinase 2 (MMP-2) and MMP-9 have been identi ed in large quantities in tumors and related to tumor metastasis [35,36]. Herein, we discovered that the overexpression of miR-32-5p remarkably prohibited EMT-associated migration and invasion by downregulation of MMP2, MMP9, Snail, and EGFR and upregulation of E-cadherin in NB cancer cells, while the downregulation of miR-32-5p had an opposite effect. ...
Neuroblastoma (NB) is a malignant pediatric tumor requiring new treatments. Accumulating evidence has confirmed that microRNAs (miRNAs) have a major effect on NB metastasis. The present study aimed to characterize a novel miRNA, miR-32-5p, by exploring its expression, function, as well as action mechanism in NB metastasis. Real-time quantitative polymerase chain reaction (qRT-PCR) as well as Western blotting was utilized to measure the expression of miR-32-5p and its target-vacuolar protein sorting 4B (VPS4B). Transwell assay was conducted to test the cell migration as well as its invasion in vitro , and the metastasis xenograft model of nude mice was set up by injections through the caudal vein in vivo . In comparison with the normal tissue and cell line, the miR-32-5p expression showed a marked downregulation in human NB samples and cell lines. Furthermore, the miR-32-5p upregulation suppressed NB cell metastasis in culture and the tumor xenograft model. Although in NB cell lines, VPS4B was found to be miR-32-5p’s direct target, its overexpression would reverse miR-32-5p’s suppressive effects. Moreover, miR-32-5p increased the sensitivity to dihydroartemisinin in neuroblastoma. The current outcomes implicated that the axis of miR-32-5p/VPS4B can be NB metastasis’s novel therapeutic target.
... In the normal bladder wall, these two types are mainly expressed in the lamina propria and around smooth muscle bundles and nerves (28). Interstitial collagens are thought to be involved in infiltration by individual rat BC cell lines (28,30). Mori and colleagues suggest that inactivation of the collagen type I α2 (COL1A2) gene (which encode type I collagen) via CpG hypermethylation, may contribute to proliferation and migration of BC cells (31). ...
Urothelial cell carcinoma (UCC) of the bladder comprises a mixture of heterogeneous tumor cell populations, the surrounding stroma (which is populated by different types of mesenchymal cells), and the extracellular matrix (ECM). Bladder cancer (BC) has certain characteristics that distinguish it from other cancers. First, BCs are categorized into two groups: non-muscle invasive BC (NMIBC) and muscle invasive BC (MIBC). The overall survival rate of patients with NMIBC is excellent compared with that of patients with other malignancies; however, some patients have a high risk of recurrence and a variable risk of progression despite administration of local therapies. The oft-cited "50% overall survival at 5 years" for MIBC has remained relatively unchanged over the last 20 years. Second, BC is a highly immunogenic cancer that shows a high rate of mutation due to the fact that more mutations are associated with a higher chance of tumor antigens triggering an appropriate immune response. The host immune response to tumor cells is based on the interactions that take place within the tumor microenvironment (TME). Intravesical bacillus Calmette-Guerin (BCG) therapy is the first U.S. Food and Drug Administration (FDA)-approved immunotherapy for BC and is the most successful immunotherapy for any established human neoplasm. Recently, the FDA approved the use of atezolizumab (Tecentriq®) and nivolumab (Opdivo®), which mimic programmed cell death ligand-1 inhibitor, to treat patients with locally advanced or metastatic UCC. Combination therapies involving cytotoxic chemotherapy, antiangiogenic agents, alternative immune checkpoint inhibitors, immunostimulatory cytokines, and cancer vaccines are currently under clinical investigation. This review summarizes recent studies of tumor-stromal crosstalk during pathogenesis of UCC of the bladder, and discusses the emerging roles of functionally important cellular components that interact within the cancer cell-TME. In addition, TME-targeted anticancer strategies such as chemotherapy, chemo-immunotherapy, and immunotherapy are briefly reviewed.
... Wound healing assays were performed as previously described, but with minor modifications. 15 Briefly, cells were seeded in P60 culture dishes and cultured to 100% confluency. The cells were then incubated with serumfree RPMI culture medium for 24 hours, and scraped with a 10 µl extra-long micro-pipette tip, thereby denuding a round of the monolayer approximately 500 µm in diameter (0 hour time point). ...
Background and aims:
Healing of the intestinal mucosal epithelium was found to be a critical factor in the treatment of inflammatory bowel disease (IBD). In this study, we provide further evidence that partially hydrolyzed dietary fiber (PHGG) enhances colonic epithelial cell wound healing, and partially characterize the mechanism that governs this process.
