Die Finanzierung dieser Forschung ist von Cancer Research UK.

1. Retrovirale Markierung von Zellen mit fluoreszierenden Proteinen <ol><li> Platte retroviralen Verpackung Zellen (zB Phoenix) bei 0,25 x 10 6 Zellen pro Vertiefung einer 6-well Gericht in 10% fötalem Rinderserum (FBS) / DMEM. </li><li> Transfektion von Zellen mit retroviralen DNA 48 Stunden später Zellen mit Effectene nach den Anweisungen des Herstellers. </li><li> Spülen Sie die Taschen zweimal mit Medium 24 Stunden später, dann fügen Sie 1,5 ml 10% FBS / DMEM pro Loch. </li><li> Sammeln verpackt Virus in Zellkulturmedium 48 Stunden später durch Pipettieren und die Übertragung auf 2 mL Mikrozentrifugenröhrchen. </li><li> Zentrifugation bei 1600 rpm für 5 min auf Pellet alle Zellen. </li><li> Entfernen Sie den Überstand in ein sauberes Röhrchen und lagern bei -80 ° C. </li><li> Cells um retroviral transduziert werden werden mit 1,5 x 10 5 Zellen pro Loch einer 6-Well Schale überzogen und in einem befeuchteten Inkubator bei 37 ° C über Nacht. </li><li> Am nächsten Tag, entfernen Sie die Medien aus den Zellen und add 1 ml Virus-Aktie mit 4 ul Polybren (2 mg / ml) in jede Vertiefung ergänzt. Ersetzen Platten im Brutschrank. </li><li> Nach Inkubation für 5-6 Stunden, 2 ml 10% FBS / DMEM in jede Vertiefung und Platten sind in 37 ° C ersetzt inbubator über Nacht. </li><li> Am nächsten Tag, ersetzen Sie die Medien und damit Zellen 24 Stunden vor dem Zusatz von geeigneten selektiven Medien. </li><li> Wenn konfluent (das hängt von der Wachstumsrate der Zellen, und die Effizienz der retroviralen Transduktion, sondern nimmt in der Regel 4 bis 14 Tage), trypsinize Zellen und sort für die Fluoreszenz unter Verwendung von nicht-transduzierten Zellen als Referenz, zu sammeln und Pool-Zellen mit Fluoreszenz größer als nicht-transduzierten Zellen und frieren Aliquots bei -80 ° C für eine spätere Verwendung. </li></ol> 2. Inverse Matrigel Invasion Assays <ol><li> Langsam tauen ein Aliquot von kompletten Matrigel (dh mit Wachstumsfaktoren) auf Eis (Abbildung 1A). </li><li> Nach dem Auftauen, verdünnter Matrigel 1:1 in eiskaltem PBS (zusammen mit allen anderen zusätzlichenBehandlungen mit 2x-Konzentration in der PBS vor der Verdünnung; Abbildung 1B). Um es einfacher zu Matrigel vor der Polymerisation Griff, alle Kunststoffprodukte (zB Tipps, Rohre, etc.) sollte eiskalt sein. </li><li> Legen Sie so viele 8 Mikron Porengröße 6,5 mm Durchmesser unbeschichteten Transwells als in den Vertiefungen einer 24-Well-Gewebekulturplatten erforderlich, dann vorsichtig Pipette 100 ul der verdünnten Matrigel in die Vertiefungen und Urlaub für ~ 30 min bei 37 ° C inkubieren sich zu verfestigen (Abbildung 1C). </li><li> Während dieser Zeit bereiten eine oder mehrere fluoreszenzmarkierten Zellsuspensionen auf zwischen 1 bis 4 x 10 5 Zellen pro ml, je nach Zelllinie, aus jedem vorbehandelten Zustand (e. g. SiRNA, medikamentöse Behandlung) in ihr normales Wachstum Medium (Abbildung 1D). </li><li> Wenn die Matrigel erstarrt ist, kehren die Transwells und Pipette 100 ul der Zellsuspension auf der nach oben weisenden Unterseite des Filters (Abbildung 1E). </li><li> Sorgfältig decken die Transwells mit der Basis eines 24-Well-GewebeKultur Teller, den Kontakt mit jedem Tropfen der Zellsuspension (Abbildung 1F). </li><li> Die Platte in den invertierten Zustand für 4 Stunden für Zelladhäsion (Abbildung 1G) zu ermöglichen. </li><li> Nach dieser Zeit werden die Platten right-side-up und waschen jedes Transwell durch sequentielle Eintauchen in 2 x 1 ml serumfreiem Medium (Abbildung 2A). </li><li> Verlassen Sie die Transwells in einem dritten auch mit Drogen oder Behandlungen erforderlich (Abbildung 2B). </li><li> Gently Pipette 100 ul 10% FBS / DMEM plus Lockstoff (e. g. EGF bei 25 ng / ml) in die Transwell auf der Oberseite des erstarrten Matrigel / PBS-Mischung, ersetzen Sie den Deckel ab und inkubieren für 3-5 Tage bei 37 ° C mit 5% CO 2 (Abbildung 2C). </li></ol> 3. Färbung und Visualisierung <ol><li> Zellen, die fluoreszierende Proteine ​​können die folgenden Schritte vor der konfokalen Mikroskopie überspringen </li><li> Um das Bild nicht fluoreszierenden Zellen eindringen Matrigel, Ort Transwells in frischen 24-Well-Gerichte und Pipette 1 ml 4 uMCalcein AM Färbelösung übereinander Matrigel-Stecker, so dass sie übergreifen den Seiten und Flecken von oben und unten. Calcein AM (Acetoxymethylester von Calcein) ist ein Live-Cell-Farbstoff, färbt die ganze Zellen grün und erfordert keine Fixierung. </li><li> Inkubieren 1 Stunde bei 37 ° C in 5% CO 2 befeuchteten