Finanzierungsquellen dieser Forschung: ein gemeinsamer Fonds von der Universität Tel Aviv Technologietransfer Firma, RAMOT und dem Sheba Medical Center-(M. CA und SI.), ICRF - Israeli Cancer Research Foundation (M. CA.) Und Israel Science Foundation ( SI).

1. Cell Culture Vorbereitung MDA-MB-231-Zellen wurden von ATCC (American Type Culture Collection) erworben und in flüssigem Stickstoff gelagert. <ol><li> Seed 10 6 MDA-MB-231 Zellen in 92 mm Durchmesser Petrischale in 10 ml Vollmedium mit Dulbeco Modified Eagle Medium (DMEM), 10% Pferdeserum, 1% L-Glutamin und 1%-Penstrep Amphotericin B. Zellen zulassen bis zu etwa 80-100% Konfluenz vermehren. </li><li> Entfernen Kulturmedium aus Schale und verwerfen. </li><li> Waschen der Zellschicht kurz mit 0,25% (w / v) Trypsin-EDTA-Lösung, um alle Spuren von Serum zu entfernen. </li><li> 2,0 ml Trypsin-EDTA-Lösung, austeilen und beobachten Zellen durch inverse Mikroskop bis Zellschicht verteilt ist (in der Regel innerhalb von 5 bis 15 min). </li><li> In 18 ml komplettem Wachstumsmedium und sanft saugen die Zellen mit einer Pipette. Transfer zu einem Rohr. </li><li> Zentrifuge die Zellsuspension bei 1.200 Umdrehungen pro Minute. </li><li> Re-suspend das Zellpellet in 24 ml culture Medium. </li><li> 2 ml Zellsuspension in 35 mm Petrischalen mit Glasboden (ca. 25% Konfluenz) und Platz im Inkubator (5% CO 2, 37 ° C). </li><li> Lösungen: <ol><li> Komplette Medium für Zellen Zellproliferation: DMEM mit 10% FBS, 1% Antibiotika (100 Einheiten / ml Penicillin G, 100 ug / ml Streptomycin, Pen-Strep-Ampho Lösung) und 2 mM L-Glutamin. </li><li> Trypsin-EDTA-Lösung, die 0,25% Trypsin-EDTA. </li></ol></li><li> Gerichte: 92 mm Durchmesser Petrischalen. 35 mm Durchmesser Poly-D-Lysin beschichteten Glasboden Kulturschalen. </li></ol> 2. Vorbereitung der Zellen für die Live-konfokale Imaging <ol><li> Seed 2 x 10 5 MDA-MB-231-Zellen in Glasbodenkulturschalen in 2 ml Vollmedium, wie in Kapitel 1 erwähnt. Wenn Zellkultur Zusammenfluss von 60-70% (ca. 3-4 x 10 5 Zellen pro Schale) erreicht, mit Transfektion gehen. </li><li> Transfektion der Zellen mit zwei Plasmiden TonkanäleING die Fusionsproteine ​​γ-Tubulin-GFP (für Fluoreszenzdetektion Zentrosome) und Histone-RED (H2b-RED, für Fluoreszenzdetektion der Chromosomen) mit dem liposomalen Transfektionsreagenz Jet-PI nach der Herstellung Protokoll. Kurz gesagt, mischen 2 ug von jedem Plasmid in einem Rohr mit 100 ul NaCl (150 mM). Mischen Sie das Transfektionsreagenz (100 ul) mit 100 ul NaCl (150 mM) in einem zweiten Rohr, und Inkubation 5 min bei Raumtemperatur (RT). Dann kombinieren Sie die beiden Lösungen, Mix (mit milden Vortex) und Spin-down. Inkubieren für 30 min bei RT. </li><li> Während der Inkubation der Transfektionsgemisch, waschen Sie die Zellen einmal mit PBS und ersetzen Sie die Zell-Medium mit 2 ml warmem DMEM ohne Zusätze (37 ° C). </li><li> Sanft fügen Sie die Transfektion Mischung zu den Zellen in DMEM und dann wieder die Zellen in den Inkubator (37 ° C, 5% CO 2) für 8 Stunden. </li><li> Nach 8 h Inkubation, ersetzen Sie das DMEM mit 2 ml Vollmedium und Inkubation der Zellen im Brutschrank für 24hr. </li><li> 24 Stunden nach der Transfektion, ersetzen Sie das Medium der Zellen mit 2 ml Vollmedium mit 20 uM PJ-34. </li><li> Inkubieren Sie die Zellen für weitere 18 Stunden (37 ° C, 5% CO 2). </li><li> Betrifft die Zellen konfokale Bildgebung für mindestens 16 Stunden zu leben in der Bildgebung Kammer halten die Zellen bei 5% CO 2 und 37 ° C. </li><li> Parallel dazu untersuchen Transfektion Wirksamkeit 36 ​​Stunden nach Transfektion unter Verwendung von Fluoreszenzmikroskopie wie folgt: <ol><li> Seed 2 x 10 5 MDA-MB-231-Zellen in 6-Well-Platte mit 1 Deckglas pro Vertiefung in 2 ml Vollmedium. </li><li> Transfektion die Zellen wie in den Abschnitten 2.2-2.5 erwähnt. </li><li> 36 Stunden nach Transfektion beheben die transfizierten Zellen auf einem Deckglas durch Inkubation montiert in kaltem Methanol: Aceton (1:1)-Lösung, 7 min, -20 ° C </li><li> Saugen Sie die Fixierung Lösung und lassen das Deckglas mit den montierten Zellen in einer chemischen Haube trocknen. </li><li> Bewerben ProLong Gold-antifade Reagenz mit DAPI und lassen the Deckglas im Dunkeln für 6 Stunden trocknen. </li><li> Untersuchen Sie den Schieberegler unter dem Fluoreszenzmikroskop und berechnen den Anteil der transfizierten Zellen (rote und grüne Signale) von der gesamten Population von Zellen (DNA-Färbung durch DAPI). Die gewünschte Transfektion Prozentsatz etwa 20-40% bei 100-200 Zellen gezählt. </li></ol></li></ol> 