Wir sind dankbar, dass Ken Iwata (OSI Pharmaceuticals, New York, USA) für Reagenzien und Fred Miller (Barbara Ann Karmanos Cancer Intitute, Detroit, USA) für die Zell-Linien.

1. Vorbereitung der Methocel-Lösung (500 mL) <ol><li> Autoclave 6 g Methylcellulose in einer 500 ml Flasche mit einem Magnetrührer. </li><li> Lösen Sie die Methylcellulose in 250 ml vorgewärmter DMEM/F12 ohne Serum (60 ° C) für 20 min. </li><li> Add 250 ml DMEM/F12 (RT) mit 10% Pferdeserum. Mix über Nacht (4 ° C). </li><li> Deaktivieren Sie die Lösung durch Zentrifugation (5000g, 2h, 4 ° C). </li><li> Nehmen Sie die klare hochviskose Lösung in ein neues Röhrchen (etwa 90-95% der Stammlösung). </li><li> Lagerung bei 4 ° C bis zur Verwendung. </li></ol> 2. Spheroid Kultur <ol><li> Bereiten Sie eine 20% Methocel-Lösung (10 ml Methocel und 40 ml Wachstumsmedium). </li><li> Wash Zellen zweimal mit PBS, fügen 0,1% Trypsin und inkubieren Zellen bei 37 ° C </li><li> Die Zellen in Wachstumsmedium und zählen die Zellen. </li><li> Eine Suspension von 10 000 Zellen pro ml in 20% Methocel. </li><li> Fügen Sie die Zellsuspension auf die 96-Well-Rundboden sUSSETZUNG Platten (100 ul pro Well). </li><li> Am nächsten Tag, überprüfen Sie das Sphäroid Bildung in der Suspension Platten. Sphäroide werden bereit sein für den Einsatz nach 1-3 Tagen. </li></ol> 3. Herstellung von Kollagen <ol><li> Bereiten Sie auf dem Eis eine Lösung von 8 ml Kollagen, 1 ml 10x PBS, 1 ml 0,1 N NaOH und 3 Tropfen 0,1 N HCl. Prüfen Sie, ob pH beträgt 7,4 von Spotting einige der Lösung auf pH-Indikator-Streifen. Der pH-Wert, wenn außerhalb des Bereichs. </li><li> Add 50 ul neutralisiert Kollagenlösung in die Vertiefungen einer 96-Well-Platte </li><li> Bei 37 ° C, bis die Kollagen verfestigt (90 min). </li><li> Bereiten Methocel-Kollagen-Ligand-Lösungen auf Eis: für jeden Liganden Pipette 1 Teil von Kollagen-Lösung. Add 20 ng pro ml Kollagen TGF-β. Nach dem Verdünnen mit Methocel und Verbreitung über die verschiedenen Schichten wird die endgültige Konzentration von TGF-β 5 ng / ml Mix durch Vortexen werden. 1 Teil Methocel-Lösung. Mix durch Vortexen. </li></ol> 4. Spheroid einbettending <ol><li> Legen Sie eine 200 ul Spitze, die etwa 5 mm von der Spitze abgeschnitten hat, auf einem 200 ul Pipette. Saugen Sie up one Sphäroid mit der Pipette und sorgfältig zu verzichten das Medium in einen leeren Teller. Sehen Sie, wenn das Sphäroid bleibt in der Spitze. Das Sphäroid sichtbar ist als weißer Punkt. Ohne die Freigabe der Kolben, saugen 100 ul Kollagen-Methocel Mischung und verzichtet das Sphäroid in das Kollagen-Gemisch in eine Kollagen-beschichtete 96-Well. Tun Sie dies für 12 Sphäroide für jede Bedingung. </li><li> Lassen Sie Kollagen festigen mindestens 30 Minuten bei 37 ° C </li><li> Luftblasen mit einer 200 ul