This research is supported by funding from CBCF (to ZW).

1. Vorbereitung der Zellen für Live-Imaging <ol><li> Zellkultur und Seeding <ol><li> Verwenden MCF-7 Brustkrebszellen , ausgewählt für die Resistenz gegen Docetaxel (MCF-7 TXT) und deren nicht-resistenten parentalen Zelllinie (MCF-7 CC). Die detaillierten Auswahlprozess und die Charakterisierung dieser ausgewählten Zelllinien wurden zuvor 24 beschrieben. </li><li> Wachsen alle Zellen in 10 cm-Kulturschalen bei 37 ° C in einem Medium, 90% Dulbecco besteht modifiziertem Eagle-Medium (DMEM) und 10% fötales Rinderserum (FBS) und supplementiert mit nicht-essentiellen Aminosäuren. Pflegen Sie das Medium in einer 5% igen CO 2 -Atmosphäre. Pflegen MCF-7 TXT Zellen bei 5 nM Docetaxel. </li><li> Seed-Zellen auf Deckgläser für die Live-Darstellung. Sterilisieren 24 mm poly-L-Lysin beschichteten Glasplättchen, indem sie mit 70% -igem Alkohol gespült und dann über Nacht sie UV ausgesetzt wird. Legen Sie die Deckgläser in einer 6-Well-Kulturplatte von 35 mm. Place ein Deckglas in jeder Vertiefung. </li><li> Entfernen Sie das Medium aus dem 10-cm-Kulturschale und waschen Sie die Zellen mit PBS. Mit 0,5 ml Trypsin-EDTA in die Schale um die Zellen von der Schale zu lösen. 5 ml Kulturmedium in die Schale um das Trypsin nach der Ablösung der Zellen zu neutralisieren. </li><li> Zähle die Zellen mit einem Hämozytometer die Anzahl der Zellen pro Volumeneinheit zu ermitteln. </li><li> In etwa 10 5 Zellen in jede Vertiefung auf der Basis der Zellzahl pro Volumeneinheit. sanft, legte die Platte wieder in den Inkubator Nach Schütteln, damit die Zellen an den Deckgläsern zu befestigen. </li></ol></li><li> Expression von GFP-markierten α-Tubulin <ol><li> Zu testen Dynamik der Mikrotubuli, Transduktion der Zellen mit GFP-markierten α-Tubulin mit dem handelsüblichen GFP Transduktion Steuerung (zB BacMam) nach dem Protokoll des Herstellers. </li><li> Kultur der Zellen in der Vertiefung für 36 h in einem 37 ° C Zellkulturinkubator com zu ermöglichenplete Adhäsion und 60% Konfluenz. </li><li> Berechne den geeigneten Volumen GFP-markierten α-Tubulin zu jeder Vertiefung hinzugefügt, entsprechend der folgenden Gleichung: <img alt="Gleichung" src="/files/ftp_upload/55027/55027eq1.jpg" /> Die Anzahl der Zellen ist, die geschätzte Gesamtzahl der Zellen in der Vertiefung zum Zeitpunkt der Markierung und der gewünschten PPC ist die Anzahl der Partikel pro Zelle. HINWEIS: Die MCF-7 - Zellen Seeding sollte die Zellzahl 10 5 bis 2 x 10 5 mit einem gewünschten PPC 20 verdoppelt haben, das Volumen für jede Deck benötigt daher 40 &amp; mgr; l. Für verschiedene Zellen, berechnet das Volumen auf der obigen Formel basiert möglicherweise nicht die beste sein. Das genaue Volumen sollte entsprechend der tatsächlichen Expression von GFP-Tubulin eingestellt werden. </li><li> Mische die GFP-markierten α-Tubulin mehrfach durch Umdrehen um eine homogene Lösung zu gewährleisten. Nicht mit dem Vortex. HINWEIS: Für die Steuerung, die Null (Kontrolle) Reagens fehlt jede Säugetier genetischeElemente und können potentielle Baculovirus-vermittelte Effekte und Hintergrundfluoreszenz verwendet werden, bestimmen zu helfen. Eine nicht gezielt GFP Transduktion Kontrolle kann auch verwendet werden, die die gesamte Zelle leuchtet. </li><li> Ersetzen Sie das Kulturmedium mit frischem Medium. 