Supportato dal NIH sovvenzioni P30 CA051008 e T32 CA009686 (ATR).

1. Generazione di Spheroids <ol><li> Preparare cella singola sospensione per appendere le culture goccia staccando colture di cellule di cancro aderenti del ~ 70% di confluenza con un PBS lavaggio seguito da esposizione a 0,05% soluzione di tripsina-EDTA. <ol><li> Neutralizzare la soluzione di tripsina con colture cellulari e contare le cellule utilizzando una aliquota della sospensione cellulare. Nota: I mezzi di coltura cellulare specifico dipenderà dalla linea cellulare in fase di test. Seguire ATCC raccomandazioni dei media, dove i media di coltura cellulare è tipicamente DMEM + 10% FBS. Ulteriori informazioni si possono trovare nella tabella dei materiali. </li><li> Eseguire una diluizione per consentire la semina di 500-1.000 cellule per 20 microlitri goccia di colture cellulari. </li></ol></li><li> Acquisire 10 centimetri piatto e aggiungere 5 ml di PBS sterile fino in fondo. Nota: Questo passaggio protegge l&#39;impiccagione scende da evaporazione. A costi inferiori &quot;batteriologiche grade&quot; piatti può essere utilizzato al posto di piatti di coltura cellulare di grado, come le cellule non sarà contagire sulla superficie cultura. </li><li> Utilizzare una pipetta multicanale per trasferire 20 gocce microlitri della sospensione cellulare diluita alla superficie interna del coperchio. <ol><li> Pipettare 40 gocce (5 righe di 8 gocce) sul coperchio del piatto 10 cm. </li></ol></li><li> Capovolgere il coperchio e posizionare sopra la capsula di Petri. Incubare le culture goccia che pendono a 37 ° C per 72 ore per generare sferoidi. <ol><li> Capovolgere il coperchio in un fiducioso, ancora modo controllato. Nota: Inversione il coperchio troppo veloce o troppo lento può causare le goccioline a spostare. Alcune linee cellulari possono formare sferoidi in 48 ore o meno, mentre altri possono richiedere più di 72 ore per la produzione di aggregati compatti. </li></ol></li></ol> 2. Integrazione di Spheroids in 3D Matrix <ol><li> Scongelare una aliquota di crescita seminterrato materiali per membrane fattore ridotto a 4 ° C per una notte prima di incorporare sferoidi. <ol><li> Opzionale: pozzi di livello con ECM in anticipo per evitare una possibile interazione con il sferoide cultu tessutire di superficie. <ol><li> Pipettare 200 ml di ECM per pozzetto su una piastra da 24 pozzetti. </li><li> La piastra di assicurare che l&#39;ECM copre l&#39;intera superficie ben. </li><li> Rimuovere con cura l&#39;eccesso ECM con una pipetta. </li><li> Incubare la piastra fino a pozzi sono asciutti: 3 ore a temperatura ambiente o per una notte a 4 ° C. </li></ol></li></ol></li><li> Raccogliere sferoidi inclinando il coperchio del piatto di coltura tissutale e condivisione del media. Trasferire i media con sferoidi in una provetta da 1,5 ml microcentrifuga. </li><li> Lasciare 10 min per sferoidi di depositarsi sul fondo della provetta. Spheroids dovrebbero essere visibili ad occhio. </li><li> Mescolare 100 ml di materiali membrana basale con 100 ml di freddo (4 ° C), collagene di tipo I in un tubo di pre-raffreddata separata. Tenere la miscela a 4 ° C per prevenire o gel dalla solidificazione prematuramente. <ol><li> Utilizzare puntali pre-refrigerati se il trasferimento di piccoli volumi. </li><li> Dare attenzione quandomescolando il tipo di materiali membrana basale e collagene che per evitare la formazione di bolle d&#39;aria. Nota: Le bolle d&#39;aria possono ostacolare l&#39;imaging più tardi nel protocollo, se esse sono incorporati