In Vitro Invasion Assay Using Matrigel™: A Reconstituted Basement Membrane Preparation

2013 ◽  
pp. 1-11 ◽  
Author(s):  
Debbie M. S. Hall ◽  
Susan A. Brooks
Keyword(s):  
Basement Membrane ◽  
Membrane Preparation ◽  
Invasion Assay ◽  
In Vitro Invasion

In Vitro Invasion Assay Using Matrigel®

2003 ◽  
pp. 061-070 ◽  
Author(s):  
Debbie M S Hall ◽  
Susan A Brooks
Keyword(s):  
Invasion Assay ◽  
In Vitro Invasion

Early alterations of microRNA expression to predict and modulate melanoma metastasis.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 8550-8550
Author(s):  
Eva Hernando ◽  
Douglas Hanniford ◽  
Shulian Shang ◽  
Miguel F. Segura ◽  
Anna C. Pavlick ◽  
...  
Keyword(s):  
Clinical Staging ◽  
Melanoma Metastasis ◽  
Invasion Assay ◽  
Discovery Cohort ◽  
Aberrant Expression ◽  
Primary Tumors ◽  
In Vitro Invasion

8550 Background: Melanoma is curable for most patients whose tumors are surgically removed early in disease progression; however, many primary melanomas recur and progress to metastasis. Clinical staging is insufficient to robustly classify patients at highest risk of recurrence, and prognostic molecular biomarkers have not yet been identified. Methods: We performed miRNA profiling of 92 FFPE primary melanomas to discover metastasis relevant miRNAs and develop predictive models of recurrence, which were then validated in an independent cohort. We identified miRNAs differentially expressed between primary tumors that did and did not recur (3-year minimum follow-up) and between thick and thin lesions. We selected candidate miRs for screening in a fluorescence-based in vitro invasion assay, and prioritized a subset for in vivo testing. Potential downstream mediators of these miRNAs were selected by mRNA array analysis and tested in a secondary invasion screen. mRNA candidates that mimicked the miR’s invasion-suppressive effect were tested in 3’UTR reporter assays to confirm them as direct targets. Results: Using the discovery cohort we identified a 20 miRNA signature that can distinguish Stage I/II primary tumors (n=70) that did from those did not recur with 3-year minimum follow-up with an AUC=94%, 95% CI: (0.88, 0.99). Applying this model to predict risk for recurrence in the independent validation cohort yielded an AUC = 96%, 95% CI: (0.90, 1) in discriminating recurrent versus non-recurrent stages I/ II patients (n=45). From the discovery cohort, 40 candidates were selected for invasion assay screening, of which 5 miRNAs robustly inhibit in vitro invasion in 5 melanoma cell lines. Three miRs (miR-382, miR-516b, and miR-7) strongly suppressed metastasis in a mouse model. Moreover, multiple mRNAs tested as potential mediators mimicked the invasion-suppressive effects of candidate miRs. Of those, NCAPG2 and CDC42 were identified as miR-516b targets, CTTN as a miR-382 target, and PIK3CD as a miR-7 target. These genes have been linked to metastasis in melanoma or other tumors. Conclusions: Our data demonstrate that aberrant expression of specific miRNAs at diagnosis is predictive of and functionally impacts melanoma progression.

scholarly journals Correlation between an In Vitro Invasion Assay and a Murine Model of Burkholderia cepacia Lung Infection

2002 ◽  
Vol 70 (3) ◽  
pp. 1081-1086 ◽  
Author(s):  
Martin V. Cieri ◽  
Nicole Mayer-Hamblett ◽  
Adam Griffith ◽  
Jane L. Burns
Keyword(s):  
Burkholderia Cepacia ◽  
Statistical Approach ◽  
A549 Cells ◽  
Invasion Assay ◽  
In Vitro Invasion ◽  
Gentamicin Protection Assay ◽  
Lung Infections ◽  
Model Correlation

ABSTRACT Our understanding of the virulence of Burkholderia cepacia complex lung infections in cystic fibrosis patients is incomplete. There is a great deal of variability in the clinical course, from simple colonization to severe and often fatal necrotizing pneumonia, termed cepacia syndrome. Multiple subspecies (called genomovars) have been identified, and these genomovars may hold the key to understanding the variable pathogenicity. Thirty-one B. cepacia complex isolates belonging to five of the seven genomovars were examined by using a gentamicin protection assay of invasion with A549 cells. The level of epithelial cell invasion by B. cepacia in the A549 model was relatively low compared with the data obtained for other pathogens and was often variable from assay to assay. Thus, a statistical approach was used to determine invasiveness. When this model was used, one of four genomovar I strains (25%), three of eight genomovar II strains (37.5%), seven of nine genomovar III strains (77.8%), one of four genomovar IV strains (25%), and none of the four genomovar V strains examined were defined as invasive. All other strains were categorized as either noninvasive or indeterminate. Invasive, noninvasive, and indeterminate isolates belonging to genomovars II and III were subsequently tested for splenic invasion with the mouse agar bead model. Correlation between the models for six strains was demonstrated. Our results indicate that a statistical model used to determine invasiveness in an in vitro invasion assay can be used to predict in vivo invasiveness.

