Name: characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics
Uploaded: 2018-03-26T14:30:00.000+00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Die Ohse Grant Foundation finanziert diese Arbeit.

1. Erweiterung der Zelleigenschaften
<ol><li>Pflegen Sie SW 13, SW-Neo und SW-DKK3 Zellen in einem standard befeuchteten Inkubator bei 37,0 ° C und 5 % CO2 in Dulbeccos geändert Eagle Medium mit 10 % fötalen Rinderserum und 10.000 U/mL Penicillin und Streptomycin (bezeichnet als "Wachstumsmedium") ergänzt.<ol>
<li>Zählen von Zellen mit einem Hemocytometer und 5.000 Zellen pro Bohrloch für SW 13, SW-Neo und SW-DKK3 Linien in separaten 6-Well-Platten und wachsen über Nacht im Wachstumsmedium auf sterile Glasdeckgläser.</li>
	</ol>
</li>
	<li>Nachdem über Nacht Wachstum, für jedes gut Aspirieren das Wachstumsmedium, waschen Sie Zellen mit 1 mL warmen Phosphat-gepufferte Kochsalzlösung (PBS) und befestigen Sie die Zellen mit 1 mL von 3,7 % Formaldehyd für 10 min.</li>
	<li>Aspirat Formaldehyd und Fleck Zellen in jedem Bohrloch mit 1 mL 0,05 % Kristallviolett für 30 min. Waschen überschüssige Farbstoff mit drei wäscht von 1 mL entionisiertem Wasser. Hinweis: Abhängig von der Höhe der Farbstoff-Aufnahme, können mehrere zusätzliche Waschungen erforderlich sein, zur Förderung der Zelle Visualisierung Färbung Hintergrund zu entfernen.<ol>
<li>Mit feinen Spitzen Pinzette, Abrufen von Deckgläsern und legen Sie sie nach unten auf einen beschrifteten Objektträger mit einem Tropfen klar Montage Reagenz.</li>
	</ol>
</li>
	<li>Wählen Sie unter einem Lichtmikroskop nach dem Zufallsprinzip 20 Ansichten von nicht überlappenden Zellen aus jeder Deckglas für die Zelle-Erweiterung-Assay.<ol>
<li>Nehmen Sie Vergößerung bei 400 X Vergrößerung von jedem Sichtfeld. Direkt die Anzahl der jede Art von Erweiterung für jede der 20 repräsentative Zellen. Hinweis: als solche sollte 20 isolierte Zellen für jede getestete Bedingung um zuverlässige Ergebnisse zu gewährleisten untersucht werden. Filopodien wurden durch zelluläre Erweiterungen mit einem Sockel von 5 μm oder weniger und einem einzigen Spitze definiert. Pseudopodien wurden durch zelluläre Erweiterungen mit einer Grundfläche von 5 bis 20 µm Länge enthält Erweiterungen mit mehreren Spitzen definiert. Lamellipodia wurden durch Zelle-Erweiterungen, die größer als 20 Mikrometer lang und mit oder ohne Spitzen sind definiert. Getestete Zelle abhängig die Größe der Zelle Erweiterungen abweichen und Verfeinerung der Identifikation Parameter erfordern.</li>
	</ol>
</li>
	<li>Bestimmen Sie die durchschnittliche Anzahl pro Zelle-Verlängerung in jeden Zelltyp (d.h., SW 13, SW-Neo, SW-DDK) über die 6 Brunnen untersucht.</li>
	<li>Mit einem One-Way Varianzanalyse (ANOVA) statistische Test, vergleichen Sie Unterschiede in den durchschnittlichen Anteil der Zellmembran Erweiterungen unter die verschiedenen Zelltypen.</li>
</ol>(2) Haftung Assay
<ol><li>SW 13, SW-Neo und SW-DKK3 Zellen in einem standard befeuchteten Inkubator bei 37,0 ° C und 5 % CO2 im Wachstumsmedium zu erhalten.<ol>
<li>Mit einem Hemocytometer, zählen und 100.000 Zellen von SW 13, SW-Neo und SW-DKK3 pro Bohrloch in separaten 6-Well-Platten und wachsen über Nacht im Wachstumsmedium.</li>
		<li>Samen 6-Well-Platten für jeden Zelltyp mit einer Platte für jede der 6 bezeichneten Zeitpunkten getestet unter getestet. Hinweis: Die Zeitpunkte können für verschiedene Zelltypen variieren.</li>
	</ol>
</li>
	<li>Nach der Inkubation über Nacht einmal waschen Sie Zellen mit warmen PBS, dann fügen Sie 0,5 mL 1 x nicht-enzymatische Zelle Dissoziation Lösung in jede Vertiefung.<ol>
<li>Schwenken Sie die Platte, um die Zelle Dissoziation Lösung zu verbreiten. Aspirieren Sie die dafür vorgesehenen Platte zu definierten Zeitpunkten (z. B. 1, 2, 3, 5, 10 und 15 min) und dann mit warmen PBS-Lösung 1 mL waschen Sie dreimal.</li>
		<li>Zwischen den Waschgängen klopfen Sie leicht die Platten um lose beigefügte Zellen zu lösen.</li>
	</ol>
</li>
	<li>Aspirieren Sie alle verbleibenden PBS und Korrektur, die die Zellen bleiben auf der Platte mit 1 mL von 3,7 % Formaldehyd für 10 min angebracht.</li>
	<li>Beflecken Sie Zellen mit 1 mL 0,05 % Kristallviolett für 30 min, dann waschen Sie überschüssige Farbstoff Weg mit 1 mL entionisiertem Wasser. Titrieren Sie waschen Zelle Visualisierung zu fördern. Hinweis: Abhängig von der Höhe der Farbstoff-Aufnahme, können mehrere zusätzliche Wäschen benötigt, um Zelle Visualisierung zu fördern.</li>
	<li>Mit einem Lichtmikroskop bei 100 X Vergrößerung, Graf befestigt die restlichen Zellen in jede Vertiefung für jede Platte. Hinweis: Verwendung von transparenten Herrscher oder Kennzeichnung feinen Stift Führer auf der Unterseite der Platte hilft zur Vermeidung wiederholter zählen die gleichen Zellen.</li>
	<li>Bestimmen Sie die durchschnittliche Anzahl der angeschlossenen Zellen pro Bohrloch für jede Platte.</li>
	<li>Vergleichen Sie die Rate der Zelle Ablösung zwischen SW 13, SW-Neo und SW-DDK3 Zellen im angegebenen Zeitraum mit einem zwei-Wege-ANOVA statistischen Test.</li>
</ol>