2 mL des neuen Mediums und 40 ul GFP-markierten α-Tubulin zu jeder Vertiefung. </li><li> Schütteln Sie die Platte vorsichtig vollständige Durchmischung von GFP-markierten α-Tubulin mit dem Kulturmedium zu ermöglichen. Bringen Sie die Platte mit dem Kultur-Inkubator und Inkubation für 24 h, um die Expression von GFP-Tubulin zu ermöglichen. </li></ol></li><li> Treat Zellen mit Docetaxel Hinweis: Dieses Experiment wird die Wirkung von Docetaxel auf Mikrotubuli dynamische Instabilität zu testen. Somit werden die Zellen mit der gewünschten Konzentration von Docetaxel behandelt. <ol><li> Bereiten Sie ein Medium, 90% DMEM und zu 10% der FBS und ergänzt mit nicht-essentiellen Aminosäuren. Verwenden Sie DMEM ohne Phenol rot, weil Phenolrot mit dem fluorescen störence von GFP-Tubulin. Fügen Sie die gewünschte Konzentration von Docetaxel (im Bereich von 10 nm bis 10 &amp; mgr; M). Die Stammlösung von Docetaxel ist 10 mM in Dimethylsulfoxid (DMSO). </li><li> Vorwärmen der Docetaxel enthaltende Medium auf 37 ° C in der Gewebekultur-Inkubator. </li><li> Ersetzen Sie die normalen Kulturmedium mit der Docetaxel-haltigen Medium und Inkubation für 30 min in der Gewebekultur-Inkubator. Es sollte für jeden Docetaxel-Konzentration mindestens 3 Deck pro Vertiefung sein. </li></ol></li></ol> 2. Live-Imaging zu untersuchen Mikrotubuli dynamische Instabilität <ol><li> Einrichten des Systems <ol><li> Führen Experimente in einer Kammer bei 37 ° C und 5% CO 2 gehalten. Erwerben Fluoreszenzbilder von Zeitablauf mit einem mikroskopischen System mit einem 60X versehen, 1,42 NA Öl Objektivlinse auf einem invertierten Fluoreszenzmikroskop und mit einer CCD-Kamera. </li><li> Legen Sie die Zellen für Livebild. Vorwärmen sowohl der Probenhalter unddas Medium auf 37 ° C. Wählen Sie ein Deckglas zu untersuchen. Montieren Sie das Deckglas von Interesse auf einem Probenhalter und Inkubation mit 1 ml der Docetaxel-haltigen Medium hergestellt in Schritt 1.3.1. </li></ol></li><li> Live - Bildgebung von Mikrotubuli dynamische Instabilität <ol><li> Identifizieren Sie die Zellen, die für die Bildgebung verwendet wird. Sie sollten flach sein, haben eine hohe Fluoreszenzintensität ausgedrückt GFP-Tubulin und haben klare Mikrotubuli-Strukturen. </li><li> Wählen einer Zelle aus der Gruppe der identifizierten Zellen aus Schritt 2.2.1 zunächst zu untersuchen. Konzentrieren sich auf den Randbereich der identifizierten Zelle. Stellen Sie das Programm für die Belichtungszeit und die Häufigkeit der Bildaufnahme auf. Als Belichtungszeit gewöhnlich 0,5 s ist, werden die Bilder alle zwei s erfasst werden. </li><li> Lassen Sie eine zusätzliche 20 Minuten für die Schritte von 1,33 bis 2.2.3. Somit genau 