3. Technische Parameter der Live-konfokale Imaging Scanner-Einstellungen <ol><li> ScanMode xyzt; Pinhole [luftig] 1,00; Zoom 3,5; Auflösung 8 Bit; Laser DPSS 561 nm; Argon, sichtbaren Laser 488 nm; Laser He / Ne 633 nm sichtbar; Ziel HCX PL APO CS 63X 1.40 OIL UV; Numerische Apertur 1,4; Scan-Geschwindigkeit 700 Hz; Brechungsindex 1,52. </li><li> Bild 3-D Präsentation wurden von Imaris Imaging-Software 7.0 vorbereitet. </li></ol> 4. Konfokalen Imaging der Mitose in fixierten Zellen <ol><li> Seed 2 x 10 5 MDA-MB-231 Brustkrebszellen (ATCC), normale embryonale Maus-Fibroblasten (MEF), oder PARP1 defizienten MEF (PARP - / -, hergestellt von Dr. Francoise Dantzer) auf Deckgläser in 6-Well-Platte in 2 ml Vollmedium. Die Deckgläser wurden mit 96% Ethanol gewaschen, gefolgt von Waschen mit sterilem DD Wasser gewaschen, 2 Stunden, und in jeder Vertiefung der 6-Well-Platte. </li><li> In PJ-34 (10-30 pM) auf das Medium und Inkubation der Zellen für den gewünschten Zeitraum (in der Regel bis zu 96 Stunden). </li><li> Waschen der Deckgläser einmal mit PBS (phosphate buffered saline), und fixieren die Zellen unter Verwendung von Inkubation in eiskaltem Methanol: Aceton (1:1)-Lösung, 7 min, 20 ° C </li><li> Saugen Sie die Fixierung Lösung und lassen Sie die Deckgläser in chemischen Haube trocknen lassen (in diesem Stadium können die Deckgläser in -20 ° C für mehrere Wochen aufbewahrt werden). </li><li> Waschen der Deckgläser einmal mit PBST (PBS mit 0,1% Tween-20 ergänzt), um die Zellmembranen durchlässig und blockieren die Zellen mit 10% NDS (Normale Eselserum) in PBST (&quot;Blocking Solution&quot;) für 1 h bei RT. </li><li> Inkubation der permeabilisierten fixierten Zellen mit primären Antibodies für 2 Stunden bei RT (für Spindeln und Zentrosome Färbung). Anti-α Tubulin (1:250 Verdünnung) und anti-γ Tubulin (1:200 Verdünnung): Die Antikörper werden in der Blocking-Lösung wie folgen verdünnt. Primäre Antikörper wie folgt angewendet: Anwendung 100 ul (in einem Tropfen) einer Mischung der Antikörper in Blocking-Lösung für jedes Deckglas auf 6-Well-Platte Abdeckung (der Deckel auf dem Kopf steht). Sanft legte das Deckglas auf dem Antikörper Tropfen, mit Blick auf die ausgesäten Zellen Drop. Inkubieren Deckgläser zugewandten Antikörper für 2 Stunden bei Raumtemperatur. </li><li> Legen Sie die Deckgläschen wieder in den Brunnen und die Zellen 3-mal mit PBST. Dann nutzen Sie die gleiche Prozedur in 4.6 für die Markierung von Zellen auf den Deckgläsern mit den fluoreszierenden Sekundärantikörper beschrieben. Inkubieren der Zellen auf den Deckgläschen mit den sekundären Antikörpern für 1 Stunde, RT im Dunkeln. Die Antikörper werden in der Blocking-Lösung wie folgt verdünnt: und Alexa Fluor 568 (1:1000 Verdünnung Alexa Fluor 488 (grün 1:1000 Verdünnung);rot). </li><li> Montieren Sie die Deckgläser mit ProLong Gold-antifade Reagenz mit DAPI (für die Chromosomen-Färbung) und Inkubation über Nacht bei RT in Dunkelheit, um zu trocknen. </li><li> Untersuchen Sie die Deckgläser durch konfokale Mikroskopie. </li></ol> 5. Zellviabilitätsmessungen von ATP Produktion gemessen ATP-Produktion wird durch eine leuchtende ATP Detektionsassay Kit gemessen. <ol><li> Seed die Zellen in 96-Well-Platte etwa 20.000 Zellen in 800 ul Medium in jeder Vertiefung. Drei leere Vertiefungen sollten für die Bestimmung der Hintergrundlumineszenz des Mediums verwendet werden. </li><li> Bereiten ATP Standardverdünnungsreihen von etwa 10 um bis 100 um und halten auf dem Eis. </li><li> In 50 ul Reinigungsmittel in jede Vertiefung und schütteln Sie die Platte für 5 min in Schüttler, 700 min. </li><li> Den Inhalt jeder Flasche des &quot;lyophilisierte Substrat &#39;mit 5 ml&#39; Substratpuffers &#39;in dem Kit. </li><li> In 50 ul des rekonstituierten Substrat-Lösung in die Vertiefungen, und schüttelndie Platte für 5 min auf Schüttler, 700 min. </li><li> Halten Sie die Platte in Dunkelheit für 10 min. </li><li> Messen Lumineszenz von jeder Vertiefung durch ELISA Mikroplatten-Reader. </li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Live-konfokale Bildgebung bot eine Echtzeit-Dokumentation der zytotoxischen Wirkung der PJ-34 im Live-Multi-zentrosomale Zellen während der Mitose (Abbildung 3 und ergänzende Informationen). Dies war die erste Live-Dokumentation zuschreibt die Zytotoxizität von PJ-34 in menschlichen Krebszellen, um zusätzliche Zentrosome de-Clustering und Zelltod, was darauf hindeutet, Induktion von Mitotic Catastrophe Zelltod durch PJ-34 5-9. Im Gegensatz dazu wurde bi-fokale Clustering von super-Numerar...

