Name: lung tumor cell recruitment assay
Uploaded: 2019-02-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Alle Verfahren, die mit Mäusen durchgeführt wurden durch das Tier Forschung Ausschuß von Tokyo Medical Frauenuniversität genehmigt.
1. Rute Vene Injektion von Lewis Lungenzellen Karzinom (LLC)
<ol><li>Pflegen LLC Zellen in DMEM mit 10 % FCS, in einer befeuchteten 5 % ergänzt CO2 Inkubator bei 37 ° C. Zellen sollten ein fluoreszierendes Protein Expressionssystems (z. B. GFP) enthalten.</li>
	<li>Entfernen Sie Zellen von Kulturschale mithilfe eines nicht-enzymatische ...

<ol>
	<li>Valastyan, S., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Kaplan, R. N., 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=VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche.">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438 (7069), 820-827 (2005).</li><li>Hiratsuka, S., Watanabe, A., Aburatani, H., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-mediated+upregulation+of+chemoattractants+and+recruitment+of+myeloid+cells+predetermines+lung+metastasis.">Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis.</a> Nature Cell Biology. 8 (12), 1369-1375 (2006).</li><li>Hiratsuka, S., 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=The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase.">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nature Cell Biology. 10 (11), 1349-1355 (2008).</li><li>Tomita, T., Sakurai, Y., Ishibashi, S., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imbalance+of+Clara+cell-mediated+homeostatic+inflammation+is+involved+in+lung+metastasis.">Imbalance of Clara cell-mediated homeostatic inflammation is involved in lung metastasis.</a> Oncogene. 30 (31), 3429-3439 (2011).</li><li>Deguchi, 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=Serum+amyloid+A3+binds+MD-2+to+activate+p38+and+NF-&#954;B+pathways+in+a+MyD88-dependent+manner.">Serum amyloid A3 binds MD-2 to activate p38 and NF-&#954;B pathways in a MyD88-dependent manner.</a> Journal of Immunology. 191 (4), 1856-1864 (2013).</li><li>Erler, J. T., 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=Hypoxia-induced+lysyl+oxidase+is+a+critical+mediator+of+bone+marrow+cell+recruitment+to+form+the+premetastatic+niche.">Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.</a> Cancer Cell. 15, 35-44 (2009).</li><li>Huang, 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=Pulmonary+vascular+destabilization+in+the+premetastatic+phase+facilitates+lung+metastasis.">Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis.</a> Cancer Research. 69 (19), 7292-7237 (2009).</li><li>Sceneay, J., Smyth, M. J., M&#246;ller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pre-metastatic+niche:+finding+common+ground.">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Reviews. 32 (3-4), 449-464 (2013).</li><li>Castle, J., Shaker, H., Morris, K., Tugwood, J. D., Kirwan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+circulating+tumor+cells+in+breast+cancer:+A+review.">The significance of circulating tumor cells in breast cancer: A review.</a> The Breast. 23, 552-560 (2014).</li><li>Wolf, M. J., 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=Endothelial+CCR2+signaling+induced+by+colon+carcinoma+cells+enables+extravasation+via+the+JAK2-Stat5+and+p38MAPK+pathway.">Endothelial CCR2 signaling induced by colon carcinoma cells enables extravasation via the JAK2-Stat5 and p38MAPK pathway.</a> Cancer Cell. 22 (1), 91-105 (2012).</li><li>Hiratsuka, S., 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=Primary+tumours+modulate+innate+immune+signalling+to+create+pre-metastatic+vascular+hyperpermeability+foci.">Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci.</a> Nature Communications. 4, 1853 (2013).</li><li>Deguchi, 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=Eritoran+inhibits+S100A8-mediated+TLR4/MD-2+activation+and+tumor+growth+by+changing+the+immune+microenvironment.">Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.</a> Oncogene. 35 (19), 1445-1456 (2016).</li><li>Ieguchi, K., 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=ADAM12-cleaved+ephrin-A1+contributes+to+lung+metastasis.">ADAM12-cleaved ephrin-A1 contributes to lung metastasis.</a> Oncogene. 33 (17), 2179-2190 (2014).</li></ol>

Vorbehandlung von Mäusen mit Antikörpern, Drogen, eine fettreiche Diät oder Tumor konditioniertes Medium vor der Tumor Zelle Injektion ist möglich. Der schwierigste Schritt in diesem Test ist das Heck Vene einspritzen. Unvollständiger Injektion führt nicht schlüssig Daten. Die Schweif Ader im C57BL/6 ist besonders schwer zu erkennen, was fehlerhafte Injektionen. Platzieren die Mäuse auf ein Heizkissen (37 ° C) erweitern hilft die Rute Vene so dass Injektion einfacher wird.
Dieser Assa...