50 min nach der Zugabe von Docetaxel, wie in Schritt 1.3.3 beschrieben, beginnen imaging (dies für Normung). </li><li> Notieren Sie sich die Mikrotubuli-dynamics für 2 Minuten, die ein Foto alle 2 s. Insgesamt erwerben 60 Bilder für die identifizierte Zelle. </li><li> Gehen Sie zum nächsten identifizierten Zelle und wiederholen Sie die Schritte 2.2.2 bis 2.2.4. Wiederholen Sie den Vorgang, bis 5 Zellen abgebildet wurden. Es ist zwingend notwendig, dies zu tun, bevor die Zeit 70 min nach der Zugabe von Docetaxel erreicht, wie es in Schritt 1.3.3 beschrieben. </li><li> Für jede Behandlungsbedingung, wiederholen Sie die Schritte 2.2.1 für 3 verschiedene Deck bis 2.2.5. Insgesamt Bild 15 Zellen, die genug Daten Mikrotubuli dynamische Instabilität zu messen. </li></ol></li></ol> 3. Bildverarbeitung und Datenanalyse <ol><li> Deconvolution der Bilder <ol><li> Entfalten die aufgenommenen Bilder eine hohe Qualität zu erreichen. Öffnen Sie das Entfaltungsprogramm mit dem Mikroskopsystem ausgestattet. </li><li> Klicken Sie auf &quot;Bearbeiten&quot; und wählen Sie &quot;entfalten.&quot; Ziehen Sie die Datei in den &quot;Input&quot; Fenster und klicken Sie auf &quot;Do&quot; Die Bilddatei wird entfalteten und dieentfalteten Datei wird automatisch als neue Datei gespeichert werden. </li></ol></li><li> Die Erzeugung von Videos <ol><li> Klicken Sie auf die entfalteten Bilddatei mit ausgestatteter Software zu öffnen. </li><li> Klicken Sie auf &quot;Datei&quot; und wählen Sie &quot;als Film speichern&quot;. Wählen Sie den &quot;Filmformat&quot; als &quot;AV&quot; und &quot;Animation-Stil&quot; als &quot;Forward&quot;. Stellen Sie die &quot;Komprimierungsqualität&quot; auf &quot;100%&quot; und der &quot;Frame Rate&quot; bis &quot;5 / Sekunde.&quot; </li><li> Überprüfen Sie die Schachtel &quot;Animieren durch die Zeit&quot; und klicken Sie auf &quot;Do it.&quot; Das Video wird erzeugt werden. </li></ol></li><li> Die Messung der Mikrotubuli - Verkürzung und Wachstumsrate <ol><li> Identifizieren Sie die Mikrotubuli, die für die Messung verwendet wird. Für jede Zelle wird nur wenige Mikrotubuli Lage sein, ihre Verkürzung oder Wachstum haben klar und vollständig folgt. </li><li> Untersuchen jeden Frame des Videos den Rahmen zu identifizieren, die die Ausgangslänge des Mikrotubuli a zeigtnd der Rahmen mit den meisten verlängert oder verkürzt Mikrotubuli. Messen der Änderung in der Länge zwischen den beiden Mikrotubuli und dem zwischen den beiden Rahmen verstrichene Zeit. </li><li> Berechnen Sie die Geschwindigkeit des Wachstums oder der Verkürzung durch die Längenänderung durch Zeitteilung (die Einheit &amp; mgr; m / s). Für jede Behandlungsbedingung, folgen mindestens 10 Zellen und mindestens 20 Mikrotubuli messen. </li><li> Berechnen Sie den Mittelwert und der Standardfehler für jede Behandlungsbedingung. Analysieren Sie den statistischen Unterschied zwischen den Bedingungen des Student-Test. Melden Sie die Daten in einer Tabelle und / oder in einem Diagramm. </li></ol></li></ol>

