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 width="653">
	<tbody>
		<tr height="63">
			<td height="63" style="width:216px;height:63px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:115px;">ThermoFisher</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:203px;">Supplemented with fetal bovine serum and penicllin and streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Deulbeco&#39;s Phosphate Buffered Saline</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td>1X solution, used directly</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">3.7% formaldehyde&#160;</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">F8775</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">0.05% crystal violet&#160;</td>
			<td style="width:115px;">Harleco</td>
			<td style="width:119px;">192-12</td>
			<td>Dilute stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:216px;height:21px;">De-ionized water</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:216px;height:42px;">Microscope with 400X magnification&#160;</td>
			<td style="width:115px;">NA</td>
			<td style="width:119px;">NA</td>
			<td>NA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td>NA</td>
		</tr>
		<tr height="43">
			<td height="43" style="width:216px;height:43px;">Cell dissociation solution non-enzymatic 1X</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5914&#160;</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 ...

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Cancer Research

7.4K 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

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Ein Protokoll für schnelle Post-mortem-Zellkultur von Diffuse Intrinsic Ponsgliom (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Forschung

Krebsforschung

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Erkennung von Mitochondrien-Membran-Potential, CLIC4 Knockdown-induzierte HN4 Zelle Apoptosis In-vitro- Studie

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Ziel Zelle Voranreicherung und ganze Genome Amplifikation für einzelne Zelle nachgelagerten Charakterisierung

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

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. ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Mesenchymale Stammzellen isoliert von Pulpagewebe und Kokultur mit Krebszellen ihre Interaktionen zu untersuchen

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

Etablierung von vom Magenkrebs abgeleiteten Xenograft-Modellen und Primärzelllinien

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Halbautomatische PD-L1-Charakterisierung und Aufzählung zirkulierender Tumorzellen von nicht-kleinen Lungenkrebspatienten durch Immunfluoreszenz

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

Strahlenempfindlichkeit von Krebsstammzellen in Lungenkrebszelllinien

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Comparing Metastatic Clear Cell Renal Cell Carcinoma Model Established in Mouse Kidney and on Chicken Chorioallantoic Membrane

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, one third of ccRCC patients will develop metastases to the lung, which is related to a very poor outcome for patients. Unfortunately, no therapy is available for this deadly stage, because the molecular mechanism of metastasis remains unknown. It has been known for 25 years that the loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is pathognomonic of ccRCC. However, no clinically relevant transgenic mouse model of ccRCC has been generated. The purpose of this protocol is to introduce and compare two newly established animal models for metastatic ccRCC. The first is renal implantation in the mouse model. In our laboratory, the CRISPR gene editing system was utilized to knock out the VHL gene in several RCC cell lines. Orthotopic implantation of heterogeneous ccRCC populations to the renal capsule created novel ccRCC models that develop robust lung metastases in immunocompetent mice. The second model is the chicken chorioallantoic membrane (CAM) system. In comparison to the mouse model, this model is more time, labor, and cost-efficient. This model also supported robust tumor formation and intravasation. Due to the short 10 day period of tumor growth in CAM, no overt metastasis was observed by immunohistochemistry (IHC) in the collected embryo tissues. However, when tumor growth was extended by two weeks in the hatched chicken, micrometastatic ccRCC lesions were observed by IHC in the lungs. These two novel preclinical models will be useful to further study the molecular mechanism behind metastasis, as well as to establish new, patient-derived xenografts (PDXs) toward the development of novel treatments for metastatic ccRCC.

Metastatic clear cell renal cell carcinoma is a disease without a comprehensive animal model for thorough preclinical investigation. This protocol illustrates two novel animal models for the disease: the orthotopically implanted mouse model and the chicken chorioallantoic membrane model, both of which demonstrate lung metastasis resembling clinical cases.

Vergleich von metastasierenden klaren Zell Nierenzellkarzinom Modell in Maus Niere und auf Huhn Chorioallantoic Membran etabliert

Metastatic clear cell renal cell carcinoma (ccRCC) is the most common subtype of kidney cancer. Localized ccRCC has a favorable surgical outcome. However, ...

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

Quantifizierung der Tumorzelladhäsion in Lymphknoten-Kryosektionen

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...