Tumor Metastasen entfallen hohe Mortalität bei Krebs-Erkrankungen. Aus der Sicht der Untersuchungen auf molekularer Ebene, kann Metastasen in mehrere Schritte unterteilt werden: Tumorentstehung am Standort der Primärtumor, Primärtumor Wachstum und Invasion in das umgebende Gewebe, Intravasation, Zirkulation durch die Blutgefäße , Extravasation auf entfernte Organe und Tumor nachwachsen. Jeder Schritt umfasst unterschiedliche Moleküle/Signale1.
Bisherigen Studien haben besorgt, mit der Ereignisse, die in entfernten Organen vor die Tumorzellen im Blut, die auch bekannt als die bereits metastasierendem Phase2,3,4,5, 6. Unsere Maus Modellstudien gezeigt, dass die bereits metastasierendem Phase Lungenkrebs Metastasen erleichtert durch lang andauernde und Low-Level-Entzündungsreaktionen in der Lunge zu schaffen. Die bereits metastasierendem Phase zeichnet sich durch Hyperpermeability von Themicrovasculature, Rekrutierung von abgeleitet Knochenmarkzellen (BMDC) und Hochregulation der Zytokin/Chemokin-ähnliche Moleküle, einschließlich S100A8 und SAA33,4 . Es wurde berichtet, dass die Lysyl-Oxidase modifiziert die extrazelluläre Matrix in der Lunge, BMDCs7zu rekrutieren. Ein weiterer Bericht hat die Bedeutung von Angpt2, MMP3 und MMP10 in der Pre-metastasiertem Phase8ergeben. Das heißt, die komplizierten Wechselwirkungen zwischen Primärtumoren, dem Knochenmark und entfernte Organe müssen verstanden und richtig (z. B. durch Drogen, die auf Proteinen, die in diesen Interaktionen) moduliert werden um zu verhindern, dass künftige Metastasen9 . Um die bereits metastasierendem Phase zu regulieren, sollte es zunächst visualisiert und ausgewertet für diagnostische oder therapeutische Interventionen. Hier berichten wir über eine Lunge Tumor Zelle Rekrutierung Assay, der die Studie bereits metastasierendem Phase in der Lunge ermöglicht.
Zirkulierende Tumorzellen bei Krebspatienten, obwohl es möglicherweise Unterschiede zwischen kultivierten Zellen und zirkulierenden Tumorzellen ähneln Tumorzellen direkt in das Blutgefäß der Maus injiziert. Zirkulierenden Tumorzellen unterziehen sich einige charakteristischen Veränderungen, wenn sie aus dem primären Tumor in Umlauf. Dennoch können kultivierte Zellen Tumore in immungeschwächte Mäuse bilden, wenn sie in die Rute Vene injiziert werden. Darüber hinaus kann im Maus-Modell-System Fluoreszenz beschriftet Tumorzellen verwendet werden, die Verfolgung dieser Zellen im Körper zu ermöglichen. Das Verhalten der zirkulierenden Tumorzellen ist wichtig, da sie die letzten Konsequenzen in Krebs Patienten10bestimmen. Blut ist kein guter Ort für das Überleben von Tumor-Zellen-2. Sie müssen vor Angriffen durch das Immunsystem des Wirts zu entkommen und brauchen Anti-Apoptotic Signale Apoptose aufgrund mangelnder körperlicher Unterstützung zu verhindern. Tumorzellen mit höheren Fähigkeiten der Tumorentstehung bei metastasiertem Websites generieren mehr Tumor Knötchen. Wenn die Tumorzellen spezifisches Organ Tropismus zeigen, Metastasen in den einzelnen Organen zu beachten. In diesem Test die ersten Schritte der metastatischen Kaskade (Primärtumor Wachstum und Invasion) sind nicht im Preis inbegriffen, und so im Mittelpunkt steht das Zusammenspiel von zirkulierenden Tumorzellen und die stromale Zellen (Endothelzellen, Epithelzellen und Blutzellen). In herkömmlichen Tumor Zelle Injektion Assays haben Forscher warten, bis der Tumor Knötchen wachsen auf eine konkrete Größe der Metastasen zu erkennen. In diesem Fall werden nicht die genaue Anzahl der Tumorzellen in den Blutgefäßen quantifiziert. Darüber hinaus enthält dieses Prozesses eine Tumor Regrowth Phase, darauf hinweist, dass andere Faktoren in den Tumorzellen das Ergebnis beeinflussen können.
Zählen ist beschriftet Tumorzellen unter eine Fluoreszenz Stereomikroskop ein einfacher Prozess. Das Stereomikroskop bietet ein breites Sichtfeld, so dass die ganze Lunge gescannt werden kann. Durchflusszytometrie ist auch ein nützliches Tool, das sofort eine genaue Anzahl von Tumorzellen in das Gewebe gibt. Ist auch möglich und zeigt die genauesten Bilder von metastatischen Tumorzellen zählen fluoreszierende Zellen in das Gewebe unter einem confocal Mikroskop, aber es ist zeitaufwändig und verzerrte Ergebnisse geben kann, wenn nur ein ausgewählter Bereich gezählt wird. Das Endziel dieser Assay ist zu beobachten, wie leicht die Tumor-Zellen in der Lunge ansammeln. Wie bereits berichtet3,4, wenn die Lunge durch entzündliche Signalisierung aus dem primären Tumor bereits Metastasen phasengleich sind, neigen die injizierten Tumorzellen in die Lunge, so impliziert höhere Chancen auf Lungenkrebs Metastasen ansammeln. Andere Bedingungen (rheumatoide Arthritis, Asthma, etc.) und deren Behandlung mit entzündungshemmenden Mitteln (z. B. Anti-TNF) kann Lungenkrebs Metastasen dämpfen. So schließen wir, dass dieser Test ein mächtiges Werkzeug ist um die Möglichkeit der Lunge Metastasen zu bewerten.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>DN-25</td>
			<td>trace amount</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td>40 micrometer mesh</td>
		</tr>
		<tr>
			<td>Fluorescence labeling kit</td>
			<td>Sigma</td>
			<td>MIN26-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>17597K</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
	</tbody>
</table>