Atmosphäre, an welcher Stelle Zellen sind voll gefärbt und bereit, durch konfokale Mikroskopie abgebildet werden. </li><li> Alternativ können nicht-fluoreszierenden Zellen eindringen Matrigel fixiert und gefärbt werden, wie unten in 3,5 bis 3,9 beschrieben. </li><li> Übertragen Sie jede Transwell einen frischen 24-Well-Platte. Overlay 1 ml 4% para-formaldehyde/0.2% Triton-X 100. </li><li> Inkubieren bei Raumtemperatur für 0,5 Stunden. </li><li> Entfernen Fixiermittel und 3 x waschen mit 1 ml PBS. </li><li> Entfernen zytoplasmatische RNA mit 30 min RNase-Behandlung mit 100 pg / ml RNase. Wash 2 mal mit PBS. </li><li> Für die Visualisierung hinzu 0,01 mg / ml Propidiumiodid (PI) in PBS verdünnt und lassen bei Raumtemperatur in den dark für 0,5 Stunden. 3 x waschen mit PBS. In diesem Stadium, gebeizt PI Transwell bei Raumtemperatur im Dunkeln für mindestens 1 Monat aufbewahrt werden. </li><li> Präzise bildgebenden Verfahren durch konfokale Mikroskopie sind abhängig von verfügbaren Ressourcen. Mit Hilfe eines invertierten konfokalen Mikroskop, Ort Transwell mit einer kleinen Menge an Rest-PBS auf große Deckglas (sicherstellen, dass keine Luftblasen vorhanden sind) über nicht-Immersions-20 X objektiv und erfassen optische Schnitte alle 10-15 um von der Unterseite der Matrigel-Plug. Individuelle optische Schnitte können verwendet werden, um das Ausmaß der Invasion (Abbildung 3A) zu quantifizieren oder in 3-dimensionale Objekte mittels geeigneter Software wie Volocity (Abbildung 3B) zu bauen. </li></ol> 4. Repräsentative Ergebnisse Ein Beispiel für eine Z-Serie von optischen Schnitten ist in Abbildung 3A gezeigt. In diesem Fall wurden die Zellen mit Calcein AM angefärbt und die Anzahl der Zellen eindringen aus dem Filter zu sehen, mit Abstand zu verringern. Die Quantifizierung der invasion kann durch die Analyse des Verhältnisses von Calcein AM positive Pixel, negative Punkte bei jedem Intervall, oder über die Fixierung / Färbemethode oben beschriebene Zählen PI positiven Kerne an jeder Position möglich. Ein Vorteil von Calcein AM Färbung ist, dass 3-dimensionale Rekonstruktion der Zellinvasion zusammengebaut mit einer Software wie Volocity, was eine visuelle Darstellung der Modus der Invasion (Abbildung 3B) werden. Wenn Zellen durch die Expression von fluoreszierenden Proteinen markiert sind, dann die Positionen der einzelnen Farben Zelle kann in 3-dimensionale Rekonstruktion visualisiert, entweder von der Seite (Abbildung 3C) oder durch Schnitte durch die Rekonstruktion (Abbildung 3D) angesehen. <img alt="Abbildung 1" src="/files/ftp_upload/3525/3525fig1.jpg" /> Abbildung 1. Schematische Darstellung der Schritte beim Aufbau einer inversen Invasion Assay beteiligt. A) Matrigel ECM auf Eis aufgetaut. B) Matrigel ist 1:1 mit PBS, das jede medikamentöse Behandlung mit der doppelten Endkonzentration verdünnt. C)<html> Transwell-Einsätze sind in Mikrotiterplatten gegeben und Matrigel in jede pipettiert. D) Zellsuspensionen hergestellt in der gewünschten Konzentration. E) Nach dem Matrigel gesetzt hat, die Platte umgedreht ist und entfernt werden, werden die Zellen auf die Unterseite Filter der Transwell-Einsätze vernickelt. F) In der invertierten Position ist das mehrlagig Platte vorsichtig über Transwell-Einsätze platziert, in Kontakt mit der Zellsuspension. G) Zellen werden dürfen, um den Filter für 4 Stunden einzuhalten. <img alt="Abbildung 2" src="/files/ftp_upload/3525/3525fig2.jpg" /> Abbildung 2. Fortführung der Schaltplan für die inverse Invasion Assay. A) Wenn Zellen eingehalten haben, tauchen jede Transwell in serum-freien Medien zweimal zu verlieren Zellen zu entfernen. B) Ort gewaschen Transwell in eine endgültige und mit Medien sowie Behandlungen nach Bedarf. C) Medien mit Lockstoff (zB 10% fötalem Rinderserum) mit Behandlungen nach Bedarf wird vorsichtig auf Matrigel geschichtet. ENT &quot;&gt; <img alt="Abbildung 3" src="/files/ftp_upload/3525/3525fig3.jpg" /> Abbildung 3. Repräsentative Bilder der Ergebnisse der inversen Invasion Assay. A) Optische Schnitte von Zellen mit Calcein gefärbten AM eindringenden Matrigel, aufgenommen bei 15 mu m Abständen durch konfokale Mikroskopie. B) Rekonstruktion eines 3-dimensionalen Rekonstruktion der Zelle Invasion aus einem Stapel von konfokalen Z-Serie Bilder von der Seite angesehen. Wiedergabe aus Lit. 10. C) Dreidimensionale Rekonstruktion des GFP und RFP-markierter Zellen eindringen Matrigel von der Seite betrachtet. D) Optischer Schnitt durch die 3-dimensionale Rekonstruktion der GFP und RFP-markierter Zellen. Wiedergabe aus Lit. 10.

