Gel Invasion Research Articles

3D Gel Invasion Assay of Gastric Cancer Cells with Fibroblasts

3D Gel Invasion Assay of Gastric Cancer Cells with Fibroblasts

Muscleblind-like 1 is required for normal heart valve development in vivo.

BackgroundDevelopment of the valves and septa of the heart depends on the formation and remodeling of the endocardial cushions in the atrioventricular canal and outflow tract. These cushions are populated by mesenchyme produced from the endocardium by epithelial-mesenchymal transition (EMT). The endocardial cushions are remodeled into the valves at post-EMT stages via differentiation of the mesenchyme and changes in the extracellular matrix (ECM). Transforming growth factor β (TGFβ) signaling has been implicated in both the induction of EMT in the endocardial cushions and the remodeling of the valves at post-EMT stages. We previously identified the RNA binding protein muscleblind-like 1 (MBNL1) as a negative regulator of TGFβ signaling and EMT in chicken endocardial cushions ex vivo. Here, we investigate the role of MBNL1 in endocardial cushion development and valvulogenesis in Mbnl1∆E3/∆E3 mice, which are null for MBNL1 protein.MethodsCollagen gel invasion assays, histology, immunohistochemistry, real-time RT-PCR, optical coherence tomography, and echocardiography were used to evaluate EMT and TGFβ signaling in the endocardial cushions, and morphogenesis, ECM composition, and function of the heart valves. ResultsAs in chicken, the loss of MBNL1 promotes precocious TGFβ signaling and EMT in the endocardial cushions. Surprisingly, this does not lead to the production of excess mesenchyme, but later valve morphogenesis is aberrant. Adult Mbnl1∆E3/∆E3 mice exhibit valve dysmorphia with elevated TGFβ signaling, changes in ECM composition, and increased pigmentation. This is accompanied by a high incidence of regurgitation across both inflow and outflow valves. Mbnl1∆E3/∆E3 mice also have a high incidence of ostium secundum septal defects accompanied by atrial communication, but do not develop overt cardiomyopathy.ConclusionsTogether, these data indicate that MBNL1 plays a conserved role in negatively regulating TGFβ signaling, and is required for normal valve morphogenesis and homeostasis in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0087-4) contains supplementary material, which is available to authorized users.

Impact of tumor necrosis factor-α activated human bone marrow mesenchymal stem cells on proliferation and invasion of colon cancer cell HT29

Objective To explore the effects of tumor necrosis factor (TNF)-α activated human bone marrow mesenchymal stem cell (MSC) on proliferation and invasion of colorectal cancer (CRC) cell line HT29. Methods The conditional medium of MSC (CM) and TNF-α activated MSC (T-MSC) (TCM) were collected. The proliferation rates of HT29 cells in α-minimum essential medium (α-MEM), CM and TCM for two, four and six days were detected by methyl thiazol tetrazolium (MTT) method. Based agar were maken in plates without seeding or seeded with MSC or T-MSC and HT29 cells were seeded on the top agarose. The colony forming ability was tested after culturing with fresh α-MEM for 30 days. Tumor sphere of HT29 cells was formed by suspension method. And three-dimensional matrix gel was immplemented for invasion examination. The invasion ability of HT29 cells in α-MEM, CM and TCM was measured. The expression of E-cadherin of HT29 cells in α-MEM, CM and TCM at mRNA level was determined by real-time polymerase chain reaction (PCR). The t test was used for two groups comparison and single factor analysis of variance was performed for multiple groups comparison. Results After cultured for four to six days, the proliferation rates of HT29 cells in CM or TCM were higher than that in α-MEM (the absorption value of each group at four days: 0.57±0.06 and 0.51±0.02, 0.22±0.01; at six days 0.45±0.01 and 0.73±0.04, 0.15±0.02), and the differences were statistically significant (tCM=8.090 and 21.140, tTCM=21.190 and 17.950, all P<0.05). After cultured in soft agarose medium for 30 days, the colony number HT29 in T-MSC was more than other two groups. And in three-dimensional matrix gel invasion examination, the invasion ability of HT29 in T-MSC was significantly higher than that in α-MEM ((82±2)% and (65±8)%, t=2.848, P <0.05). The expression of E-cadherin of HT29 cells in TCM at mRNA level was lower than that in α-MEM (0.32±0.02 vs 1.00±0.00), and the difference was statistically significant (t=35.190, P <0.05). Conclusion TNF-α activated human bone marrow MSC may promote proliferation and invasion of CRC cells. Key words: Mesenchymal stem cells; Colorectal neoplasms; Cell line; Neoplasms; Invasion