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Es gibt zwei Hauptmethoden Mikrotubuli dynamische Instabilität zu messen: in vitro und in vivo. In dem in vitro - Verfahren wird gereinigtes Tubulin verwendet Mikrotubuli dynamische Instabilität mit Computer-enhanced Zeitrafferdifferentialinterferenzkontrastmikroskopie zu messen. In dem in vivo - Verfahren, mikroinjiziert Fluoreszenz Tubulin oder Tubulin-GFP exprimiert, in Mikrotubuli eingebaut. Die Dynamik (Wachstum und Verkürzung) der Mikrotubuli wird dann durch Zeitraffer unter ...

<html> Die Haupttodesursache Brustkrebs ist durch Metastase 1, 2. Taxane wie Docetaxel und Paclitaxel, werden derzeit als First-Line - Therapien bei der Behandlung von metastasierendem Brustkrebs 2, 3, 4, 5, 6 verwendet. Sie sind Teil einer Gruppe von Mikrotubuli-Targeting-Mittel (MTAs), die die Dynamik der Mikrotubuli stören. Allerdings ist eine der größten Herausforderungen für die Taxane in kurativen Therapie ist die Entwicklung von Taxan - Resistenz von Krebszellen unter Verwendung, die 7 bis Wiederauftreten der Krankheit führt. Arzneimittelresistenz ist für mehr als 90% aller Todesfälle bei Patienten mit metastasiertem Brustkrebs , 7. Mikrotubuli sind durch die Polymerisation von α- und β-Tubulin-Heterodimeren gebildetclass = &quot;xref&quot;&gt; 8, 9. Die genaue Regelung der Dynamik der Mikrotubuli ist wichtig für viele zelluläre Funktionen, einschließlich der Zellpolarisation, Zellzyklus, den intrazellulären Transport und Zellsignalisierung. Dysregulation von Mikrotubuli und deren Dynamik werden die Zellfunktion stören und führen in den Zelltod 10, 11. Je nachdem , wie sie diese Dysregulation verursachen kann MTA Medikamente entweder als Mikrotubuli stabilisierende Mittel eingestuft werden (dh Taxane) oder Mikrotubuli-destabalizing Mittel (dh, Vinca - Alkaloiden oder Colchicin-Bindungsstelle Mittel) 20. Trotz ihrer gegenläufigen Effekte auf Mikrotubuli Masse, bei einer ausreichenden Dosierung, beide Klassen können 21 Krebszellen durch ihre Auswirkungen auf die Dynamik der Mikrotubuli töten. Taxane funktionieren in erster Linie durch die Mikrotubuli Spindel 12 zu stabilisieren, was zuchromosomale Fehlausrichtung. Die anschließende unaufhörlichen Aktivierung der Spindelanordnung checkpoint (SAC) hemmt die Zelle in die Mitose. Längerer mitotischen Arrest bewirkt dann , dass die Apoptose 13, 14. Taxan interagiert mit Mikrotubuli durch die Taxan - Bindungsstelle auf β-Tubulin - 8, 15, die im zusammengebauten Tubulin 16 nur vorhanden ist. Mehrere Mechanismen für Taxan Widerstand haben 9 vorgeschlagen, 17. Diese Mechanismen umfassen sowohl allgemeine Multidrug - Resistenz durch die Überexpression von arzneimittel Efflux Proteine und Taxan-spezifischen Widerstand 5, 9, 18, 19. Beispielsweise Taxan-resistenten Krebszellen Expression und Funktion bestimmter β-Tub möglicherweise verändertulin Isotypen 5, 9, 19, 20, 21, 22, 23. Durch die Verwendung eines in vivo - Verfahren Mikrotubuli dynamische Instabilität zu messen, zeigen wir , dass, wenn im Vergleich zu nicht-resistenten, elterliche MCF-7 - Zellen CC 17 die Mikrotubuli - Dynamik von Docetaxel-resistenten MCF-7 - Zellen zu TXT Docetaxel Behandlung unempfindlich sind. Zum besseren Verständnis der Funktion von MTAs und der genaue Mechanismus der Taxan-Resistenz in Krebszellen zu verstehen, ist es wichtig, die Dynamik der Mikrotubuli zu messen. Hier berichten wir über eine in vivo - Methode, dies zu tun. Durch die Verwendung von Echtzeit- Bildgebung in Kombination mit der Expression von GFP-markierten Tubulin in Zellen, können wir die Dynamik der Mikrotubuli von MCF-7 TXT und MCF-7 CC - Zellen mit und wi messenthout Behandlung mit Docetaxel. Die Ergebnisse können uns wirksamer Medikamente helfen entwerfen, die Taxan Widerstand überwinden kann.