<ol>
	<li>Olson, M. 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=The+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., 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=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., 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=The+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</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. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>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=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

Die Autoren haben nichts zu offenbaren.

Matrigel Invasion Assays wurden traditionell mit Zellen auf eine Schicht aus extrazellulären Matrix-Proteins mit Lockstoff-induzierten Motilität in Richtung und durch einen Filter am unteren Rand platziert gesetzt. Invasivität wurde als Funktion der, wie viele Zellen könnte auf der Unterseite des Filters gezählt werden gewertet. Obwohl praktisch gibt es kaum einen Unterschied mit dem &quot;inverse&quot; Invasion Assay beschrieben, gibt es wesentlich mehr Informationen über den Prozess der Invasion, die durch die V...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th></tr><tr><td> DMEM (Dulbecco modifiziertem Eagle-Medium) </td><td> GIBCO </td><td> 21969 </td></tr><tr><td> Fetal Bovine Serum </td><td> PAA </td><td> A15-101 </td></tr><tr><td> Penicillin Streptomycin </td><td> GIBCO </td><td> 15140 </td></tr><tr><td> 200 mM L-Glutamin (100x) </td><td> GIBCO </td><td> 25050-032 </td></tr><tr><td> Puromycin </td><td> Sigma-Aldrich </td><td> P8833 </td></tr><tr><td> 0,05% Trypsin EDTA </td><td> GIBCO </td><td> 25300 </td></tr><tr><td> Polybrene </td><td> Sigma-Aldrich </td><td> AL-118 </td></tr><tr><td> Lipofectamine 2000 Reagent </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> 6.5mm Transwells, 8,0 um Porengröße </td><td> Corning </td><td> 3422 </td></tr><tr><td> Komplette Matrigel </td><td> BD Biosciences </td><td> 354234 </td></tr> &lt;tr&gt; <td> Calcein AM </td><td> Invitrogen </td><td> C1430 </td></tr><tr><td> RNase </td><td> Qiagen </td><td> 19101 </td></tr><tr><td> Propidiumiodid </td><td> Sigma-Aldrich </td><td> P4864 </td></tr><tr><td> Konfokale microcope </td><td> Leica </td><td> SP2MP </td></tr></tbody></table>

modeling and imaging 3-dimensional collective cell invasion

Ein charakteristisches Merkmal von Krebs Malignität Invasion und Metastasierung 1. In einigen Krebsarten (e. g. Gliom 2), lokale Invasion in das umgebende gesunde Gewebe ist die Ursache von Krankheit und Tod. Für andere Krebsarten (e. g. Brust, Lunge, etc.), Wird der Prozess der Metastasierung ist, in die Tumorzellen von einem Primärtumor Masse zu bewegen, zu kolonisieren distalen Seiten und letztendlich zum Organversagen beitragen, dass führt schließlich zu Morbidität und Sterblichkeit 3. Es wurde geschätzt, dass der Invasion und Metastasierung verantwortlich für 90% der Krebstodesfälle 4 sind. Als Ergebnis gab es reges Interesse bei der Identifizierung der molekularen Prozesse und kritische Protein Mediatoren der Invasion und Metastasierung für die Zwecke der Verbesserung von Diagnose und Behandlung 5 gewesen. Eine Herausforderung für Krebsforscher ist Invasionsassays hinreichend ähneln den in-vivo-Situation zu entwickelngenaue Krankheit Modellierung 6 zu ermöglichen. Zweidimensionale Zellmotilität Assays sind nur informativ über einen Aspekt der Invasion und berücksichtigen nicht die extrazelluläre Matrix (ECM)-Protein Umbau, die auch ein kritisches Element zu nehmen. In jüngster Zeit hat die Forschung unser Verständnis von Tumorzellinvasion verfeinert und gezeigt, dass einzelne Zellen kann durch längliche oder runde Modi 7 zu bewegen. Darüber hinaus hat es eine größere Wertschätzung des Beitrags der kollektiven Invasion in die Zellen eindringen in Stränge, Platten und Clustern, insbesondere in hoch differenzierten Tumoren, die epithelialen Eigenschaften beizubehalten, um die Ausbreitung von Krebs 8 wurden. Wir präsentieren eine raffinierte Methode 9 für die Prüfung der Beiträge der Kandidaten-Proteine, um kollektive Invasion 10. Insbesondere durch technische separate Pools von Zellen auf verschiedene fluoreszierende Proteine ​​exprimieren, ist es möglich, molekular sezieren die Aktivitäten und Proteins in führenden Zellen im Vergleich zu denen in folgenden Zellen erforderlich erforderlich. Die Verwendung von RNAi stellt die molekulare Werkzeug, um experimentell zu zerlegen die Prozesse in einzelne Zelle Invasion sowie in verschiedenen Positionen der kollektiven Invasion beteiligt. In diesem Verfahren, Mischungen von fluoreszenzmarkierten Zellen auf dem Boden eines Transwell einfügen zuvor mit Matrigel ECM-Protein gefüllt sind vernickelt, dann erlaubt, &quot;nach oben&quot; durch den Filter und in den Matrigel Invasion. Rekonstruktion der z-Serie Bildstapeln durch konfokale Bildgebung erhalten, in dreidimensionale Darstellungen ermöglicht Visualisierung von kollektiv eindringenden Stränge und Analyse der Darstellung von fluoreszenzmarkierten Zellen in führenden gegenüber folgenden Positionen.

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

Models of Tumorzellinvasion in dreidimensionale extrazelluläre Matrix besser widerspiegeln<em&gt; In vivo</em&gt; Situation als zweidimensionale Beweglichkeit Assays. Mit Matrix-Invasion Assays mit konfokale Bildgebung von Fluoreszenz-markierten Zellen, detaillierte Informationen über die Invasion Modi und die einzelnen Beiträge zu den führenden im Vergleich zu folgenden Zellen kombiniert erreicht werden kann.

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 ...

modeling-and-imaging-3-dimensional-collective-cell-invasion

Research

JoVE Journal

Medicine

16.6K Aufrufe.  University of Strathclyde. Models of Tumorzellinvasion in dreidimensionale extrazelluläre Matrix besser widerspiegeln In vivo Situation als zweidimensionale Beweglichkeit Assays. Mit Matrix-Invasion Assays mit konfokale Bildgebung von Fluoreszenz-markierten Zellen, detaillierte Informationen über die Invasion Modi und die einzelnen Beiträge zu den führenden im Vergleich zu folgenden Zellen kombiniert erreicht werden kann.

Serie: Modeling and Imaging 3-Dimensional Collective Cell Invasion

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-Photonen-Imaging der Tumorzellinvasion in einem orthotopen Mausmodell des oralen Plattenepithelkarzinoms

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Forschung

Medizin

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.

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

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 ...

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

3-Dimensional Resin Casting und Imaging of Mouse Pfortader oder intrahepatische Gallengang-System

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

Molecular Imaging Transplantierte Muscle Progenitor Zielzellen

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Generation und 3-Dimensional Quantifizierung der arterielle Läsionen in Mäuse mit optischen Projektionstomographie

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Molecular Profiling von der invasiven Tumor-Microenvironment in einem 3-Dimensional Model of Colorectal Cancer Cells und<em&gt; Ex-vivo-</em&gt; Fibroblasten

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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 ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Modellierung Spontane metastasiertem Nierenzellkarzinom (mRCC) in Mäusen nach Nephrektomie

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolierung und Immortalisierung von Patienten stammende Zelllinien von Muskelbiopsie for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Eine Krebszelle Spheroid Assay zur Invasion in einem 3D-Rahmen Beurteilen

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...