nel gel. </li></ol></li><li> Aspirare sferoidi dal 40 microlitri porzione di fondo della provetta, e si combinano con il materiale della membrana basale / collagene di tipo I miscela. Fare attenzione per evitare la formazione di bolle d&#39;aria in fase di missaggio. Nota: La soluzione contenente 100 microlitri materiali membrana basale, 100 ml di tipo collagene I, e 40 ml di sferoidi creerò materiale sufficiente per 4 culture 3D indipendenti. Questo può essere scalata su o giù. </li><li> Pipettare 40 gocce microlitri della miscela viscosa in centri di pozzetti in una piastra da 24 pozzetti. Mantenere il livello piastra per evitare che il composto scorre verso il lato del pozzo. Nota: Una singola colonna a 24 pozzetti - 4 pozzi - è consigliato per ogni condizione da testare. </li><li> Posizionare la piastra di intoa 37 ° C incubatore e lasciare riposare per 30 minuti. La cultura 3D dovrà polimerizzare durante questo periodo. </li><li> Lentamente immergere le culture 3D in 1 ml di coltura cellulare multimediale. <ol><li> Assicurarsi che il supporto è caldo quando aggiungerlo nei pozzetti per promuovere ulteriormente il gel di polimerizzazione. </li><li> PUNTO CRITICO: aggiungere delicatamente il supporto alle culture 3D. Pipettaggio i media troppo in fretta nei pozzetti può portare al distacco delle culture 3D dalla superficie coltura di tessuti. </li></ol></li></ol> 3. Monitoraggio e analisi Spheroid Invasion <ol><li> Invasione Immagine da sferoidi nella matrice 3D circostante a punti temporali decisi dallo sperimentatore. Acquisire fotografie utilizzando un microscopio invertito con la lente dell&#39;obiettivo 20x. Nota: ideali punti temporali variano a seconda della linea cellulare in fase di test. Linee cellulari più invasive inizieranno la loro uscita dagli sferoidi poco dopo la placcatura e, di conseguenza, prendere fotografia iniziales entro un paio d&#39;ore dopo la placcatura. In generale, le fotografie sono prese a 0 ore (dopo la placcatura), 24 ore e 48 ore. Invasione conclude tipicamente 24-48 ore dopo la placcatura. </li><li> Quantificare la capacità invasiva utilizzando il software di analisi di immagine come immagine J. <ol><li> Analizzare invasione come la distanza cella dal bordo dello sferoide. <ol><li> Utilizzare lo strumento Disegna Dritto per segnare il raggio e / o massima distanza invasivo. </li><li> Fai clic su &quot;Analizza&quot; nel menu in alto, e quindi fare clic su &quot;Misura&quot; per visualizzare la misura della lunghezza. </li></ol></li><li> Analizzare invasione come l&#39;area totale invasivo al di fuori della sferoide. <ol><li> Utilizzare lo strumento Disegno a mano libera per tracciare il confine dello sferoide e / o area totale invasivo. </li><li> Fai clic su &quot;Analizza&quot; nel menu in alto, e quindi fare clic su &quot;Misura&quot; per visualizzare la misura dell&#39;area. </li></ol></li><li> Utilizzare un plugin come ROI Manager Tools per creare misure per una pila di immagini. </li></ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Questo studio ha valutato le prestazioni di un pannello di linee cellulari di cancro in un saggio di invasione sferoide (Tabella 1). Generalmente, troviamo che la formazione sferoide è migliorata in più linee cellulari epiteliali cercando, dove la presenza di giunzioni cellula-cellula promuove la formazione di un&#39;architettura sferoide-like. Linee cellulari noti che hanno subito una transizione epitelio-mesenchimale, come MDA-MB-231 cellule non formano sferoidi nella cultura goccia appeso molto pro...