Materials and methods:
Young adult mouse colonic (YAMC) epithelial cells were scraped with a 10 μl micro-pipette tip to denude a round of the monolayer and were incubated with PHGG. The area of cell migration was measured using Image J software. Meanwhile, Rho activation assays were utilized to monitor Rho activation levels. To assess in vivo effects, C57B6 mice were treated with DSS for 7 days and then provided food supplemented with PHGG for 8 days.
Results:
YAMC cells treated with PHGG exhibited significantly enhanced wound healing compared to the control cells; however, this enhancement was inhibited by both Y-27632 (RhoA inhibitor) and U0126 (ERK1/2 inhibitor). Likewise, there was a PHGG-dependent increase in F-actin accumulation and Rho kinase activity that was blocked by U0126. Meanwhile, PHGG-dependent ERK1/2 activity was not inhibited by Y-27632. In the DSS-induced mouse colitis model, animals that received food supplemented with PHGG exhibited significant recovery of the colonic mucosa.
Conclusions:
In this study, we demonstrate that PHGG promotes colonic epithelial cell wound healing via activation of RhoA, which occurs downstream of ERK1/2 activation. These findings indicate that PHGG could be utilized as a therapeutic agent for patients with intestinal mucosal damage such as those with IBD.
... Wound healing assays were performed as previously described, but with minor modifications. 15 Briefly, cells were seeded in P60 culture dishes and cultured to 100% confluency. The cells were then incubated with serumfree RPMI culture medium for 24 hours, and scraped with a 10 µl extra-long micro-pipette tip, thereby denuding a round of the monolayer approximately 500 µm in diameter (0 hour time point). ...
... For a more accurate evaluation of cell migration in a second screening, we modified the screening method as follows. Fibronectin, one of the components of the transfection mixture, is essential for efficient transfection on cell chips, but NBT-II cells [9] and the derivative NBT-L2b cells [5] migrate more slowly on fibronectin than on collagen. Slower migration on a spot on the chip might, therefore, affect the accurate evaluation of cell speed. ...
Background
Cell migration plays a major role in a variety of normal biological processes, and a detailed understanding of the associated mechanisms should lead to advances in the medical sciences in areas such as cancer therapy. Previously, we developed a simple chip, based on transfected-cell microarray (TCM) technology, for the identification of genes related to cell migration. In the present study, we used the TCM chip for high-throughput screening (HTS) of a kinome siRNA library to identify genes involved in the motility of highly invasive NBT-L2b cells.ResultsWe performed HTS using TCM coupled with a programmed image tracer to capture time-lapse fluorescence images of siRNA-transfected cells and calculated speeds of cell movement. This first screening allowed us to identify 52 genes. After quantitative PCR (qPCR) and a second screening by a conventional transfection method, we confirmed that 32 of these genes were associated with the migration of NBT-L2b cells. We investigated the subcellular localization of proteins and levels of expression of these 32 genes, and then we used our results and databases of protein-protein interactions (PPIs) to construct a hypothetic but comprehensive signal network for cell migration.Conclusions
The genes that we identified belonged to several functional categories, and our pathway analysis suggested that some of the encoded proteins functioned as the hubs of networks required for cell migration. Our signal pathways suggest that epidermal growth factor receptor (EGFR) is an upstream regulator in the network, while Src and GRB2 seem to represent nodes for control of respective the downstream proteins that are required to coordinate the many cellular events that are involved in migration. Our microarray appears to be a useful tool for the analysis of protein networks and signal pathways related to cancer metastasis.
... NBTII is a rat-derived carcinoma cell line [18]. A normal cultured NBTII cell shows typical epithelial morphology; however, when NBTII cells were cultured on type I collagen-coated plastic cell culture dishes for 4–12 h, they acquired a polarized shape and migrate individually, exhibiting an epithelial to mesenchymal transition (EMT) [19,20,21,22]). During our experiments, we observed that NBTII cells on collagen had medium-sized lamellae (Marked with ''LM'') and lamellipodia (Marked with ''LP''), some filopodia (Marked with ''FP'') dynamically formed at the leading edge of the cell, and multiple retraction fibers (Marked with ''RF'') formed at the trailing edge of the cell. ...
The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.
... Where extracellular matrix components are concerned, an induction of EMT by collagens (types I, III, IV, and V) was early suggested by using NBT-II rat bladder carcinoma cells (260). Guarino then proposed (89) that peritumoral ECM may favor EMT and then cancer cell invasion, suggesting the contact of epithelial cells with an interstitial type of collagen as the putative relevant switch. ...