Phenanthrene abgeleitet PARP1 Hemmern, einschließlich PJ-34, wurden entwickelt, um ruhende Zellen von Apoptose durch die Energie verbrauchen PARP1 vermittelten DNA-Reparatur unter Stressbedingungen (Schlaganfall oder Myokardinfarkt) 1 induziert schützen. Doch vor kurzem entdeckten wir, dass PJ-34, bei zweimal höhere Konzentration als die induzierende PARP1 Hemmung kann ausschließlich Zelltod verursachen in menschlichen Krebszellen 2,3. Je schneller die Proliferation der Zelle war, desto effizienter ist die Tilgung der Zellen. Die zytotoxische Aktivität von PJ-34 wurde extra Zentrosome de-Clustering in Mitose 2 zugeschrieben. Viele menschliche Krebszellen Hafen multicentrosomes 4,5. Inkubation von menschlichen Brustkrebszellen MDA-MB-231, die überzähligen Centrosomen Hafen, mit 20 uM PJ-34 effizient beseitigt diese Zellen innerhalb 72-96 Stunden ohne Beeinträchtigung ruhenden Zellen oder einige gutartige proliferierende Zellen beherbergen zwei Centrosomen in der Mitose <sup&gt; 2,3. Benigne Zellen enthalten humanen Mamma-Epithelzellen MCF-10, Humane Endothelzellen (HUVEC) und primäre mesenchymale Zellen aus menschlichen Thymus hergestellt. Diese Zellen waren resistent gegen die zytotoxische Aktivität der PJ-34. PJ-34 nicht mit ihren Zellzyklus stören während 96 h Inkubation oder auf ihre Centrosomen und bi-fokale Spindelbildung 2,3. Bipolar Zentrosoms Montage ist entscheidend für bipolare Spindel Bildung in der Mitose 4,5. Daher wurden Zellen mit mehr als zwei Centrosomen eine kaum verstanden molekularen Mechanismus entwickelt, Clustering ihre zusätzliche Zentrosome an zwei Pole 4-9. Ausfall der bipolaren Anordnung ihrer Zentrosome kann multipolare Spindeln und aberrante verzerrten Chromosomen Segregation, dass die Verhaftungen Zellzyklus in der G2 / M Verhaftung und zum Zelltod führt zu mitotischen Ausfall 4,5 zugeschrieben. Die molekularen Mechanismen, die extra Zentrosome de-Clustering werden intensiv untersucht &lt;sup&gt; 10. Das Verständnis dieses Mechanismus Tod ermöglichen exklusive Beseitigung von Krebszellen unter Schonung gesunden Gewebes 5,10. So bieten Verbindungen, die mitotische Katastrophe Zelltod aktivieren einen neuen Modus eines selektiven Krebs-Therapie, die wirksam sein kann in einem breiten Spektrum von humanen soliden cancers.Our Ergebnisse legen nahe, dass konfokale Bildgebung verwendet werden, um Moleküle beeinflussen extra Zentrosome Clustering in identifizieren Mitose 2,3, wodurch diese Verbindungen Krebs Targeting Wirkstoffkandidaten. Wir haben die zytotoxische Aktivität der Phenanthridin PJ-34 durch Abtasten festen und lebenden menschlichen Krebszellen (mit hohem Auftreten der extra-Zentrosome in Mitose) gegenüber normalen Zellen dokumentiert. Eine Schritt-für Schritt-Beschreibung der bildgebenden Verfahren verwendet, um die zytotoxische Aktivität der PJ-34 in menschlichen Krebszellen zu identifizieren sind unten aufgeführt.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (GIBCO)</td>
			<td>41965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine Serum)</td>
			<td>Invitrogen (GIBCO)</td>
			<td>12657</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep-Ampho solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-033-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Tripsin-EDTA</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92 mm Petri dishes</td>
			<td>Nunc,Thermo scientific</td>
			<td>150350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-D-lysine coated glass bottom culture dishes</td>
			<td>MatTek Corporation, USA</td>
			<td>P35GC-0-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminescent ATP detection assay kit</td>
			<td>Abcam</td>
			<td>ab113849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NDS (Normal Donkey Serum)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti &alpha;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>1:250 dilution (IF)</td>
		</tr>
		<tr>
			<td>Anti &gamma;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5192</td>
			<td>1:200 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-10042</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI (mounting)</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JetPEI (liposomal transfection reagent )</td>
			<td>Polyplus</td>
			<td>101-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica (Mannheim, Germany)</td>
			<td>TCS SP5II</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