È accertato che la motilità delle cellule tumorali è predittiva di potenziale metastatico, dato che tale comportamento facilita l&#39;invasione attraverso la membrana basale e l&#39;ingresso nel sistema circolatorio 1. Maggior parte delle ricerche sulla motilità si è concentrata su come le cellule si comportano in un ambiente bidimensionale (2D), anche se sta diventando ampiamente riconosciuto che il movimento delle cellule in una tridimensionale (3D) matrice è più rappresentativo di come queste stesse cellule saranno effettivamente comportarsi in vivo 2. Sistemi di coltura 3D sono sempre più utilizzati per studiare i comportamenti cellulari che vanno dalla morfologia delle cellule, di cinetica di crescita e sensibilità ai farmaci 3. Il desiderio di controllare l&#39;invasione delle cellule del cancro nel contesto di un ambiente 3D ha portato alla sintesi di tecniche precedentemente stabilito che comportano la generazione di aggregati di cellule 3D (sferoidi) tramite il metodo di coltura goccia pendente 4, seguita da incorporare questi sferoidi in un extracellulare 3D matrice (ECM), composto di collagene e membrana basale materiali 5. Questo metodo cerca di migliorare queste tecniche precedentemente stabiliti fornendo un approccio semplificato che può essere facilmente utilizzato per confrontare invasione sotto una varietà di condizioni sperimentali. Altri modi tradizionali per valutare la motilità cellulare in vitro sono il saggio di graffi ferita e il dosaggio transwell 6. Il primo test raffigura motilità cellulare in un ambiente 2D, ed è quindi indipendente di una varietà di caratteristiche critiche per l&#39;invasione in vivo, ad esempio attività della proteasi 7. Il test può transwell invasione delle cellule modello migliore quando gli inserti e sono rivestiti con i substrati ECM, ma solo un unico parametro, cioè la comparsa di cellule sulla superficie della membrana opposta è misurata, e molte sfumature di invasione delle cellule non sono così facilmente osservabile. In contrasto con queste tecniche, il saggio delle cellule del cancro invasione sferoide (Figura 1 &lt;/ strong&gt;) permette per il monitoraggio in tempo reale di invasione delle cellule in un ambiente che non è solo fisiologicamente rilevanti, ma permette anche importanti caratteristiche specifiche della linea di cellule per essere visualizzati, come la migrazione delle cellule individuale contro collettiva 8. Questo metodo permette anche vantaggi rispetto ai saggi di crescita della cultura 3D standard. La generazione di aggregati cellulari tramite il metodo goccia appeso limita inizialmente il movimento delle cellule, in modo che le cellule saranno incentivati ​​ad invadere dopo questo vincolo viene sollevato. Inoltre, una volta che vincolo viene sollevato, l&#39;uscita cella procederà in una direzione uniforme, che può quindi essere quantificata convenientemente. I materiali ECM più popolari utilizzati per cellulari tumorali sferoidi test sono Matrigel e collagene di tipo I, dove ognuna di queste componenti ha ruoli importanti e distinti a influenzare il comportamento metastatico. Matrigel è una miscela di secreto proteine ​​prodotte da cellule di sarcoma Engelbreth-Holm Sciame di topo, e si arricchisce in basemeproteine ​​di membrana nt quali laminina, entactina, e collagene di tipo IV 9. Per questo motivo Matrigel è seguito definita come &quot;materiali della membrana basale.&quot; Questi materiali forniscono membrana basale ligandi essenziali necessari per integrina adesione durante l&#39;invasione delle cellule del cancro 10 oltre a molte altre proteine ​​che esercitano una gamma di effetti sul comportamento cellulare 11. In confronto, collagene di tipo I, comunemente preparato da acido digest di tendini e altre strutture collagene dense 12, è un materiale di matrice molto più semplice che serve come un importante elemento strutturale dei tessuti e stroma sostegno tessuti connettivi ed organi del corpo. È stato dimostrato che le caratteristiche fisiche di collagene possono regolare un numero di caratteristiche di motilità cellulare; per esempio, l&#39;allineamento delle fibrille di collagene all&#39;interfaccia tumore stromale permette alle cellule tumorali di migrare successivamente lungo queste fibrille quando invadere nel stroma 13. Nel saggio qui presentato, sia di tipo I collagene e di materie membrana basale sono utilizzate come strumenti per studiare le interazioni cellula-stroma cancro 3D. L&#39;effetto di inibizione o stimolazione di vie che controllano invasione può essere monitorato dopo le cellule sono stati incorporati nella matrice 3D. Le celle possono essere pretrattati durante la crescita in gocce sospese o al momento del trasferimento alla cultura 3D, a seconda che un trattamento prolungato sarà richiesto per modulare invasione. Per i trattamenti brevi, si raccomanda che il farmaco essere miscelato con la sospensione sferoide dopo la raccolta, così come i mezzi che circonderà le culture 3D, per facilitare un&#39;adeguata esposizione al farmaco alle cellule. Successivamente, le cellule stromali normali o tumore-associati possono essere mescolati con il materiale di matrice per valutare il loro ruolo nel modulare l&#39;invasione delle cellule tumorali, o per determinare come il comportamento paracrino e autocrino cellule influenze segnalazione. Questa idea è stata mostrata in uno studio in cui la coculture di colsul cancro e le cellule endoteliali in appesi gocce portato ad una rete vascolare all&#39;interno sferoidi 14. Infine, i costituenti ECM possono anche essere modificati, come l&#39;invasione delle cellule tumorali è influenzata da diversi substrati 15. Il metodo presentato qui di seguito vi offre pertanto un quadro per la valutazione invasione delle cellule di cancro in una varietà di condizioni. In generale, si è riscontrato che non tutte le linee cellulari creeranno sferoidi nelle gocce appesi e linee cellulari epiteliali cercando tipicamente formare sfere regolare.