Epithelial to mesenchymal transition (EMT) is a fundamental process, paradigmatic of the concept of cell plasticity, that leads epithelial cells to lose their polarization and specialized junctional structures, to undergo cytoskeleton reorganization, and to acquire morphological and functional features of mesenchymal-like cells. Although EMT has been originally described in embryonic development, where cell migration and tissue remodeling have a primary role in regulating morphogenesis in multicellular organisms, recent literature has provided evidence suggesting that the EMT process is a more general biological process that is also involved in several pathophysiological conditions, including cancer progression and organ fibrosis. This review offers first a comprehensive introduction to describe major relevant features of EMT, followed by sections dedicated on those signaling mechanisms that are known to regulate or affect the process, including the recently proposed role for oxidative stress and reactive oxygen species (ROS). Current literature data involving EMT in both physiological conditions (i.e., embryogenesis) and major human diseases are then critically analyzed, with a special final focus on the emerging role of hypoxia as a relevant independent condition able to trigger EMT.
... In addition, it is still unclear what the downstream effecter of PIP 3 is, and what the molecular basis of keratocytelike movement of amiB 2 cells is. Since a small fraction of wild type cells as well as certain mammalian cells [Tucker et al., 1990; also move in a keratocyte-like manner, understanding the mechanism of keratocyte-like movement in amiB 2 cells and the roles PIP 3 plays, would have a general impact on amoeboid movements. ...
Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) to gain insight into roles PIP(3) plays in those spontaneous motilities. In keratocyte-like cells, PIP(3) showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP(3) repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP(3) waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP(3) waves by coupling steady lamellipodial protrusions to the PIP(3) waves. Simultaneous live observation of actin filaments and PIP(3) in wild type or stalled amiB(-) cells indicated that the PIP(3) waves were correlated with wave-like distributions of actin filaments. Most notably, PIP(3) waves often followed actin waves, suggesting that PIP(3) induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP(3) was often correlated with local retraction of the periphery. We propose that the waves of PIP(3) and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity.
... NBT-II cells grown on glass or plastic surfaces in standard medium show an epithelial morphology. However, when they are cultured on collagen-coated substrates, they migrate at about 100 µm/h with a large lamellipodium in a manner similar to fish keratocytes (Tucker et al., 1990). We report here that the addition of n-butanol and depletion of PLD by RNAi inhibited cell migration, and that GFP fused with PLD2 localized to protruding regions in the lamellipodia of migrating NBT-II cells. ...
NBT-II cells on collagen-coated substrates move rapidly and persistently, maintaining a semi-circular shape with a large lamellipodium, in a manner similar to fish keratocytes. The inhibitor of phospholipase D (PLD), n-butanol, completely blocked the migration and disturbed the characteristic localization of actin along the edge of lamellipodia. To investigate the functional difference between the two isozymes of PLD (PLD1 and PLD2), we transfected NBT-II cells with vectors expressing shRNA to deplete PLD1 or PLD2. Depletion of both PLD1 and 2 by RNA interference reduced the velocity of the migration, but depletion of PLD2 inhibited motility more severely than that of PLD1. Furthermore, GFP-PLD2 was localized to the protruding regions of lamellipodia in migrating cells. Thus, PLD is essential for the maintenance of keratocyte-like locomotion of NBT-II cells, presumably by regulating the actin cytoskeleton.
... In the normal bladder wall, these two types are mainly expressed in the lamina propria and around smooth muscle bundles and nerves (Wilson et al. 1996). Interstitial collagens have been suggested to be involved in rat bladder cancer cell lines single cell infi ltration (Tucker et al. 1990). The role of collagens in UC has not been extensively studied, except for collagens IV and VII, known to be basal membrane components; collagen VII playing a role in the formation of hemidesmosomes with the basal cells of the urothelium (Daher et al. 1987;Schapers et al. 1990;Liebert et al. 1994). ...
The extracellular matrix (ECM) plays a key role in the modulation of cancer cell invasion. In urothelial carcinoma of the bladder (UC) the role of ECM proteins has been widely studied. The mechanisms, which are involved in the development of invasion, progression and generalization, are complex, depending on the interaction of ECM proteins with each other as well as with cancer cells. The following review will focus on the pathogenetic role and prognostic value of structural proteins, such as laminins, collagens, fibronectin (FN), tenascin (Tn-C) and thrombospondin 1 (TSP1) in UC. In addition, the role of integrins mediating the interaction of ECM molecules and cancer cells will be addressed, since integrin-mediated FN, Tn-C and TSP1 interactions seem to play an important role during tumor cell invasion and angiogenesis.