lung tumor cell recruitment assay

Um die molekularen Mechanismen Tumor Metastasen zu untersuchen, wurden verschiedene Tests mit der Maus als Modell Tier vorgeschlagen. Hier zeigen wir eine einfache Assays Tumor Zelle Extravasation oder Micrometastasis zu bewerten. In diesem Test Tumorzellen durch die Rute Vene injiziert wurden, und nach kurzer Zeit die Lunge wurden seziert und verdaut die angesammelten beschrifteten Tumorzellen zu zählen. Dieser Assay überspringt den ersten Schritt der primäre Tumorinvasion in das Blutgefäß und erleichtert die Untersuchung der Ereignisse in der entfernten Organ wo Tumor Metastasen auftreten. Die Anzahl der Zellen in das Blutgefäß injiziert kann optimiert werden, um eine begrenzte Anzahl von Metastasen zu beobachten. Es wurde berichtet, dass Stromazellen Zellen in den entfernten Organ zur Metastasierung beitragen. So könnte dieser Assay ein nützliches Werkzeug, um mögliche therapeutische Medikamente oder Geräte für die Prävention von Tumor Metastasen zu erkunden.

Die Lungen präsentieren viele GFP-LLC Zellen 2 h nach der Injektion (Abbildung 1A). Es sei darauf hingewiesen, dass die fluoreszierende Punkte auch in den roten Filter erkannt aus der Nummer Zellenzahl ausgeschlossen werden sollen. Die überwiegende Mehrheit der LLC Zellen verschwinden aus den Lungen 24 h nach der Injektion (Abbildung 1).
Um die Anzahl der GFP-LLC in d...

Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Lung Tumor Cell Recruitment Assay

Lung Tumor Zelle Rekrutierung Assay

Dieses Manuskript beschreibt eine Methode zur Quantifizierung der Tumor Zelle Ansammlung in der Lunge in einem Tiermodell der Tumor Metastasen.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Diese Methode kann helfen, wichtige Fragen im Bereich der Tumorbiologie über die molekularen Mechanismen der Metastasierung zu beantworten. Der Hauptvorteil dieser Technik ist, dass die Rekrutierung von Tumorzellen innerhalb eines entfernten Organs quantitiert werden kann. Beginnen Sie mit einem nicht-enzymatischen Zelldissoziationsreagenz, um fluoreszierendes Protein-exzierendes Lewis-Lungenkarzinom oder LLC- oder LLC-Zellen gemäß Standardprotokollen aus ihrem Kulturbehälter zu entfernen.Übertragen Sie die resultierende Zelllösung vor der Zentrifugation in ein 15-Milliliter-Konikonröhre, und Zentrifugen waschen Sie die Pellets zweimal in fünf Milliliter PBS pro Waschgang. Nach der zweiten Wäsche die Zellen durch ein 40-Mikrometer Porenzellsieb filtern und die Suspension auf ein mal 10 bis sechs Zellen pro Milliliter Konzentration verdünnen. Dann laden Sie die Zellen in eine Ein-Milliliter-Spritze mit einer 29-Spur-Nadel ausgestattet, und injizieren 100 Mikroliter Zellen in die Schwanzvene jeder acht bis zehn Wochen alten, C57-Black-6, männliche Maus.Verwenden Sie am entsprechenden experimentellen Endpunkt eine Schere, um die Haut von der ventralen Oberfläche jeder Maus zu entfernen, und schneiden Sie die Rippen und das Zwerchfell, um die Brusthöhle freizulegen. Verwenden Sie eine 25-Spur geflügelte Infusionsnadel und Schläuche, um 15 Milliliter PBS durch den rechten Ventrikel zu spülen und das Herz und den Thymus zu entfernen. Die Luftröhre mit Zangen greifen, hochziehen, das Bindegewebe um die Luftröhre sezieren, um die Lunge zu ernten.Dann waschen Sie die Lunge in PBS, und lösen Sie einen Lappen für die Visualisierung der fluoreszierenden Zellen in situ unter einem Fluoreszenzmikroskop. Um das Lungengewebe zu verdauen, würfeln Sie zuerst den kaudalen Lappen mit einer Schere und legen Sie die Lungenstücke in eine 10-Milliliter-Spritze. Schließen Sie die Spritze mit dem Kolben, und aspirieren Sie fünf Milliliter Verdauungslösung in die Spritze, bewegen Sie den Kolben in und aus der Spritzenwelle, bis das Lungengewebe in der gesamten Lösung verbreitet wird.Die Lungenschlämme in eine 50-Milliliter-Röhre geben und das Gewebe 15 Minuten bei 37 Grad Celsius und 150 U/min auf einem wechselseitigen Shaker verdauen. Am Ende der Inkubation das verbleibende Gewebe trituieren, bis die Lungenzellen vollständig dispergiert sind, und geben Sie das Rohr für weitere 30 Minuten an den Shaker zurück. Filtern Sie dann die Zellen durch ein 40-Mikrometer Porensieb und sammeln Sie die Zellen durch Zentrifugation.Um die isolierten Lungenzellen durch Durchflusszytometrie zu analysieren, setzen Sie das Pellet in zwei Millilitern roter Blutzelllysepuffer für eine Minute bei Raumtemperatur wieder auf und sammeln sie die Zellen durch Zentrifugation. Dann Zentrifugieren Sie das Pellet zwei Mal in fünf Milliliter frischen PBS mit 1%rinderserum albumin pro Wäsche ergänzt. Nach der zweiten Zentrifugation das Pellet in einem Milliliter frischer PBS plus BSA wieder aufsetzen und die Zellen durch ein neues 40-Mikrometer Porensieb zur Quantifizierung der Tumorzellen durch Durchflusszytometrie filtern.Die Lunge stellt zwei Stunden nach der Injektion viele GFP-LLC-Zellen dar, wobei die überwiegende Mehrheit der Zellen innerhalb von 24 Stunden aus der Lunge verschwindet. Beachten Sie, dass alle fluoreszierenden Flecken, die im roten Filter erkannt werden, von der Zellanzahl ausgeschlossen werden sollten. Um die Anzahl der GFP-LLC in der Lunge zu bestätigen, kann einer der Lappen für die zytometrische Analyse des Durchflusses verwendet werden, wie gezeigt.Beim Versuch dieses Verfahrens ist es wichtig, sich daran zu erinnern, dass ein eingefrorener Abschnitt eine genauere Methode zur Beobachtung der genauen Position von Tumorzellen ist.

Research

JoVE Journal

Cancer Research

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

Bedingte Knockdown von Genexpression in Krebs-Zell-Linien, die Rekrutierung von Monozyten/Makrophagen, den Tumor Mikroumgebung zu studieren

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Forschung

Krebsforschung

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

Die Einrichtung eines Lunge Kolonisation Assays für zirkulierenden Tumor Zelle Visualisierung im Lungengewebe

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Azelluläre und zellulären Lunge Modell Tumor Metastasen zu studieren

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Live Cell Imaging-Assays Studie Glioblastom Gehirn Tumor Stammzellen Migration und Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

Digitalen Polymerase-Kettenreaktion-Assay für die genetische Variation bei einem sporadischen familiäre adenomatöse Polyposis Patienten mit dem Chip-in-Rohr-Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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 ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Echtzeit-Erkennung von In Vitro Tumor Zelle Apoptose induziert durch CD8+ T-Zellen, unterdrückende Immunfunktionen Tumor infiltrieren myeloische Zellen zu studieren

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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 ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

In Vitro-Assay zur Untersuchung der Tumor-Makrophage-Interaktion

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Nachweis der Lungentumorprogression bei Mäusen durch Ultraschall-Bildgebung

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...