LPP inhibits collective cell migration during lung cancer dissemination.

Lipoma preferred partner (LPP) is a LIM domain protein, which has multiple functions as an actin-binding protein and a transcriptional coactivator, and it has been suggested that LPP has some roles in cell migration or invasion, however, its role in cancer cells remains to be elucidated. Here, we showed that LPP degraded N-cadherin in lung cancer, PC14PE6 cells via regulating the expression of matrix metalloproteinase 15 (MMP-15), and loss-of-LPP increases collective cell migration (CCM) and dissemination consequently. Knockdown of LPP and its functional partner, Etv5, markedly restores the full-length N-cadherin and increases cell-cell adhesion. We investigated the common target of LPP and Etv5, and found that MMP-15 is transcribed as their direct transcriptional target. Furthermore, MMP-15 could directly digest the N-cadherin extracellular domain. LPP knockdown in PC14PE6 cells increases N-cadherin-dependent CCM in the three-dimensional collagen gel invasion assays, and promoted the dissemination of cancer cells when they were orthotopically implanted in nude mice. Immunohistochemistry of lung adenocarcinoma specimens revealed the heterogeneity of LPP intensity and complementary expression of LPP and N-cadherin in the primary tumors. These findings suggest that loss-of-LPP, Etv5 or MMP-15 can be a prognostic marker of increasing malignancy.

The role of matrix metalloproteinases in retinal pigment epithelial cell-induced gel contraction and collapse

Purpose: To determine the role of matrix metalloproteinases (MMP)-1, -2, and -9 in directing the behavior of human retinal pigment epithelial (HRPE) cells during contraction of three-dimensional collagen gels. Materials and Methods: The effect of HRPE cells on collagen gel invasion and contraction were quantified by using phase contrast microscopy. Immunohistochemistry of the gel using monoclonal antibodies against MMP-1, -2, and -9 was performed. The effects of MMP inhibitor, Batimastat (BB-94) on cultured HRPE cells, and gel contraction were observed. Results: HRPE cells mostly proliferated as sheets of cells on the surface of the collagen gel and only minimally invaded the gel. However, some HRPE cells were seen to invade the gel as single cells detaching from the surface sheets. Surface-located sheets of cells exerted a dose-dependent contraction on the gel and generally failed to express MMPs. Single cells invading the gel expressed MMP-2 and -9. No expression of MMP-1 was observed by HRPE cells. BB-94, at a dose of 500 nM and 5 μM, reduced the amount of gel contraction. Conclusions: These data indicate that MMPs are selectively involved in HRPE invasion and collagen gel contraction. Both these processes may be implicated in the pathogenesis of similar conditions in vivo in which contraction of collagen gel is a feature, for example, proliferative vitreoretinopathy.