<ol>
	<li>Libe, R., Fratticci, A., Bertherat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+cancer:+pathophysiology+and+clinical+management.">Adrenocortical cancer: pathophysiology and clinical management.</a> Endocrine-related cancer. 14 (1), 13-28 (2007).</li><li>Veeck, J., Dahl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Wnt+pathway+in+cancer:+the+emerging+role+of+Dickkopf-3.">Targeting the Wnt pathway in cancer: the emerging role of Dickkopf-3.</a> Biochimica et biophysica acta. 1825 (1), 18-28 (2012).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nat Rev Mol Cell Biol. 10 (7), 445-457 (2009).</li><li>Jacquemet, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=L-type+calcium+channels+regulate+filopodia+stability+and+cancer+cell+invasion+downstream+of+integrin+signalling.">L-type calcium channels regulate filopodia stability and cancer cell invasion downstream of integrin signalling.</a> Nat Commun. 7, 13297 (2016).</li><li>Machesky, L. M., Li, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fascin:+Invasive+filopodia+promoting+metastasis.">Fascin: Invasive filopodia promoting metastasis.</a> Commun Integr Biol. 3 (3), 263-270 (2010).</li><li>Petrie, R. J., Harlin, H. M., Korsak, L. I., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activating+the+nuclear+piston+mechanism+of+3D+migration+in+tumor+cells.">Activating the nuclear piston mechanism of 3D migration in tumor cells.</a> J Cell Biol. 216 (1), 93-100 (2017).</li><li>Korah, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+RASSF1A+deregulates+cytoskeleton+and+promotes+malignant+behavior+of+adrenocortical+carcinoma.">Epigenetic silencing of RASSF1A deregulates cytoskeleton and promotes malignant behavior of adrenocortical carcinoma.</a> Molecular cancer. 12, 87 (2013).</li><li>Cheng, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+FOXO1-mediated+dedifferentiation+blocking+role+for+DKK3+in+adrenocortical+carcinogenesis.">A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis.</a> BMC cancer. 17 (1), 164 (2017).</li><li>Barry, D. J., Durkin, C. H., Abella, J. V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> J Cell Biol. 209 (1), 163-180 (2015).</li><li>Cliffe, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+analysis+of+complex+single+border+cell+behaviors+in+coordinated+collective+cell+migration.">Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration.</a> Nat Commun. 8, 14905 (2017).</li></ol>