<table>
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-treated Dishes</td>
			<td>Corning Incorporated</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC-treated Plates</td>
			<td>NEST Biotechnlology</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX-71 Inverted Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td>
		</tr>
	</tbody>
</table>

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.

Usando il saggio di invasione sferoide (figura 1), un pannello di linee cellulari di tumore è stato testato per la loro capacità di formare sferoidi, così come per la quantità di fuoriuscita di cellule esposte dopo l&#39;impianto in una matrice 3D costituiti da materie membrana basale e collagene di tipo I (Tabella 1). Questi risultati dimostrano che non ogni linea cellulare di tumore creerà sferoidi ben formate, dove le linee cellulari che possiedono una morfologia epiteliale ...

Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

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

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.

Un Cancer Cell Saggio sferoide valutare Invasion in un Ambiente 3D

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

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32.4K Views.  Georgetown University. 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. 

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

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.

Un tempo reale impedenza elettrica tecnica basata su Misura Invasione di cellule endoteliali monostrato dalle cellule tumorali

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

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Medicina

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-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

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

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

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

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

Saggio sferoide Misura TGF-β-indotta Invasion

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

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modellazione e imaging 3-Dimensional Invasion cella Collettiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro test Clearance mesoteliali che modella i primi passi di metastasi del cancro ovarico

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificazione del cancro al seno Cellula Invasività Utilizzando una tridimensionale (3D) Modello

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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

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

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

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

A Modified<em&gt; In vitro</em&gt; Invasion test per determinare il ruolo potenziale di ormoni, citochine e / o fattori di crescita nella mediazione Cancer Invasion cellulare

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.

Valutazione del cancro ovarico Spheroid attaccamento e l&#39;invasione delle cellule mesoteliali in tempo reale

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

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Concentrarsi Formazione: Un test cellulare per determinare il potenziale oncogeno di un gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Infusi carotide di farmacocinetica e di farmacodinamica Analisi dei taxani nei topi

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...