Phenanthrenderivate als potente Inhibitoren PARP1 verhinderte die bi-fokale Clustering überzähliger Centrosomen in multi-zentrosomale menschliche Krebszellen in der Mitose. Die Phenanthridin PJ-34 war die stärkste Molekül. Declustering der extra-Zentrosome verursacht mitotischen Versagen und Zelltod in multi-zentrosomale Zellen. Die meisten soliden menschlichen Krebserkrankungen eine hohe Auftreten von extra Zentrosome. Die Aktivität der PJ-34 wurde in Echtzeit durch konfokale Bildgebung von lebenden menschlichen Brustkrebszellen MDA-MB-231-Zellen mit Vektoren, die für fluoreszierende γ-Tubulin, die sehr häufig in den Centrosomen ist und für Leuchtstofflampen Histone H2B in transfiziert dokumentiert die Chromosomen. Aberrante Chromosom Anordnungen und de-Cluster γ-Tubulin Brennpunkte darstellt Declustered Zentrosome wurden in den transfizierten MDA-MB-231-Zellen nach der Behandlung mit PJ-34 erfasst. Un-Cluster extra Centrosomen in den beiden vorangegangen Spindelpole ihren Zelltod. Diese Ergebnisse verknüpft for das erste Mal, das kürzlich entdeckt exklusiven zytotoxische Aktivität von PJ-34 in menschlichen Krebszellen mit extra Zentrosome de-Clustering in der Mitose und mitotischen Ausfall zum Zelltod führen. Nach den bisherigen Erkenntnissen durch konfokale Bildgebung von fixierten Zellen, PJ-34 ausschließlich ausgerottet Krebszellen mit Multi-Zentrosome ohne Beeinträchtigung normalen Zellen Mitose mit zwei Centrosomen und bi-fokale Spindeln beobachtet. Dieser zytotoxische Aktivität von PJ-34 wurde nicht durch andere potente Inhibitoren PARP1 geteilt, und wurde in PARP1 defizienten MEF beherbergen extracentrosomes beobachtet, was seine Unabhängigkeit von PARP1 Hemmung. Live-konfokale Bildgebung bot ein nützliches Werkzeug für die Identifizierung neuer Moleküle beseitigen Zellen während der Mitose.