Spitze. </li><li> Dann werden 50 ul Medium mit 1,6% Serum und Inhibitoren in DMSO gelöst. Für SB-431542 add 4 ul Stammlösung (10 mM) zu 1 ml Medium. Nach der Diffusion wird die endgültige Konzentration des Inhibitors 10 uM werden. TGF-β und Inhibitoren bleibt der Verlauf des Experiments. Machen Sie Fotos von der Sphäroide unter dem Mikroskop mit 40-facher Vergrößerung. </li><li> Nach 48h der Stimulation mit TGF-β und / oder Inhibitoren, machen Sie Fotos von der Sphäroide unter dem Mikroskop mit 40-facher Vergrößerung. </li><li> Man misst die Fläche des eindringenden Sphäroide nach 2 Tagen und subtrahieren den Startplatz. Die Vermessung der Fläche eines Sphäroid: Öffnen Sie das Bild aus dem Sphäroid in Adobe Photoshop Extended (Abbildung 2a) und wählen Sie die Schnellauswahl-Werkzeug (Abb. 2B). Der Mauszeiger verwandelt sich in einen Kreis mit einem &quot;Plus&quot;-Zeichen (+) (Abbildung 2C). Während des Ziehens den Cursor über das Sphäroid, wird Photoshop erkennt die Grenzen der Sphäroid. (-)-Zeichen in der Symbolleiste Wenn ein Bereich außerhalb der Sphäroid ausgewählt ist, kann diese Region von der Auswahl durch Drücken der Alt-Taste oder über die negative Selektion Werkzeug, das durch eine angegeben ist ausgeschlossen. Der Cursor verwandelt sich in einen Kreis mit einem &quot;minus&quot; (-)-Zeichen und durch Ziehen der Maus über die falsch gewählten Bereich, wird dieser Bereich von der Auswahl (Abbildung 2d) entfernt. Wenn nötig, mehrere Regionen können durch Drücken der Sh gewählt werdenift-Taste. Um Arbeit genauer kann die Größe des Mauszeigers, indem Sie die Bürste Durchmesser in der Symbolleiste (Abbildung 2E) eingestellt werden. Wenn die ganze Sphäroid ausgewählt ist, wird die Fläche der Auswahl über ANALYSE-Maßnahme in der Menüleiste oder durch Drücken von Strg + Shift + M (Abbildung 2f) berechnet. Die Messwerterfassung Fenster erscheint und zeigt den Dateinamen und den Bereich der Auswahl in Pixel, sowie andere Daten (Abb. 2G). Wenn Mehrfachauswahl gemessen werden, zeigt die erste Zeile die Summe aller Bereiche. Die Messungen der einzelnen Einstellungen sind in den Zeilen dargestellt. Die Daten aller Messungen können in eine Textdatei exportiert werden und in Excel geöffnet. </li></ol> 5. Repräsentative Ergebnisse: Ein Beispiel für das Sphäroid-Assay mit MCF10A1 (M1) normal Brustepithelzellen verwandelte H-RAS MCF10AneoT (M2)-Zellen und M2-derived MCF10CA1a (M4) ist in Abbildung 3 angegeben. M1-Zellen zeigten nur schwache Invasion ohneStimulation, sondern fielen deutlich besser nach Stimulation durch TGF-β. Im Gegensatz dazu die RAS-transformierten M1-derivative M2 überfallen bereits effizient und ohne Zusatz von Stimuli, und das war weitere 4-5 fach auf TGF-β Behandlung erhöht. M4-Zellen zeigten den stärksten Einbruch, mit und ohne TGF-β