Die Autoren haben nichts preisgeben.

Hier beschreiben wir eine in-vitro- quantitative Methode zur Charakterisierung von Zelle Erweiterungen mit Leichtigkeit, paar Grube fällt und zuverlässige Reproduzierbarkeit, die für verschiedene Testbedingungen angewendet werden können. Darüber hinaus können einfache, quantifizierbare Haftung und/oder Motilität Assays gleichzeitig durchgeführt werden, um mögliche funktionale Bedeutung der beobachteten Veränderungen in der Zellmembran Erweiterungen zu korrelieren.
Diese Meth...

Dysregulated WNT signalisieren spielt eine entscheidende Rolle in der NNR Malignome1. Die in dieser Studie verwendeten Methoden untersuchen, ob Schweigen der DKK3, ein negativer Regulator der WNT signalisieren, stellt eine Entdifferenzierung Ereignis in der Nebennierenrinde und fördert Tumorbildung im Zusammenhang mit der Zelle-Erweiterung Repertoire Änderungen. DKK3 ist eine 38 kDa Glykoprotein mit einer N-terminalen Signalpeptid abgesondert und frühere Studien haben gezeigt, dass seine erzwungene Ausdruck Zellzyklus Festnahme führte, aggressiven bösartigen Verhaltens gehemmt und epitheliale-mesenchymale umgekehrt Übergang2.
Das bösartige Verhalten der NNR-Karzinom (ACC) und anderen Krebsarten ist, im Teil, beeinflusst durch die Fähigkeit der Tumorzellen, Schnittstelle mit der umgebenden Flächen, einschließlich die extrazelluläre Matrix, die wiederum Tumorinvasion Zelle erleichtert und Migration-3. Die Rolle der spezifischen Zellmembran Erweiterungen in Tumorprogression ist zunehmend in verschiedenen Kontexten, vor allem über die Bildung von Filopodien vorgeführt. Zum Beispiel wurde Überexpression des L-Typ-Kalziumkanäle zu induzieren Filopodien Bildung und Förderung der Tumor Zelle Invasion4gefunden. Ebenso ist Fascin, ein Aktin-bindende Protein minimal ausgedrückt in normalem Gewebe auch überexprimieren in Krebszellen in Verbindung mit Filopodien Bildung5. Pseudopodien Bildung ermöglicht nicht-malignen Fibroblasten, effektiv durch die extrazelluläre Matrix zu migrieren, jedoch wurde nachgewiesen, dass Fibrosarkom Zellen abhängig Metalloproteinase Aktivität anstelle von Pseudopodien Zellwanderung zu erleichtern und Invasion-6. Wir haben gezeigt, dass Tumor Entstörer, einschließlich Ras Verband Domäne Familie 1 Isoform (RASSF1A) und DKK3, kann die Funktion zum Zellskelett Elemente zu verändern und Lamellipodia Bildung und stymie invasive Eigenschaften7,8.
Als solches ist es wichtig, die Auswirkungen von Genen, die in der Karzinogenese und ihre Beziehung zur Zellmembran Erweiterung Änderungen, insbesondere Beurteilung Filopodien, Pseudopodien und Lamellipodia Bildung unter Testbedingungen zu charakterisieren. Aktuellen State-of-the-Art-Techniken umfassen die Verwendung von immer ausgefeilteren Mikroskopieverfahren, fluoreszierende Kennzeichnung und/oder komplexe Computer-Algorithmen für die Datenerfassung und Auslegung. Während diese Methoden neue, leistungsstarke Analysetools bieten, schränkt ihre Komplexität ihrer Verbreitung und Anpassungsfähigkeit in Zelle Biologie Experimente. Darüber