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	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma-Aldrich</td>
			<td>D5796</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Life Technologies, Invitrogen</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco, Invitrogen</td>
			<td>12483</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Anti (100x)</td>
			<td>Life Technologies, Invitrogen</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>docetaxel</td>
			<td>Sigma-Aldrich</td>
			<td>01885-5mg-F</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM phenol red-free</td>
			<td>Gibco, Invitrogen</td>
			<td>21063</td>
			<td></td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td>
			<td>ThermoFisher Scientific</td>
			<td>C10613</td>
			<td>Key reagent for expressing GFP tubulin in cells</td>
		</tr>
		<tr>
			<td>CellLight Reagent *BacMam 2.0* GFP</td>
			<td>ThermoFisher Scientific</td>
			<td>B10383</td>
			<td>Control</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich+B9:AA9</td>
			<td>472301</td>
			<td>for dissoving decetaxel</td>
		</tr>
		<tr>
			<td>22-mm glass coveslip</td>
			<td>Fisher Scientifics</td>
			<td>12-545-101</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>Greiner Bio-One International</td>
			<td>6 Well Celi Culture Plate</td>
			<td></td>
		</tr>
		<tr>
			<td>DeltaVision Microscopy Imaging Systems</td>
			<td>GE Health</td>
			<td></td>
			<td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>ThermoFisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemacytometer Set, Hausser Scientific</td>
			<td>Hausser Scientific, Distributed by VWR</td>
			<td>Supplier No.: 1492 VWR No.:15170-172</td>
			<td></td>
		</tr>
	</tbody>
</table>

live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

Mit dem Protokoll hier vorgestellten, untersuchten wir die Wirkung von Docetaxel auf die Mikrotubuli - Dynamik der normalen (MCF-7 CC) und Docetaxel-resistenten (MCF-7 TXT) Brustkrebszellen. Zwei Sätze von Aufnahmen zeigen die Wirkung von Docetaxel (0,5 uM) auf Mikrotubuli Wachstum und Verkürzung in MCF-7 CC und MCF-7 - TXT - Zellen (1A). Wir auch die Geschwindigke...

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

Live-Imaging zur Untersuchung Mikrotubuli dynamische Instabilität in Taxan-resistenten Mammakarzinome

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

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7.5K Aufrufe.  University of Alberta. In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. 

Serie: Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

Functional Genomics Screening Verwendung zur Identifizierung möglicher neuer Wirkstoff-Targets in Cancer Cell Sphäroid-Kulturen

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Forschung

Krebsforschung

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Ein Portal Veneninjektion Modell zur Untersuchung der Leber Metastasierung von Brustkrebs

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

Live-Darstellung der Brust Epithelzelle Migration nach der vorübergehenden Erschöpfung der TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Langfristige Leben-Cell Imaging zu Zelle Schicksal als Reaktion auf Paclitaxel bewerten

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

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

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

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

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

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

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Studieren von dreifach negativem Brustkrebs mit orthotopischem Brustkrebsmodell

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.

We present the chicken chorioallantoic membrane model as an alternative, transplantable, in vivo model for the engraftment of gynecological and urological cancer cell lines and patient-derived tumors.

Verwendung des Chicken Chorioallantoic Membrane In Vivo Modells zur Untersuchung gynäkologischer und urologischer Krebsarten

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Verwendung computerbasierter Bildanalyse zur Verbesserung der Quantifizierung von Lungenmetastasen im 4T1-Brustkrebsmodell

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Modellierung von Brustkrebs im menschlichen Brustgewebe mit einem mikrophysiologischen System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Ein murines Ommaya Xenograft-Modell zur Untersuchung der direkten zielgerichteten Therapie von Leptomeningealerkrankungen

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...