Biphasic response of cell invasion to matrix stiffness in three-dimensional biopolymer networks

When cells come in contact with an adhesive matrix, they begin to spread and migrate with a speed that depends on the stiffness of the extracellular matrix. On a flat surface, migration speed decreases with matrix stiffness mainly due to an increased stability of focal adhesions. In a three-dimensional (3-D) environment, cell migration is thought to be additionally impaired by the steric hindrance imposed by the surrounding matrix. For porous 3-D biopolymer networks such as collagen gels, however, the effect of matrix stiffness on cell migration is difficult to separate from effects of matrix pore size and adhesive ligand density, and is therefore unknown. Here we used glutaraldehyde as a crosslinker to increase the stiffness of self-assembled collagen biopolymer networks independently of collagen concentration or pore size. Breast carcinoma cells were seeded onto the surface of 3-D collagen gels, and the invasion depth was measured after 3days of culture. Cell invasion in gels with pore sizes >5μm increased with higher gel stiffness, whereas invasion in gels with smaller pores decreased with higher gel stiffness. These data show that 3-D cell invasion is enhanced by higher matrix stiffness, opposite to cell behavior in two dimensions, as long as the pore size does not fall below a critical value where it causes excessive steric hindrance. These findings may be important for optimizing the recellularization of soft tissue implants or for the design of 3-D invasion models in cancer research.

Bortezomib protects from varicose-like venous remodeling.

Despite the high prevalence of venous diseases that are associated with and based on the structural reorganization of the venous vessel wall, not much is known about their mechanistic causes. In this context, we demonstrated that the quantity of myocardin, a transcriptional regulator of the contractile and quiescent smooth muscle cell phenotype, was diminished in proliferating synthetic venous smooth muscle cells (VSMCs) of human and mouse varicose veins by 51 and 60%, respectively. On the basis of the relevance of proteasomal activity for such phenotypic changes, we hypothesized that the observed VSMC activation is attenuated by the proteasome inhibitor bortezomib. This drug fully abolished VSMC proliferation and loss of myocardin in perfused mouse veins and blocked VSMC invasion in collagen gels by almost 80%. In line with this, topical transdermal treatment with bortezomib diminished VSMC proliferation by 80%, rescued 90% of VSMC myocardin abundance, and inhibited varicose-like venous remodeling by 67 to 72% in a mouse model. Collectively, our data indicate that the proteasome plays a pivotal role in VSMC phenotype changes during venous remodeling processes. Its inhibition protects from varicose-like vein remodeling in mice and may thus serve as a putative therapeutic strategy to treat human varicose veins.

MP21-17 EFFECTS OF ADIPOSE TISSUE STROMAL CELLS ON APOPTOSIS, GROWTH AND INVASION OF SUPERFICIAL AND INVASIVE TYPE UROTHELIAL CARCINOMAS OF THE BLADDER

INTRODUCTION AND OBJECTIVES: Cancer-stromal interaction is critical for cancer biology. Bladder cancer develops within the mucosa, and invades into muscle layer surrounded by abundant adipose tissue. Adipose tissue has stromal cell types, such as preadipocytes and mesenchymal stem cells (MSCs). Thus, the adipose tissue stromal cells (ATSCs) seem critical for bladder cancer biology. The aim of this study is to address the effects of ATSCs on the apoptosis, growth and invasion of the superficial and invasive types. METHODS: RT4 and RT112 were used as human superficial urothelial carcinoma type of bladder, while EJ and HT1376 were done as invasive type. ATSCs were isolated from subcutaneous adipose tissue of 1-week old male Wister rats. We examined the apoptosis, growth and invasion of cancer cells, using collagen gel invasion assay system, in which the tumor cells were co-cultured on ATSC-embedded gel layer. The cellular behavior, and the apoptosis-, growthand ivasion-related molecules were analyzed by morphometry, immunohistochemistry, Western blot and real-time RT-PCR. RESULTS: ATSCs promoted and inhibited the apoptosis and growth of superficial type cancer cells, respectively. In contrast, ATSCs inhibited and promoted the apoptosis and growth of invasive type cancer cells. ATSCs had no effect on the invasion of superficial type, whereas they promoted the invasion of invasive type. ATSCs promoted the expression of MAPK proteins (raf-1, MEK-1 and ERK1/ERK2) in all cancer cell types. In turn, superficial type promoted [alpha]-SMA-positive myofibroblastic differentiation in ATSCs, while invasive type did lipid dropletand S-100 protein-positive adipogenic differentiation in ATSCs. CONCLUSIONS: The data suggest the following issues: 1) ATSCs suppress the progression of superficial type urothelial carcinoma through the growth inhibition and apoptosis promotion, whereas ATSCs promote that of invasive type through the enhancement of the growth and invasion, and the apoptosis prohibition; 2) ATSCinduced MAPK pathway activation plays differential roles in biological behavior of superficial and invasive types; and 3) Superficial type induces myofibroblastic differentiation in ATSCs as cancer-associated phenotype, while invasive type does adipogenic differentiation in ATSCs. Source of Funding: none