hinaus ist die exakte Quantifizierung und Beobachtung der Veränderungen der Zellmorphologie Erweiterung nicht in der Regel gemessenen9,10. Im Gegensatz dazu stellen wir Ihnen hier eine Technik, die Zelle Erweiterung Änderungen mit mikroskopischen Standardtechniken und leicht anpassbar in-vitro- Methoden genau quantifiziert. Diese Methoden auch quantifizieren jeden Erweiterungstyp Zelle gleichzeitig für jede Zelle analysiert und Veränderungen in der Zellmembran Erweiterung Repertoire zu bestimmen. Wir zeigen auch, wie diese Veränderungen auf Zelle Adhäsionseigenschaften beziehen können.
Als ein experimentelles Beispiel verwenden wir eine zuvor erstellte Zelllinie SW-13 bezeichneten SW-DDK3, das hat mit pCMV6-Eintrag/DKK3 Plasmid-Vektoren stabil transfiziert wurden und konstitutiv overexpresses DKK3, ein Unterscheidungsmerkmal in der Nebenniere. Non-transfizierten SW 13 und SW-13 Zellen mit leerer Vektor (pCMV6-Eintrag), benannten SW-Neo, stabil transfiziert werden als experimentelle Kontrollen dienen.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>ThermoFisher</td><td>11965-092</td><td>Supplemented with fetal bovine serum and penicllin and streptomycin</td></tr><tr><td>Deulbeco&#039;s Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>D8537</td><td>1X solution, used directly</td></tr><tr><td>3.7% formaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F8775</td><td>Dilute stock solution</td></tr><tr><td>0.05% crystal violet&amp;nbsp;</td><td>Harleco</td><td>192-12</td><td>Dilute stock solution</td></tr><tr><td>De-ionized water</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>Microscope with 400X magnification&amp;nbsp;</td><td>NA</td><td>NA</td><td>NA</td></tr><tr><td>6-well plates</td><td>Corning</td><td>353046</td><td>NA</td></tr><tr><td>Cell dissociation solution non-enzymatic 1X</td><td>Sigma-Aldrich</td><td>C5914&amp;nbsp;</td><td>1X solution, used directly</td></tr></tbody></table>

characterization of cell membrane extensions and studying their roles in cancer cell adhesion dynamics

Die Zellmembran Erweiterung Repertoire moduliert verschiedene bösartige Verhaltensweisen von Krebszellen, einschließlich ihrer Klebe- und wandernden Potenziale. Die Fähigkeit, genau zu klassifizieren und Zelle Erweiterungen zu quantifizieren und die Wirkung auf eine Zelle Klebstoff Kapazität zu messen ist entscheidend für die Bestimmung, wie Zelle Signalisierung Ereignisse Auswirkungen Krebs Zelle Verhalten und Aggressivität. Hier beschreiben wir die in-vitro- Gestaltung und Nutzung einer Zelle Quantifizierung Erweiterungsmethode in Verbindung mit einem Adhäsion Kapazität Assay in einem etablierten in-vitro- Modell für NNR-Karzinom (ACC). Insbesondere prüfen wir die Auswirkungen der DKK3, eine vermeintliche Tumorsuppressor und pro-Differenzierung Faktor auf der Membran Erweiterung Phänotyp der ACC-Zelllinie, SW-13. Wir schlagen diese Tests bieten relativ einfach, zuverlässig und leicht interpretierbare Metriken, misst diese Eigenschaften unter verschiedenen experimentellen Bedingungen.