hinaus. Als nächstes untersuchten wir, ob wir mit TGF-β-induzierte Invasion in dieses Sphäroid-Modell mit Hilfe chemischer Inhibitoren stören. Zu diesem Zweck benutzten wir SB-431542, einem ATP-Analogon und selektiver Inhibitor der Kinase-Aktivität von TβRI sowie Activin Typ IB Rezeptor (ActRIB) und Aktivin-Rezeptor-ähnliche Kinase (ALK) 7 12. Wie erwartet, SB-431542 inhibiert TGF-β-induzierte Invasion von M1, M2 und M4-Zellen (Abb. 4), was darauf hinweist, dass TGF-β-induzierte Invasion TβRI-Kinase abhängig. Darüber hinaus die basale Invasion der Sphäroide wurde auch stark gehemmt, was darauf hindeutet, dass die basale Invasion ist auch dehängig von TGF-β. <img alt="Abbildung 1" src="/files/ftp_upload/3337/3337fig1.jpg" /> Abbildung 1. Flussdiagramm der 3D-Sphäroid-Assay. Zunächst werden die Zellen unter nicht-adhärenten Bedingungen in u-förmigen Suspensionen ausplattiert. Cells aggregrate in multcellular Sphäroide und kann in eine Kollagen-Matrix eingebettet werden. In diese Kollagen-Matrix, sind sie in der Lage, anzugreifen. <img alt="Abbildung 2" src="/files/ftp_upload/3337/3337fig2.jpg" /> Abbildung 2. Quantifizierung der Bereich der Sphäroide mit Hilfe von Adobe Photoshop Extended. (A) Das Bild des Sphäroids ist in Adobe Photoshop Extendend (B) Auswahl des Schnellauswahl-Werkzeug (C) Ziehen Sie den Mauszeiger über das Sphäroid in den Bereich der Sphäroid (D) Entfernung eines wongly enthalten Gebiet mit der Option eröffnet negativen Schnellauswahl-Werkzeug (E) Die Einstellung der Pinselgröße des Schnellauswahl-Werkzeug, um genauer den Bereich auswählen (F) Auswahl derMessung Befehl zur Aufzeichnung (G) Beispiel für ein Messprotokoll. <img alt="Abbildung 3" src="/files/ftp_upload/3337/3337fig3.jpg" /> Abbildung 3. TGF-β-induzierte Invasion von Sphäroiden spontan verewigt MCF10A1 (M1) (A), RAS-transformierten MCF10AneoT (M2 (B) und seine Metastasen derivative MCF10CA1a (M4) (C). M1, M2 und M4 Sphäroide embedded in Kollagen wurden mit 5ng/ml von TGF-β für 48h behandelt wie angegeben. AC Vertreter aufgenommenen Bilder am Tag der Einbettung und zwei Tage später. D Relative Invasion wurde als Gebiet Unterschied am Tag 2 minus Tag 0 quantifiziert. Die Ergebnisse ausgedrückt werden als Mittelwert ± sd ab 13 angepasst. <img alt="Abbildung 4a" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="Abbildung 4b" src="/files/ftp_upload/3337/3337fig4b.jpg" /> Abbildung 4. Basal-und TGF-β-induzierte Sphäroid Invasion gehemmt by SB-431542. M1, M2 und M4 Sphäroide wurden in Kollagen eingebettet und mit TGF-β 5ng/ml und / oder 10 uM SB-431542, wie angegeben. Vertreter Bilder nach 2 Tagen (A) übernommen. Relative Invasion wurde als Gebiet Unterschied am Tag 2 minus Tag 0 (B) quantifiziert. Die Ergebnisse sind als Mittelwert ± sd von 13 Adapted ausgedrückt.