PJ-34 ist eine stabile wasserlösliche Phenanthridin 1 (Abbildung 1). Unsere bisherigen Ergebnisse zeigen, Zelltod und extra Centrosomen in verschiedene Arten von festen Multi-zentrosomale Krebszellen, die mit PJ-34 behandelt wurden, de-Cluster. Im Gegensatz dazu wurden normalen proliferierenden Zellen nicht beeinträchtigt 2,3. Zentrosomen wurden durch doppelte Markierung mit Antikörpern gegen centrine1 und γ-Tubulin in den festen extra zentrosomale Zellen 2 gerichtet...

Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Die zytotoxische Aktivität der Phenanthridin PJ-34 in Krebszellen Mitose wurde in Echtzeit Live-konfokale Bildgebung dokumentiert. PJ-34 ausgerottet menschlichen Brustkrebszellen MDA-MB-231-Zellen, die extra Centrosomen in Mitose. Im Gegensatz zu normalen bifokalen Mitose wurden die extra Zentrosome in den beiden Spindelpole in Gegenwart von PJ-34 gruppiert.

Cell Death mit Abnormal Mitose Assoziierte Beobachtet von konfokalen Imaging im Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

cell-death-associated-with-abnormal-mitosis-observed-confocal-imaging

Research

JoVE Journal

Medicine

14.9K Aufrufe.  Sheba Medical Center. Die zytotoxische Aktivität der Phenanthridin PJ-34 in Krebszellen Mitose wurde in Echtzeit Live-konfokale Bildgebung dokumentiert. PJ-34 ausgerottet menschlichen Brustkrebszellen MDA-MB-231-Zellen, die extra Centrosomen in Mitose. Im Gegensatz zu normalen bifokalen Mitose wurden die extra Zentrosome in den beiden Spindelpole in Gegenwart von PJ-34 gruppiert.

Serie: Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

High-Efficiency Transduktion von Liver Cancer Cells von rekombinanten Adeno-assoziierten Virus Serotyp 3 Vektoren

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Forschung

Medizin

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 ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Evaluation of Cancer Stem Cell Migration Mit Abschottung Mikrofluidiksysteme und Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

MAME Modelle für die 4D-Bildgebung lebender Zellen von Tumor: Mikroumgebung Wechselwirkungen, die Einfluss auf maligne Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Modeling Colitis-assoziierten Krebs mit Azoxymethan (AOM) und Dextransulfat Natrium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolation, Charakterisierung und vergleichende Differenzierung humaner Dental Pulp Stammzellen aus bleibenden Zähne mit zwei verschiedenen Methoden abgeleitet

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Live-Imaging of Drug Responses in der Tumor-Mikroumgebung in Mausmodellen von Brustkrebs

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Ein Multiplex-Luciferase-basierte Screening-Plattform zur Abfrage Krebs-assoziierte Signaltransduktion in kultivierten Zellen

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.

Die Beurteilung Phagozytische Clearance von Cell Death in Experimental Stroke ligierbaren von Fluoreszenzsonden

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling von Östrogen-regulierte MicroRNAs in Brustkrebszellen

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...