Soluble VCAM-1 Alters Lipid Phosphatase Activity in Epicardial Mesothelial Cells: Implications for Lipid Signaling During Epicardial Formation

Epicardial formation involves the attachment of proepicardial (PE) cells to the heart and the superficial migration of mesothelial cells over the surface of the heart. Superficial migration has long been known to involve the interaction of integrins expressed by the epicardium and their ligands expressed by the myocardium; however, little is understood about signals that maintain the mesothelium as it migrates. One signaling pathway known to regulate junctional contacts in epithelia is the PI3K/Akt signaling pathway and this pathway can be modified by integrins. Here, we tested the hypothesis that the myocardially expressed, integrin ligand VCAM-1 modulates the activity of the PI3K/Akt signaling pathway by activating the lipid phosphatase activity of PTEN. We found that epicardial cells stimulated with a soluble form of VCAM-1 (sVCAM-1) reorganized PTEN from the cytoplasm to the membrane and nucleus and activated PTEN’s lipid phosphatase activity. Chick embryonic epicardial mesothelial cells (EMCs) expressing a shRNA to PTEN increased invasion in collagen gels, but only after stimulation by TGFβ3, indicating that loss of PTEN is not sufficient to induce invasion. Expression of an activated form of PTEN was capable of blocking degradation of junctional complexes by TGFβ3. This suggested that PTEN plays a role in maintaining the mesothelial state of epicardium and not in EMT. We tested if altering PTEN activity could affect coronary vessel development and observed that embryonic chick hearts infected with a virus expressing activated human PTEN had fewer coronary vessels. Our data support a role for VCAM-1 in mediating critical steps in epicardial development through PTEN in epicardial cells.

Pancreatic secretory trypsin inhibitor causes autocrine-mediated migration and invasion in bladder cancer and phosphorylates the EGF receptor, Akt2 and Akt3, and ERK1 and ERK2

Pancreatic secretory trypsin inhibitor (PSTI) is expressed in most bladder carcinomas, where its pathophysiological relevance is unclear. Using recombinant normal sequence PSTI/tumor-associated trypsin inhibitor (TATI), a variant associated with familial pancreatitis (N34S), an active site-inactivated variant (R18/V19), and immunoneutralization and RNA interference-mediated knockdown techniques, we investigated the actions of PSTI/TATI on cell migration (wounding monolayers), collagen invasion (gel invasion assays), and proliferation (Alamar blue) on 253J, RT4, and HT1376 human bladder carcinoma cell lines. All three forms of PSTI/TATI stimulated migration twofold, and normal sequence PSTI/TATI showed synergistic promigratory effects when added with EGF. Addition of structurally unrelated soybean trypsin inhibitor had no promigratory activity. Similar results were seen using collagen invasion assays, although the active site mutated variant had no proinvasive activity, probably due to reduced Akt2 activation. PSTI/TATI did not stimulate proliferation despite acting, at least partially, through the EGF receptor, as effects of PSTI/TATI were truncated by the addition of an EGF receptor blocking antibody or the tyrosine kinase inhibitor tyrphostin. Cell lines produced endogenous PSTI/TATI, and PSTI/TATI RNA interference knockdown or the addition of PSTI/TATI, EGF receptor, or tyrphostin blocking agents reduced migration and invasion below baseline. PSTI/TATI induced phosphorylation of the EGF receptor, ERK1 and ERK2, Akt2 and Akt3, JNK1, MKK3, and ribosomal protein S6 kinase 1. This profile was more limited than that induced by EGF and did not include Akt1, probably explaining the lack of proproliferative activity. Our findings of autocrine stimulation and synergistic responses between EGF and PSTI/TATI at concentrations found in urine and tissue suggest that PSTI/TATI has pathophysiological relevance.

Matrix Rigidity Activates Wnt Signaling through Down-regulation of Dickkopf-1 Protein

Cells respond to changes in the physical properties of the extracellular matrix with altered behavior and gene expression, highlighting the important role of the microenvironment in the regulation of cell function. In the current study, culture of epithelial ovarian cancer cells on three-dimensional collagen I gels led to a dramatic down-regulation of the Wnt signaling inhibitor dickkopf-1 with a concomitant increase in nuclear β-catenin and enhanced β-catenin/Tcf/Lef transcriptional activity. Increased three-dimensional collagen gel invasion was accompanied by transcriptional up-regulation of the membrane-tethered collagenase membrane type 1 matrix metalloproteinase, and an inverse relationship between dickkopf-1 and membrane type 1 matrix metalloproteinase was observed in human epithelial ovarian cancer specimens. Similar results were obtained in other tissue-invasive cells such as vascular endothelial cells, suggesting a novel mechanism for functional coupling of matrix adhesion with Wnt signaling.

Mesenchymal Stem Cells Overexpressing CXCR4 Attenuate Remodeling of Postmyocardial Infarction by Releasing Matrix Metalloproteinase-9

Myocardial infarction (MI) results in loss of myofibers in the ischemic zone of the heart, followed by scar formation. These factors increase barriers to mobilization of mesenchymal stem cells (MSC), thereby impeding their effectiveness in cardiac repair. This study examined MSC overexpressing CXCR4 (MSC(CX4)) to determine penetration into infarcted myocardium by releasing collagen degrading enzyme, matrix metalloproteinase-9 (MMP-9). In vitro, mouse MSC were utilized, including MSC using adenoviral transduction, to express CXCR4/green fluorescent protein (GFP) (MSC(CX4)), Null/GFP (MSC(Null)), MSC treated with siRNA targeting CXCR4 (MSC(siR)), MSC treated with control siRNA(MSC(Con-siR)), MSC(CX4) treated with siRNA targeting MMP-9 (MSC(CX4-siRMP9)) and MMP-14 (MSC(CX4-siRMP14)), MSC derived from MMP-9 knockout mouse with adenoviral transduction for GFP (MSC(MP9-)), or MSC(MP9-) plus overexpressing CXCR4 (MSC(MP9-CX4)). The ability to cross the basement membrane was evaluated in all MSC using a trans-collagen gel invasion assay. The CXCR4 and MMP expression were analyzed by Western blot. In vivo, MSC with various treatments were infused into mice via tail vein injections 7 days after MI. Echocardiography was performed before harvesting hearts for analysis at 4 weeks after MSC injection. Both in vitro and in vivo studies demonstrated upregulation of MMP-9 induced by MSC(CX4), promoting increased GFP(+) cell migration into the infarcted area in comparison to control group. This enhanced response was associated with reduced left ventricular (LV) fibrosis, increased LV free wall thickness, angiogenesis, and improved LV function. Under hypoxic conditions, MMP-9 is upregulated in MSC(CX4), thus facilitating cross of the basement membrane, resulting in an improved remodeling of post-MI tissue.

Interaction between adipose tissue stromal cells and gastric cancer cells in vitro

Adipose tissue exists in the gastric submucosa and subserosa. Thus, adipose tissue stromal cells (ATSCs), which include mesenchymal stem cells (MSCs), seem critical for the progression of gastric cancer but their interaction with the cancer cells is unknown. We demonstrated an interaction between these cells, using immunohistochemistry, Western blot and the collagen gel invasion assay system, in which the adenocarcinoma cells (well and poorly differentiated types, MKN28 and MKN45, respectively) were cultured on a ATSC-embedded or ATSC-non-embedded gel. ATSCs promoted the expression of the growth marker, proliferation cell nuclear antigen but inhibited that of the apoptosis marker, single-stranded DNA, in the cancer cell types. ATSCs accelerated the invasion of only MKN28 into the gel and promoted the expression of mitogen-activated protein kinase (MAPK, pERK-1/2) but decreased that of the molecularly targeted protein, HER2, in the cancer cells. ATSCs did not affect the expression of the prostaglandin biosynthetic enzyme cyclooxgenase-2 (COX-2) in the cancer cells. The COX-2 inhibitor celecoxib did not affect the morphology or invasion of the cancer cells. The cancer cell types in turn promoted the display of the myofibroblast marker, α-smooth muscle actin, whereas they decreased that of some MSC markers, e.g., CD44 and CD105, in ATSCs. The data suggest that (1) ATSCs influence the progression of gastric cancer by increasing their growth/invasion and decreasing their apoptosis through MAPK activation in a COX-2-independent way; (2) ATSCs adversely affect HER2-targeted therapy; (3) the cancer cells induce the cancer-associated myofibroblast phenotype in ATSCs.

From the laboratory to the patient and back - an interview with Marc Mareel

The career of Marc Mareel is a synthesis of scientific research and clinical activity. During his medical studies, he already made his first enthusiastic steps in research via experimental work on avian developmental biology. Later, during his training as a radiotherapist, he founded his own laboratory for experimental cancer research. There he built up his international reputation as a pioneer in invasion research. Although invasion is the hallmark of tumor malignancy, he also kept an open mind about invasion in non-cancer conditions, such as in placental behavior, developmental biology, immunology and parasitology. His contribution to our understanding of invasion mechanisms has been both technical and conceptual. A number of assays have been developed in his lab, such as the embryonic chick heart and collagen gel invasion models, that have been (and still are) useful for many other research teams. He also contributed to the discovery of a number of key elements in the process of invasion, such as the stromal influence (including its extracellular matrix) and the cadherin family of cell-cell adhesion molecules. Concerning metastasis formation, he developed the original concept that a number of interacting eco-systems are implicated, such as the primary tumor, regional lymph nodes, the bone marrow and the (pre)metastatic niches in distant organs. Since his retirement, Marc Mareel has continued to integrate clinical practice with research creativity. He favours the idea of translational research bringing the results of laboratory findings to medical applications, and exploiting the feedback to the laboratory. The team in the Laboratory of Experimental Cancer Research at Ghent University currently consists of about 25 collaborators, who continue to appreciate his inspiring ideas and suggestions.

190 Sorafenib effectively controls early-stage hepatocellular carcinoma but not visceral metastases in a preclinical orthotopic transplant model

The aim of this study was to examine the effects of fibroblast growth factor-2 (FGF-2) on cancer cell invasion on fibroblast proliferation in an in vitro model of invasion. Three kinds of human oral squamous cell carcinoma cell lines with different invasive activity were used: OSC-20, OSC-19 (lower invasive type), HOC313 (higher invasive type). FGF-2 its high-affinity receptors FGFR-1 FGFR-2 were detected by western blotting. The expression of FGF-2 FGFRs mRNA was examined in cultured human oral squamous cell carcinoma cells by reverse transcriptase polymerase chain reaction (RT-PCR). Furthermore, recombinant human FGF-2 (rhFGF-2) was reacted with each cell line, the invasion rate was determined by invasion assay. We also observed the behavior of cancer cell invasion in the collagen gel invasion model in the presence or absence of FGF-2-neutralizing antibody (anti-FGF-2). HOC313 cells showed higher expression of FGF-2 than OSC-20 OSC-19 cells. The addition of rhFGF-2 promoted not only the proliferation of fibroblasts, but also the invasion of all cancer cell lines. In contrast, the addition of anti-FGF-2 completely inhibited the invasion of OSC-20 OSC-19 cells. These results suggest that a higher invasiveness of squamous carcinoma cells is associated with higher production of FGF-2, which acts in an autocrine fashion to promote cancer cell invasion, in a paracrine fashion to promote fibroblast proliferation.

Abstract 4812: Collagen regulation of Snail expression in pancreatic cancer involves TGF-β1 signaling

Abstract Pancreatic cancer, the fourth leading cause of cancer death in the U.S., is associated with pronounced desmoplastic reaction; however, the role of this collagen-rich fibrotic reaction in pancreatic cancer progression is poorly understood. Previously, we had shown that the collagen increased expression of the key proteinase membrane type 1-matrix metalloproteinase (MT1-MMP). Now we show that collagen can also increase expression of the zinc-finger transcription factor Snail to enhance pancreatic cancer invasion. Relative to cells grown on tissue culture plastic, PDAC cells grown in 3D collagen gels induced Snail. As we had previously published that TGF-β1 increases Snail levels, we examined the contribution of TGF-β1 signaling to collagen-induced Snail expression. Inhibiting TGF-β1 signaling with siRNA against TGF-β1 receptor type I (TbRI) or with a highly specific inhibitor of TbRI kinase activity attenuated the effect of collagen on Snail levels. To further examine the role of Snail in PDAC progression, we generated Snail-inducible PDAC cells lines. Expression of Snail in PDAC cells grown in 3D collagen gels enhanced invasion, which was blocked in the presence of the MMP inhibitor GM6001. Overexpression of Snail in PDAC cells increased MT1-MMP, while siRNA against Snail decreased MT1-MMP and also attenuated collagen-induced MT1-MMP expression. Snail expression of MT1-MMP requires ERK1/2 signaling and blocking ERK1/2 phosphorylation inhibited Snail-induced invasion in 3D collagen gels. To provide in vivo support for our findings that Snail can regulate MT1-MMP, we examined the expression of Snail and MT1-MMP in human PDAC tumors and found a statistically significant positive correlation between MT1-MMP and Snail in these tumors. Overall, our data demonstrate that collagen-rich milieu can increase the expression of Snail, and suggests that the desmoplastic reaction actively contributes to PDAC progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4812.

Arsenic Exposure Perturbs Epithelial-Mesenchymal Cell Transition and Gene Expression In a Collagen Gel Assay

Arsenic is a naturally occurring metalloid and environmental contaminant. Arsenic exposure in drinking water is reported to cause cancer of the liver, kidneys, lung, bladder, and skin as well as birth defects, including neural tube, facial, and vasculogenic defects. The early embryonic period most sensitive to arsenic includes a variety of cellular processes. One key cellular process is epithelial-mesenchymal transition (EMT) where epithelial sheets develop into three-dimensional structures. An embryonic prototype of EMT is found in the atrioventricular (AV) canal of the developing heart, where endothelia differentiate to form heart valves. Effects of arsenic on this cellular process were examined by collagen gel invasion assay (EMT assay) using explanted AV canals from chicken embryo hearts. AV canals treated with 12.5-500 ppb arsenic showed a loss of mesenchyme at 12.5 ppb, and mesenchyme formation was completely inhibited at 500 ppb. Altered gene expression in arsenic-treated explants was investigated by microarray analysis. Genes whose expression was altered consistently at exposure levels of 10, 25, and 100 ppb were identified, and results showed that 25 ppb in vitro was particularly effective. Three hundred and eighty two genes were significantly altered at this exposure level. Cytoscape analysis of the microarray data using the chicken interactome identified four clusters of altered genes based on published relationships and pathways. This analysis identified cytoskeleton and cell adhesion-related genes whose disruption is consistent with an altered ability to undergo EMT. These studies show that EMT is sensitive to arsenic and that an interactome-based approach can be useful in identifying targets.

The Air Liquid-interface, a Skin Microenvironment, Promotes Growth of Melanoma Cells, but not Their Apoptosis and Invasion, through Activation of Mitogen-activated Protein Kinase

The air-liquid interface (ALI) is a common microenvironment of the skin, but it is unknown whether the ALI affects melanoma cell behaviors. Using a collagen gel invasion assay, immunohistochemistry, and Western blots, here we show that melanoma cell proliferation in cultures with an ALI is higher than melanoma cell proliferation in submerged cultures. Bromodeoxyuridine (BrdU) uptake, an indicator of cell proliferation, of melanoma cells at the ALI was about 3 times that of submerged cells, while ALI and submerged melanoma cells had similar levels of single-stranded DNA (a marker of apoptosis). The ALI enhanced the expression of Raf-1, MEK-1 and pERK-1/2 components of the mitogen-activated protein kinase (MAPK) cascade, in cells more than the submerged condition did. The increases in BrdU uptake and pERK-1/2 expression promoted by ALI was abolished by the MEK inhibitor, PD-98059. ALI-treated and submerged melanoma cells did not infiltrate into the collagen gel, and they showed no significant difference in the expression of the invasion- and motility-related molecules, matrix metalloproteinase-1 and -9, laminin 5, and filamin A. Our data indicate that the ALI, a skin microenvironment, accelerates the growth, but not the apoptosis or invasion, of melanoma cells through MAPK activation.

RhoB enhances migration and MMP1 expression of prostate cancer DU145

Rho family protein regulates variety of cellular functions as cytoskeletal organization, cell proliferation and apoptosis. In the present study, we demonstrate that RhoB-overexpressed prostate cancer cells showed an enhanced cell motility and the administration of the GSK-3 inhibitors inhibited this increase in migration. Among the extracellular matrix and adhesion-related molecules, MMP1 RNA expression was increased in RhoB-overexpressed cells, administration of MMP inhibitor suppressed the collagen gel invasion in these cells. This is the first report evaluating RhoB function and the downstream signaling events in prostate cancer cell. Our results indicate that RhoB promotes cell motility and invasion in a metastatic prostate cancer cell.

Connexin 43 regulates epicardial cell polarity and migration in coronary vascular development

Connexin 43 knockout (Cx43 KO) mice exhibit conotruncal malformations and coronary artery defects. We observed epicardial blisters in the Cx43 KO hearts that suggest defects in epicardial epithelial-mesenchymal transformation (EMT), a process that generates coronary vascular progenitors. Analysis using a three-dimensional collagen gel invasion assay showed that Cx43 KO epicardial cells are less invasive and that, unlike wild-type epicardial cells, they fail to organize into thin vessel-like projections. Examination of Cx43 KO hearts using Wt1 as an epicardial marker revealed a disorganized pattern of epicardial cell infiltration. Time-lapse imaging and motion analysis using epicardial explants showed a defect in directional cell migration. This was associated with changes in the actin/tubulin cytoskeleton. A defect in cell polarity was indicated by a failure of the microtubule-organizing center to align with the direction of cell migration. Forced expression of Cx43 constructs in epicardial explants showed the Cx43 tubulin-binding domain is required for Cx43 modulation of cell polarity and cell motility. Pecam staining revealed early defects in remodeling of the primitive coronary vascular plexuses in the Cx43 KO heart. Together, these findings suggest an early defect in coronary vascular development arising from a global perturbation of the cytoarchitecture of the cell. Consistent with this, we found aberrant myocardialization of the outflow tract, a process also known to be EMT dependent. Together, these findings suggest cardiac defects in the Cx43 KO mice arise from the disruption of cell polarity, a process that may be dependent on Cx43-tubulin interactions.