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Wir haben ein Sphäroid Modell, in dem MCF10A1 Zellen und ihre bösartigen Derivate in eine Kollagen-Matrix in einem TGF-β-abhängigen Weise zu erobern. Erstens sind Sphäroide in 96-Wells mit rundem Boden Aufhängeplatten in Gegenwart von Methylcellulose gebildet. Natürlich können auch U-förmigen Poly-HEMA beschichteten Platten für diesen Zweck verwendet werden. Methylcellulose ist absolut in dieser Schritt erforderlich, da die Zellen in Abwesenheit von Methylcellulose sterben. TGF-β ist direkt an die Kollagen-Me...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Methylcellulose </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Erweiterte Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> pH-Indikatorstreifen </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96-Loch-Aufhängung Platten </td><td> Greiner Bio-One </td><td> 650 185 </td><td></td></tr><tr><td> 96-well Haftung TC Platten </td><td> Greiner Bio-One </td><td> 655 180 </td><td></td></tr></tbody></table>

spheroid assay to measure tgf-&#946;-induced invasion

TGF-β hat entgegengesetzte Rollen bei Brustkrebs Progression, indem sie als Tumorsuppressor in der Anfangsphase, aber anregend Invasion und Metastasierung zu einem späteren Zeitpunkt 1,2. Darüber hinaus ist TGF-β häufig bei Brustkrebs überexprimiert und seine Expression korreliert mit einer schlechten Prognose und Metastasierung 3,4. Die Mechanismen, durch die TGF-β induziert Invasion nicht gut verstanden sind. TGF-β entlockt seiner zellulären Reaktionen via TGF-β Typ II (TβRII) und Typ I (TβRI)-Rezeptoren. Bei TGF-β-induzierte heteromeren Komplexbildung, phosphoryliert TβRII die TβRI. Die aktivierte TβRI initiiert seine intrazelluläre kanonischen Signalweges durch Phosphorylierung Rezeptor Smads (R-Smads), dh Smad2 und Smad3. Diese aktivierten R-Smads Form heteromeren Komplexe mit Smad4, die im Zellkern ansammeln und regulieren die Transkription von Zielgenen 5. Zusätzlich zu den zuvor described Smad Signalweg, Rezeptor-Aktivierung führt zur Aktivierung von mehreren anderen nicht-Smad Signalwege, zB Mitogen Activated Protein Kinase (MAPK) Signalwege 6. Zur Untersuchung der Rolle der TGF-β in verschiedenen Stadien des Brustkrebses, benutzten wir die MCF10A Zellsystem. Dieses System besteht aus spontan immortalisierte MCF10A1 (M1) Brustepithelzellen 7, verwandelte die H-RAS M1-derivative MCF10AneoT (M2), die Präkanzerosen produziert in Mäusen 8 und die M2-Derivat MCF10CA1a (M4), die von gegründete M2 Xenotransplantate und Formen hochgradige Karzinome mit der Fähigkeit, die Lunge 9 metastasieren. Diese MCF10A Serie bietet die Möglichkeit, die Reaktionen der Zellen mit verschiedenen Graden der Bösartigkeit, die nicht durch einen anderen genetischen Hintergrund sind voreingenommen zu studieren. Für die Analyse der TGF-β-induzierte Invasion, generierten wir homotypische MCF10A Sphäroid Zellkulturen embedded in einer 3D-Kollagen-Matrix in vitro (Abb. 1). Solche Modelle ähneln menschlichen Tumoren in vivo durch die Errichtung eines Gradienten von Sauerstoff und Nährstoffen, was aktive und invasive Zellen auf der Außenseite und Ruhe oder auch abgestorbene Zellen in das Innere des Sphäroids 10. Spheroid basierte Assays haben auch gezeigt, dass bessere rekapitulieren drug resistance als Monolayer-Kulturen 11. Diese MCF10 3D-Modell-System erlaubt es uns, die Auswirkungen von TGF-β-Signalisierung auf der invasiven Eigenschaften von Brust-Zellen zu untersuchen in verschiedenen Stadien der Bösartigkeit.

Watch this Scientific Journal Video about Spheroid Assay to Measure TGF-Î²-induced Invasion at JoVE.com

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

Ein Test zur quantitativen Messung Transforming Growth Factor (TGF)-β-induzierte Invasion im 3-dimensionalen Kollagen-Gelen beschrieben. Dieser Assay nutzt die MCF10A Reihe von Zelllinien, die verschiedenen Stadien der Entwicklung von Brustkrebs darstellen. Diese Methode kann angenommen, um mit anderen Zelllinien verwendet werden und könnte verwendet werden, um andere potenzielle Aktivatoren oder Inhibitoren der Invasion zu untersuchen sein.

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 ...