Unter Verwendung der oben genannten Assays, wurden die Auswirkungen der DKK3 Überexpression auf Verlängerung Zellmorphologie und Zelle Adhäsionseigenschaften in die etablierten ACC-Zellinie SW-13, in Vitrogetestet. DKK3 überexprimierenden Zellen wurden durch stabil transfecting SW-13 Zellen mit Myc-DDK tagged pCMV6-Eintrag/DKK3-Plasmid-Vektoren und als SW-DKK3 erzeugt. Ebenso wurden mit leerer Vektor pCMV6-Eintrag stabil transfizierten Zellen als Transfektion und Passagierung...

Watch this Scientific Journal Video about Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics at JoVE.com

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

Diese Studie zeigt den nutzen und die einfache quantitative Zellmembran Erweiterung Messung und ihre Korrelation zum Klebstoff Kapazität von Zellen. Als ein repräsentatives Beispiel zeigen wir hier der Dickkopf-related Protein 3 (DKK3) fördert erhöhte Pseudopodien Bildung und Zelle Haftfähigkeit in NNR Karzinom-Zellen in Vitro.

Charakterisierung der Zellmembran Erweiterungen und studieren ihre Rolle im Krebs Zelle Adhäsion Dynamik

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

characterization-cell-membrane-extensions-studying-their-roles-cancer

Research

JoVE Journal

Cancer Research

7.5K Aufrufe.  Yale University School of Medicine. Diese Studie zeigt den nutzen und die einfache quantitative Zellmembran Erweiterung Messung und ihre Korrelation zum Klebstoff Kapazität von Zellen. Als ein repräsentatives Beispiel zeigen wir hier der Dickkopf-related Protein 3 (DKK3) fördert erhöhte Pseudopodien Bildung und Zelle Haftfähigkeit in NNR Karzinom-Zellen in Vitro.

Serie: Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modeling and Imaging 3-Dimensional Collective Zellinvasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Forschung

Medizin

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

3-d-Zellkultursystem für die Invasion zu studieren und Bewertung Therapeutika bei Blasenkrebs

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Krebsforschung

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Analyse der Zellmigration in eine dreidimensionale Kollagenmatrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Biotechnik

Examining the Dynamics of Cellular Adhesion and Spreading of Epithelial Cells on Fibronectin During Oxidative Stress

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the homeostasis of adult tissues. Interestingly, oxidative stress can alter these processes, thus contributing to the pathophysiology of diseases such as metastatic cancer. Therefore, understanding the mechanism(s) of how cells attach and spread on the ECM during perturbations in redox status can provide insight into normal and disease states. Described below is a step-wise protocol that utilizes an immunofluorescence-based assay to specifically quantify cell adhesion and spreading of immortalized fibroblast cells on fibronectin (FN) in vitro. Briefly, anchorage-dependent cells are held in suspension and exposed to the ATM kinase inhibitor Ku55933 to induce oxidative stress. Cells are then plated on FN-coated surface and allowed to attach for predetermined periods of time. Cells that remain attached are fixed and labeled with fluorescence-based antibody markers of adhesion (e.g., paxillin) and spreading (e.g., F-actin). Data acquisition and analysis are performed using commonly available laboratory equipment, including an epifluorescence microscope and freely available Fiji software. This procedure is highly versatile and can be modified for a variety of cell lines, ECM proteins, or inhibitors in order to examine a broad range of biological questions.

This method is useful for quantifying the early dynamics of cellular adhesion and spreading of anchorage-dependent cells onto the fibronectin. Furthermore, this assay can be used to investigate the effects of altered redox homeostasis on cell spreading and/or cell adhesion-related intracellular signaling pathways.