The Migration of Human Smooth Muscle Cells In Vitro Is Mediated by Plasminogen Activation and Can Be Inhibited by α2-Macroglobulin Receptor Associated Protein

1997 ◽  
Vol 78 (02) ◽  
pp. 880-886 ◽  
Author(s):  
Monique J Wijnberg ◽  
Paul H A Quax ◽  
Nancy M E Nieuwenbroek ◽  
Jan H Verheijen
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Ldl Receptor ◽  
Migration And Invasion ◽  
Invasion Assay ◽  
Plasminogen Activation ◽  
Smooth Muscle Cell Migration

SummaryThe plasminogen activation system is thought to be important in cell migration processes. A role for this system during smooth muscle cell migration after vascular injury has been suggested from several animal studies. However, not much is known about its involvement in human vascular remodelling. We studied the involvement of the plasminogen activation system in human smooth muscle cell migration in more detail using an in vitro wound assay and a matrix invasion assay. Inhibition of plasmin activity or inhibition of urokinase-type plasminogen activator (u-PA) activity resulted in approximately 40% reduction of migration after 24 h in the wound assay and an even stronger reduction (70-80%) in the matrix invasion assay. Migration of smooth muscle cells in the presence of inhibitory antibodies against tissue-type plasminogen activator (t-PA) was not significantly reduced after 24 h, but after 48 h a 30% reduction of migration was observed, whereas in the matrix invasion assay a 50% reduction in invasion was observed already after 24 h. Prevention of the interaction of u-PA with cell surface receptors by addition of soluble u-PA receptor or α2-macroglobulin receptor associated protein (RAP) to the culture medium, resulted in a similar inhibition of migration and invasion. From these results it can be concluded that both u-PA and t-PA mediated plasminogen activation can contribute to in vitro human smooth muscle cell migration and invasion. Furthermore, the interaction between u-PA and its cell surface receptor appears also to be involved in this migration and invasion process. The inhibitory effects on migration and invasion by the addition of RAP suggests an involvement of a RAP sensitive receptor of the LDL receptor family, possibly the LDL-receptor related protein (LRP) and/or the VLDL receptor.

scholarly journals Biosynthesis of basement membrane collagen by rabbit corneal endothelium in vitro.

1976 ◽  
Vol 251 (3) ◽  
pp. 730-733 ◽  
Author(s):  
N A Kefalides ◽  
J D Cameron ◽  
E A Tomichek ◽  
M Yanoff
Keyword(s):  
Basement Membrane ◽  
Corneal Endothelium ◽  
Basement Membrane Collagen ◽  
Membrane Collagen

scholarly journals POLYMORPHONUCLEAR LEUKOCYTES IN IMMUNOLOGIC REACTIONS

1966 ◽  
Vol 124 (4) ◽  
pp. 733-752 ◽  
Author(s):  
Charles G. Cochrane ◽  
Barbara S. Aikin
Keyword(s):  
Basement Membrane ◽  
Polymorphonuclear Leukocytes ◽  
Electron Microscopic Observation ◽  
Severe Damage ◽  
Electron Microscopic ◽  
Cationic Proteins ◽  
Vascular Basement Membrane ◽  
Arthus Phenomenon ◽  
Immunologic Reactions

Vascular basement membrane was disrupted in the presence of polymorphonuclear leukocytes (PMN's) during two immunologic reactions: The Arthus phenomenon and the reaction to locally injected antibody to vascular basement membrane. This disruption was evidenced by (a) the inability of the basement membrane to retain circulating carbon, by (b) loss of antigenic constituents, and by (c) electron microscopic observation showing actual gaps in the structure of the vascular basement membrane. The factors within PMN's responsible for damage to isolated glomerular basement membrane in vitro were found by isolation procedures to be cathepsins D and E. Cationic proteins of PMN's were separable from the cathepsins. While inducing vascular permeability upon injection, these basic proteins failed to inflict the severe damage to the basement membrane observed in Arthus and antibasement membrane reactions. It is concluded that the full expression of these immunologic lesions requires destruction of the basement membrane possibly brought about by cathepsins D and E. Some of the physicochemical properties of these pathologically active leukocytic factors are given.

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