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

21.7K Aufrufe.  Leiden University Medical Centre. Ein Test zur quantitativen Messung Transforming Growth Factor (TGF)-β-induzierte Invasion im 3-dimensionalen Kollagen-Gelen beschrieben. Dieser Assay nutzt die MCF10A Reihe von Zelllinien, die verschiedenen Stadien der Entwicklung von Brustkrebs darstellen. Diese Methode kann angenommen, um mit anderen Zelllinien verwendet werden und könnte verwendet werden, um andere potenzielle Aktivatoren oder Inhibitoren der Invasion zu untersuchen sein.

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

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Eine Echtzeit-Elektrische Impedanz basierte Technik zur Invasion von Endothelzellen Monolayer von Krebs Zellen zu messen

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Forschung

Medizin

Method to Measure Tone of Axial and Proximal Muscle

The control of tonic muscular activity remains poorly understood. While abnormal tone is commonly assessed clinically by measuring the passive resistance of relaxed limbs1, no systems are available to study tonic muscle control in a natural, active state of antigravity support. We have developed a device (Twister) to study tonic regulation of axial and proximal muscles during active postural maintenance (i.e. postural tone). Twister rotates axial body regions relative to each other about the vertical axis during stance, so as to twist the neck, trunk or hip regions. This twisting imposes length changes on axial muscles without changing the body's relationship to gravity. Because Twister does not provide postural support, tone must be regulated to counteract gravitational torques. We quantify this tonic regulation by the restive torque to twisting, which reflects the state of all muscles undergoing length changes, as well as by electromyography of relevant muscles. Because tone is characterized by long-lasting low-level muscle activity, tonic control is studied with slow movements that produce "tonic" changes in muscle length, without evoking fast "phasic" responses. Twister can be reconfigured to study various aspects of muscle tone, such as co-contraction, tonic modulation to postural changes, tonic interactions across body segments, as well as perceptual thresholds to slow axial rotation. Twister can also be used to provide a quantitative measurement of the effects of disease on axial and proximal postural tone and assess the efficacy of intervention.

We have developed a device (Twister) to study the regulation of tonic muscle activity during active postural maintenance. Twister measures torsional resistance and muscular responses in standing subjects during twisting of the body axis. The device can be flexibly configured to study various aspects of tonic control across the neck, trunk, and/or hips.

Methode, um Tone von Axial-und der proximalen Muskulatur messen

The control of tonic muscular activity remains poorly understood.  While abnormal tone is commonly assessed clinically by measuring the passive resistance ...

Accuracy in Dental Medicine, A New Way to Measure Trueness and Precision

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations. The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 &#177; 1.1 &#181;m for trueness and 1.6 &#177; 0.6 &#181;m for precision in case of full arch scans. Current dental impression methods showed much higher deviations (trueness: 20.4 &#177; 2.2 &#181;m, precision: 12.5 &#177; 2.5 &#181;m) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss. The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes.

Accuracy is a major demand in dental medicine. To verify accuracy, reference scanners are needed. This article presents a new reference scanner with an adjusted scanning method to acquire a broad variety of dental morphologies with high trueness and precision.

Genauigkeit in Zahnmedizin, einen neuen Weg zu Richtigkeit und Präzision messen

Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base ...

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 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Beurteilung von Eierstockkrebs Sphäroid Bindung und Invasion von Mesothelzellen in Echtzeit

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

Intrauterine Telemetrie zur Maus Kontraktionsdruck messen<em&gt; In-vivo-</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

Three-Dimensional 3D Tumor Spheroid Invasion Assay

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

Dreidimensionale (3D) Tumor Spheroid Invasionsassay

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

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

Küken-Herz-Invasion Assay zum Testen der Invasivität von Krebszellen und die Aktivität von Potenziell Anti-invasive Verbindungen

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

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 ...

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.&#160;

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer

Perineural invasion is an aggressive phenotype for head and neck squamous cell carcinomas and other tumors. The chick chorioallantoic membrane model has been used for studying angiogenesis, cancer invasion, and metastasis. Here we demonstrate how this model can be utilized to assess perineural invasion in vivo.

Das Chick Chorioallantoic Membrane In Vivo Modell zur Beurteilung der perineuralen Invasion bei Kopf- und Halskrebs

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous ...