Name: moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol
Uploaded: 2018-03-12T13:30:00.000+00:00
Duration: 6 min 14 s
Description: Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Diese Arbeit wurde von der National Institutes of Health (NIMHD-G12MD007581) durch das RCMI-Zentrum für Gesundheit und Umwelt an Jackson State University und das US Department of Defense, Idea Award 11-1-0205 unterstützt.

Hinweis: Dieses Protokoll ist im McArdle Et Al. beschrieben. 11. eine Zeitleiste finden Sie in Abbildung 1.
1. Vorbereitung der Kultur-Platte
<ol><li>Waschen Sie die 35-mm-Kulturschale mit einem 10-mm-Glasboden mit 1 mL 1 M Salzsäure (HCl) für 15 min, Hydrophobie der Glasboden zu senken.</li>
	<li>Waschen Sie die Kultur-Platte zweimal mit 1 mL der Phosphat-Puffer Kochsalzlösung (PBS) get rid of der HCl.</li>
	<li>Waschen Sie die Platte Kultur zweimal mit 1 mL 70 % igem Ethanol.</li>
	<li>Spülen Sie die Kultur-Platte mit 1 mL der spezifischen Zellkulturmedium (hier Gebrauch α-MEM ergänzt mit 10 % Hitze inaktiviert FBS und 1 % nicht-essentiellen Aminosäuren). Hinweis: Die behandelten Kultur-Platte mit dem Kulturmedium kann erst am nächsten Tag in der CO2 Inkubator mit einem Luftbefeuchter bei 37 ° C gespeichert werden.</li>
	<li>Wachsen Sie die Zellen (MSCs und MCF-7 Co Kultur) zu 100 % Konfluenz in den behandelten Glasboden gut von der 35-mm-Kulturschale. Gemeinsam Kultur 35.000 MSC Zellen und 75.000 MCF7 Zellen für 24 h.</li>
</ol>2. Vorbereitung der Kollagen-Gel
<ol><li>Die Mikropipette Tipps für pipettieren die Kollagen-Gel in den Gefrierschrank verwendet, um zu verhindern, dass die Polymerisation des Kollagens bei Kontakt zu speichern. Hinweis: Dies ist der wichtigste Schritt.</li>
	<li>Bestimmen Sie ein Volumen von Ratte Schweif Kollagen ich verwendet werden basierend auf die Konzentration von Kollagen-Lager. Dieser Assay 100 µL Kollagen Gel vorbereiten. Hinweis: Hoher Konzentration von Ratte Schweif Kollagen, die ich auf Lager funktioniert besser. Bestimmen Sie die Lautstärke von 10 x Dulbeccos geändert Eagle Medium verwendet werden, um das Kollagen entsprechend zu verdünnen.</li>
	<li>Kombinieren Sie auf Eis Ratte Schweif Kollagen I (3,8 µg/mL Brühe), 10 X Dulbecco geändert Eagle Medium und 1 X Zellkulturmedium ergänzt um 2 % fötalen Rinderserum und die entsprechenden Lockstoffgradient auf eine Endkonzentration von Kollagen von 2,4 mg/mL.</li>
	<li>Hinzu kommt die Mischung 4,5 % Natriumbikarbonat-Lösung, die Kollagen-Gel zu neutralisieren. Hinweis: Das Volumen der Natriumbicarbonat-Lösung hinzugefügt variieren basierend auf der genauen pH-Wert der anderen Reagenzien. Es empfiehlt sich, die Mischung mit pH-Streifen, neutrale Lösung zu gewährleisten, oder verwenden Sie einen pH-Indikator in das Kulturmedium zu testen.</li>
	<li>Legen Sie über die Zellen innerhalb der Glasboden gut von der Kulturschale 80 µL der Kollagen-Mischung.</li>
	<li>Lassen Sie das Kollagen-Gel zu polymerisieren für 30 min in einem CO2 Inkubator mit einem Luftbefeuchter bei 37 ° C.</li>
	<li>Decken Sie das Kollagen-Gel mit 2 mL Zelle Medium mit reduzierten Serum (2 %) und inkubieren Sie die Zellen bei 37 ° C in einem CO2 Inkubator mit einem Luftbefeuchter für 24, 48 oder 72 h. Vorsicht: Die Platte sollte mit Sorgfalt zu verhindern, dass das Kollagen-Gel aus der Glasboden behandelt werden.</li>
</ol>3. Beispiel der Herstellung von Kollagen gel mit einer Ratte Kollagen Reitstock von 3,8 µg /mL
<ol><li>Kombinieren Sie auf dem Eis 114,5 µL Ratte Schweif Kollagen, 16,6 µL 10 x DMEM und 33,1 µL Kulturmedium, enthält die Lockstoffgradient.</li>
	<li>Neutralisieren Sie die Kollagen-Mischung mit 14,0 µL Natriumbicarbonat.</li>
	<li>Weiter wie in Schritt 2.5.</li>
</ol>4. Bildgebung der Zellen
<ol><li>Entfernen Sie vorsichtig die Medien aus der Schale.</li>
	<li>Sanft spülen Sie das Gel mit 1 mL PBS.</li>
	<li>Befestigen Sie die Zellen mit 1 mL 4 % Paraformaldehyd für 15 Minuten.</li>
	<li>Waschen Sie die Zellen zweimal mit 1 mL PBS.</li>
	<li>Färben Sie die Zellen mit DAPI-Lösung (100 ng/mL) für 20 min bei Raumtemperatur.</li>
	<li>Bild der Zellen unter Verwendung eines konfokalen Mikroskops an verschiedenen Standorten und 400 µm Z-Stapel Bilder von jedem Standort in 16 µm Schritten. Das Mikroskop verwendet hat eine Festplatte, die Scan-Einheit, die konfokale Bildgebung mit einem weißen Licht, Bogen Anregungsquelle ermöglicht. Verwenden Sie ein 20 X Objektiv mit numerischer Apertur von 0,75 genutzt.</li>
	<li>Bilder zu rekonstruieren.<ol>
<li>Öffnen Sie Bilder für ein bestimmtes Gel (ca. 25 Bilder) und erstellen Sie einem Bildstapel zu, indem Sie auf Bild → Stapel → Bilder stapeln. Das erste Bild entsprach der Unterseite des Gels (Z = 0 µm), das zweite Bild entsprach der erste Schritt in Z-Richtung (Z = 16 µm), das dritte Bild entsprach der zweite Schritt in Z-Richtung (Z = 32 µm). Beurteilen Sie jeder Kern in jedem Bildtiefe zu und benennen Sie die Tiefe, an der die Keimzelle im schärfsten Fokus als die Invasion Tiefe für diese bestimmten Zelle war.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric.">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3, 362-374 (2003).</li><li>Yarrow, J. C., Perlman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparison+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Wolf, 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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li><li>Sahai, E., Marshall, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differing+modes+of+tumour+cell+invasion+have+distinct+requirements+for+Rho/ROCK+signalling+and+extracellular+proteolysis.">Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.</a> Nat Cell Biol. 5, 711-719 (2003).</li><li>Kam, Y., Guess, C., Estrada, L., Weidow, B., Quaranta, V. <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+circular+invasion+assay+mimics+in+vivo+invasive+behavior+of+cancer+cell+lines+and+distinguishes+single-cell+motility+in+vitro.">A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.</a> BMC Cancer. 8, 198 (2008).</li><li>Staton, C. A., Reed, M. W., Brown, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90, 195-221 (2009).</li><li>Poujade, M., 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=Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound.">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci USA. 104, 15988-15993 (2007).</li><li>Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-induced+cancer+cell+fusion:+a+mechanism+of+breast+cancer+metastasis.">Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.</a> FASEB J. 29, 4036-4045 (2015).</li><li>McArdle, T. J., Ogle, B. M., Noubissi, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Inverted+Vertical+Invasion+Assay+to+Avoid+Manipulation+of+Rare+or+Sensitive+Cell+Types.">An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types.</a> J Cancer. 7, 2333-2340 (2016).</li><li>Noubissi, F. K., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Cell+Fusion:+Mechanisms+Slowly+Unravel.">Cancer Cell Fusion: Mechanisms Slowly Unravel.</a> Int J Mol Sci. 17, (2016).</li><li>Meyvantsson, I., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+models+in+microfluidic+systems.">Cell culture models in microfluidic systems.</a> Annu Rev Anal Chem. 1, 423-449 (2008).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods In Enzymol. 406, 625-643 (2006).</li><li>Yang, C., Tibbitt, M. W., Basta, L., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+memory+and+dosing+influence+stem+cell+fate.">Mechanical memory and dosing influence stem cell fate.</a> Nat Materials. 13, 645-652 (2014).</li></ol>

Die Autoren haben erklärt, dass keine konkurrierenden Interesse vorhanden ist.

Hier beschreiben wir eine einfache umgekehrte vertikale Invasion-Probe, die eignet sich für eine Vielzahl von Zelltypen, einschließlich der Zellen, die seltene und/oder ihrer Mikroumgebung abhängig sind. In diesem 3D Invasion-Assay Zellen bleiben in ihrer ursprünglichen Mikroumgebung und fallen unter das Kollagen-Gel mit einem Lockstoffgradient. Die Zellen dann migrieren und dringen in das Kollagen. In diesem Test werden die Zellen gefärbt und leicht in das Gel überwacht. Die Einfachheit und Reproduzierbarkeit des ...

Zelle Motilität ist ein normaler Entwicklungs- und physiologischen Prozess der Zellen. Allerdings sind Änderungen in der Struktur und die Fähigkeit der Zellen zur Migration und Invasion mit pathologischen Zuständen, einschließlich der entzündlichen Erkrankungen und Krebs Metastasen1,2,3verbunden. Metastasierung ist wohl der am wenigsten verstandenen Aspekt in der Krebstherapie. Um zu metastasieren, müssen Krebszellen zersetzen die extra-zellulären Matrix (ECM), erobern die lokalen Gewebe und Intravasate. Einmal im Umlauf werden die Krebszellen in den entfernten Standort verbreiten, Leber und sekundäre Tumore bilden. Daher die Fähigkeit der Zellen, die ECM zu vermindern, zu migrieren und zu erobern bezieht sich auf ihr metastatische Potential. Verschiedene Ansätze wurden entwickelt, um zu analysieren und die Migration und Invasion Funktionen von Zellen zu quantifizieren. Die am häufigsten verwendeten Ansätze umfassen die Wundheilung Assay, Time-Lapse-Mikroskopie und Boyden Kammer Assay. Die Wunde heilende Assay4 und Zeit verfallen Mikroskopie sind einfache und bequeme Assays, die ersten Einblick in die Zellwanderung geben würde. Jedoch sind beide 2D Assays, die nicht die 3D-Umgebung widerspiegeln, die wie Zellen in Vivoexistieren. Studien haben gezeigt, dass Zellen in einer 3D Umgebung im Vergleich zu einem 2D eine5,6unterschiedlich reagieren. Darüber hinaus sind heilende Wunde Assays schwer zu reproduzieren und zu quantitativen Ergebnissen7,8,9ergeben. Die Zelle Sperrzone Assay, die eine 3D Modifikation der Wundheilung Assay ist, ist ein mehr physiologisch relevanten Assay aber es ist nicht geeignet für niedrige Zahl wie z. B. Hybridzellen aus Zelle Fusion Veranstaltungen10,11 Zellen .
Boyden Kammer Assay ist ein 3-dimensionales Assay, der zelluläre Studien relevanten Mikroumgebungen vorsieht. Es erfordert jedoch Zellen aus ihrer ursprünglichen Mikroumgebung entfernt werden und in der oberen Kammer des Boyden-Testsystem zu migrieren und dringen nach unten in Richtung der Lockstoffgradient enthaltenden Medium hinterlegt werden. Diese 3D Methode eignet sich nicht bei der Analyse der Migration und Invasion Fähigkeit der Zellen, die anfällig für ihre Mikroumgebung und/oder in begrenzter Anzahl10,11,12. Ein neuer Ansatz zur Zelle Migration und Invasion zu analysieren ist der mikrofluidischen gradient Kammer Assay13. Obwohl geeignet und zuverlässig in der Migration und Invasion Studien, dieser Test ist teurer und für ein typisches Labor technisch schwierig sein könnte. Wir beschreiben hier einen einfache umgekehrte vertikale Invasion-Assay, der kompatibel mit vielen Zelltypen, einschließlich der Zellen empfindlich auf ihrer Mikroumgebung und/oder beschränkter Zahl ist. Dieser Test wurde von dem Protokoll vorgeschlagen von Hooper Et al. 14. in diesem Test die Zellen bleiben in ihrer ursprünglichen Mikroumgebung und ihrer Migration und Invasion wird überwacht, da sie nach oben in das Kollagen-Gel mit einem Lockstoffgradient11bewegen. Dieser Assay ist reproduzierbar und optimiert und validiert in der Kulturschale von 35 mm. Es könnte verschiedene Assay-Bedingungen, einschließlich der verschiedenen Zellgrößen Kultur, verschiedenen Matrizen oder Stärke der Adhäsion und verschiedenen Chemoattractants angepasst werden. Dieser Test könnte als in-vitro- Plattform für die erste Sichtung in der Arzneimittelforschung dienen.

<table><tbody><tr><td>MatTek P35G-1.5-10C</td><td>MatTek Corporation, Ashland, MA</td><td>P35G-1.5-10-C</td><td>mattek glass bottom dishes</td></tr><tr><td>Rat tail collagen I</td><td>Corning, Corning, NY, USA</td><td>354236</td><td>Collagen gel</td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>H1758</td><td>HCl</td></tr><tr><td>Phosphate buffered saline</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>10010023</td><td>PBS</td></tr><tr><td>10X Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>D2429</td><td>10X DMEM</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich, St. Louis, MO 63178 USA</td><td>S5761</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Confocal Fluorescent microscope</td><td>Olympus America, Center Valley, PA, USA</td><td>Olympus IX81</td><td>Confocal&amp;nbsp; microscope</td></tr><tr><td>Defined Fetal Bovine Serum</td><td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td><td>SH30070.03</td><td>FBS</td></tr><tr><td>Ti:Sapphire multi-photon laser scanning microscope</td><td>Prarie Technologies, Sioux Falls, SD, USA</td><td>40x NA 0.8 objective</td><td>MPLSM</td></tr><tr><td>Imaris software</td><td>Bitplane Inc. Zurich, CH</td><td>Imaris 7.5.2</td><td>Imaris software</td></tr><tr><td>DAPI</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>D9542</td><td>DAPI</td></tr><tr><td>Paraformaldehyde</td><td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td><td>50980487</td><td>PFA</td></tr><tr><td>&amp;alpha;-minimum essential medium</td><td>Sigma-Aldrich, St. Louis, MO 63178, USA</td><td>M0894</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC; Manassas, VA, USA</td><td>HBT-22</td><td></td></tr><tr><td>MSC</td><td>Trivedi and Hematti, 2007</td><td></td><td></td></tr><tr><td>20X lens UPLAPO</td><td>Olympus America, Center Valley, PA, USA</td><td>36-064</td><td>Olympus UPLSAPO 20X Objective</td></tr></tbody></table>

moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol

Obwohl 3D Invasion Assays entwickelt worden sind, bleibt die Herausforderung, um Zellen zu untersuchen, ohne die Integrität ihrer Mikroumgebung. Traditionelle 3D Tests wie der Boyden Kammer erfordern, dass Zellen verdrängt vom ursprünglichen Speicherort, Kultur und zog in eine neue Umgebung. Nicht nur stört dies die zelluläre Prozesse, die untrennbar mit der Mikroumgebung sind, aber es führt oft zu einem Verlust von Zellen. Diese Probleme sind eine besondere Herausforderung im Umgang mit Zellen, die entweder selten oder extrem empfindlich auf ihrer Mikroumgebung. Hier beschreiben wir die Entwicklung eines 3D Invasion-Assays, die beide Probleme vermeidet. In diesem Assay Zellen sind in einem kleinen gut beschichtet und eine ECM-Matrix mit einer Lockstoffgradient auf die Zellen gelegt. Dies erfordert keine Verschiebung der Zelle und ermöglicht es die Zellen, nach oben in die Matrix zu erobern. In diesem Test kann innerhalb der Kollagen-Gel Zellinvasion sowie Zellmorphologie beurteilt werden. Mit Hilfe dieser Assay, kennzeichnen wir die invasive Kapazität der seltene und empfindliche Zellen; die Hybridzellen infolge Fusion von Brustkrebs-Zellen MCF7 und mesenchymalen/multipotenten Zellen Stammzellen/Stroma (MSCs).

Wir haben eine 35-mm-Kulturschale mit einem Glas unten und Ratte Schweif Kollagen verwendet ich entwickeln und optimieren einer in-vitro- 3D Invasion assay Protokoll. Mit diesem Test, wir haben gezeigt, dass Brust Krebszellen MCF7 und MSC Zellen könnte dringen in das Kollagen-Gel in einer mittleren Entfernung von 0,04 ± 1,8 µm und 41,3 ± 76,0 µm bzw. nach 48 h bei IGF1 (18.6 ng/mL) diente als Lockstoffgradient (Abbildung 2). Noch wichtiger ist, ...

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Dieses Protokoll beschreibt einen umgekehrte vertikale Invasion-Assay, der verwendet werden könnten, um die Migration und Invasion Funktionen von Zellen in einer dreidimensionalen Umgebung unter Beibehaltung der Zelle Mikroumgebung zu quantifizieren. Dieser Test eignet sich für seltene und/oder empfindliche Zellen.

Nach oben bewegen: Eine einfache und Flexible In-vitro- dreidimensionale Invasion Assay Protokoll

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

moving-upwards-simple-flexible-vitro-three-dimensional-invasion-assay

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

6.0K Aufrufe.  University of Minnesota - Twin Cities. Dieses Protokoll beschreibt einen umgekehrte vertikale Invasion-Assay, der verwendet werden könnten, um die Migration und Invasion Funktionen von Zellen in einer dreidimensionalen Umgebung unter Beibehaltung der Zelle Mikroumgebung zu quantifizieren. Dieser Test eignet sich für seltene und/oder empfindliche Zellen.

Serie: Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

Spheroid Assay zur TGF-β-induzierte Invasion Measure

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

Forschung

Medizin

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 ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Voll menschliche Tumor-basierte Matrix im dreidimensionalen Spheroid-Invasions-Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

Krebsforschung

An In vitro FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to both secrete proteases that break down the basement membrane as well as migrate in order to be invasive. BD BioCoat Tumor Invasion System provides cells with conditions that allow assessment of their invasive property in vitro1,2. It consists of a BD Falcon FluoroBlok 24-Multiwell Insert Plate with an 8.0 micron pore size PET membrane that has been uniformly coated with BD Matrigel Matrix. This uniform layer of BD Matrigel Matrix serves as a reconstituted basement membrane in vitro providing a true barrier to non-invasive cells while presenting an appropriate protein structure to study invasion. The coating process occludes the pores of the membrane, blocking non-invasive cells from migrating through the membrane. In contrast, invasive cells are able to detach themselves from and migrate through the coated membrane. Quantitation of cell invasion can be achieved by either pre- or post-cell invasion labeling with a fluorescent dye such as DiIC12(3) or calcein AM, respectively, and measuring the fluorescence of invading cells. Since the BD FluoroBlok membrane effectively blocks the passage of light from 490-700 nm at &gt;99% efficiency, fluorescently-labeled cells that have not invaded are not detected by a bottom-reading fluorescence plate reader. However, cells that have invaded to the underside of the membrane are no longer shielded from the light source and are detected with the respective plate reader. This video demonstrates an endpoint cell invasion assay, using calcein AM to detect invaded cells.

An In vitro FluoroBlok Tumor Invasion Assay

This video demonstrates how to measure cell invasion through cell culture inserts using a fluorescence-based methodology.

Ein In-vitro- FluoroBlok Tumor Invasion Assay

The hallmark of metastatic cells is their ability to invade through the basement membrane and migrate to other parts of the body. Cells must be able to ...

Biologie

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

In-vitro-Tests zur Bewertung der Migration, Invasion und Proliferation von verewigt menschlichen Ersttrimester-Trophoblast Zelllinien

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Entwicklungsbiologie

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Eine modifizierte<em&gt; In-vitro-</em&gt; Invasion Assay, um die mögliche Rolle von Hormonen, Cytokinen und / oder Wachstumsfaktoren bei der Vermittlung der Invasion von Krebszellen fest,

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

3D-Modelle in der Krebsbiologie: Invasionsdynamik in Heterospheroiden

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 &#181;m intervals, covering a depth of 90 &#181;m in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

Autor im Rampenlicht: Entschlüsselung der Wechselwirkung zwischen Krebs und Immunzellen während des Fortschreitens von Kopf- und Halskrebs

Invasion is a major biological phenomenon in the development and progression of cancer. This process is influenced by non-neoplastic cells and components of the tumor microenvironment. The purpose of this study is to describe an alternative method for analyzing tumor cell invasion in vitro using three-dimensional (3D) cultures.

Alternative Strategie zur Analyse der in vitro Zellinvasion von 3D-Kulturen

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Dreidimensionale (3D) Tumor Spheroid Invasionsassay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Nach oben bewegen: Eine einfache und Flexible In-vitro- dreidimensionale Invasion Assay Protokoll

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Spheroid Assay zur TGF-β-induzierte Invasion Measure

Modeling and Imaging 3-Dimensional Collective Zellinvasion

Voll menschliche Tumor-basierte Matrix im dreidimensionalen Spheroid-Invasions-Assay

Ein In-vitro- FluoroBlok Tumor Invasion Assay

In-vitro-Tests zur Bewertung der Migration, Invasion und Proliferation von verewigt menschlichen Ersttrimester-Trophoblast Zelllinien

Eine modifizierte

Alternative Strategie zur Analyse der in vitro Zellinvasion von 3D-Kulturen

Alternative Strategie zur Analyse der in vitro Zellinvasion von 3D-Kulturen

Alternative Strategie zur Analyse der in vitro Zellinvasion von 3D-Kulturen

Dreidimensionale (3D) Tumor Spheroid Invasionsassay