Untersuchung der Dynamik der zellulären Adhäsion und Ausbreitung von Epithelzellen auf Fibronectin während oxidativen Stresses

The adhesion and spreading of cells onto the extracellular matrix (ECM) are essential cellular processes during organismal development and for the ...

Biochemie

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

Enthüllung des Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely F-actin-negative membrane projections in endothelial and cancer cells that can be visualized through TM4SF1 (Transmembrane-4-L-six-family-1) immunofluorescence staining. TM4SF1 clusters in 100-300 &#956;m diameter TMED (TM4SF1 enriched microdomains) containing 3 to as many as 14 individual TM4SF1 molecules. TMED are arranged intermittently along nanopodia at a regular spacing of 1 to 3 TMED per &#956;m and firmly anchor nanopodia to matrix. This enables nanopodia to extend more than 100 &#956;m from the leading front or trailing rear of polarized endothelial or tumor cells, and causes membrane residues to be left behind on matrix when the cell moves away. TMED and nanopodia have been overlooked because of their extreme fragility and sensitivity to temperature. Routine washing and fixation disrupt the structure. Nanopodia are preserved by direct fixation in paraformaldehyde (PFA) at 37 &#176;C, followed by brief exposure to 0.01% Triton X-100 before staining. Nanopodia open new vistas in cell biology: they promise to reshape our understanding of how cells sense their environment, detect and identify other cells at a distance, initiate intercellular interactions at close contact, and of the signaling mechanisms involved in movement, proliferation, and cell-cell communications. The methods that are developed for studying TM4SF1-derived nanopodia may be useful for studies of nanopodia that form in other cell types through the agency of classic tetraspanins, notably the ubiquitously expressed CD9, CD81, and CD151.

Nanopodia are thin but fragile membrane channels that extend up to 100 &#956;m from a cell's leading front or trailing rear and sense the cellular environment. Direct fixation at 37 &#176;C, gentle washing, and avoidance of organic solvents like ethanol, methanol, or acetone and of higher Triton X-100 concentrations are required to observe these cellular structures.

Nanopodia - Dünne, Zerbrechlich Membran Projektionen mit Rollen in der Zellbewegung und interzelluläre Wechselwirkungen

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

Biologie

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

Quantitative Analyse der Zellkantendynamik während der Zellstreuung

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.

Abbildung der molekularen Adhäsion im Zellrollen durch Adhäsion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

Messung der Zellkanten-Überstandsdynamik während des Streuens mittels Lebendzellmikroskopie

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

Cell Adhesion Molecules - Types and Functions

Cell adhesion molecules (CAMs) are pivotal to multicellularity and the coordinated functioning of tissues and organ systems. They enable physical interactions between cells and provide mechanical strength to tissues. They also function as receptors for signal transmission across the plasma membrane. The CAMs are broadly classified into four families - integrins, cadherins, selectins, and immunoglobulin-like CAMs (IgCAMs).
CAM Families
The Integrin family of proteins is primarily&#160; involved in a cell&#8217;s interaction with its surrounding matrix. However, some integrins can bind CAMs on another cell&#8217;s surface and participate in direct cell-cell interactions. For example, the integrins on immune cells bind IgCAMs expressed on the vascular endothelium.
Cadherins are a superfamily of calcium-dependent glycoproteins primarily involved in establishing strong cell adhesions. They compose the adherens junctions and desmosomes in tissues, such as the epithelium.
Selectins and IgCAMs are involved in transient cell interactions and participate in directing cells towards target sites. For example, they help in the selective recruitment of lymphocytes to the secondary lymphoid organs.
CAM and Multicellularity
CAMs are found&#160; across virtually all multicellular organisms - from sponges and simple nematodes to complex invertebrates and vertebrates. The complexity of the cellular interactions, and therefore of the CAMs, increases with the complexity of the organisms. For example,&#160; while the fruit fly D.melanogaster has approximately&#160; 500 genes involved in cell adhesion, complex vertebrates like mammals have over a thousand genes that code for different types of CAMs.