The authors acknowledge funding sources from D&#8217;Youville College School of Pharmacy, Buffalo, NY.

1. Préparer les différents médias et Composants supplémentaires <ol><li> Avant l&#39;expérience, préparer des milieux consistant en DMEM ou un autre support spécifiés avec l&#39;addition de FBS soit normale, FBS charbon de bois, charbon ou FBS plus le composant à tester. Notez que plusieurs composants peuvent être testés dans chaque expérience. </li><li> Peser et diluer les hormones, les facteurs de croissance, cytokines ou de façon appropriée pour être dissous dans le sérum dépouillé de charbon de bois à la concentration physiologique. </li></ol> 2. Préparer la Matrice Collagène on Ice <ol><li> Préparation 2 ml collagène I matrice, à 2,2 mg / ml en ajoutant les composants suivants stérilisées par filtration sur de la glace: 10 x 200 pi de PBS (pH 7,4), 5,4 pi de NaOH 1 N, 600 pi d&#39;eau distillée deux fois 2 O, et 1,2 ml de collagène de type I (à 3,63 mg / ml). </li><li> Conserver la solution de collagène I sur la glace jusqu&#39;au moment de l&#39;assiette. </li></ol> 3. Préparer la migration / invasion Plaques pour le dosage &lt;/ P&gt; <ol><li> Pour chaque lignée cellulaire à tester, en utilisant une plaque de chambre de 24 puits à puits qui 12 contiennent des inserts. Utilisez les 12 puits supplémentaires qui ne contiennent pas inserts pour ajouter les médias chimioattractives et le transfert des inserts pour la mise en place expérimentale. REMARQUE: une matrice de collagène sur des plaques avec un polyéthylène téréphtalate (PET) et la membrane 8 um de taille de pore est optimale pour les lignées cellulaires utilisent ici. Cependant, une matrice de matrigel dans des plaques pré-revêtues peut également être substitué par la taille des pores en fonction de la diminution de la lignée cellulaire à l&#39;étude. </li><li> Identifiez clairement la plaque, en utilisant trois puits par condition étant analysés (migration FBS, migration CS-FBS, invasion FBS, et d&#39;invasion CS-FBS ainsi que FBS-migration pour un contrôle, la lignée cellulaire non invasive). Dosage par déplacement migration à travers les pores d&#39;une membrane en PET, et l&#39;invasion de dosage par circulation à travers une matrice de collagène ou de matrigel et ensuite à travers les pores dans la membrane. Utiliser différents marqueurs de couleur pour chaque état de la cellule à unid dans le processus de placage. </li></ol> 4. Verser l&#39;invasion Matrice <ol><li> Pipetter prudemment 75 ul de la solution de matrice de collagène dans les inserts à être utilisés pour des essais d&#39;invasion. Faites preuve de prudence pour éviter les bulles. Disperser les bulles par l&#39;application d&#39;une pointe de pipette à la surface inversée. </li><li> Transférer la plaque avec les inserts revêtus de collagène à un incubateur à CO2 à 37 ° C et 5% pendant 30 minutes pour permettre au gel de se solidifier. </li></ol> 5. Plaque les cellules sur la membrane ou d&#39;invasion Matrice <ol><li> Pendant ce temps, Trypsiniser cellules et ajouter du contenu multimédia avec 10% de FBS. Spin cellules à 200 g pendant 5 min sur une centrifugeuse de table et rincer 3x dans un milieu sans sérum. </li><li> Remettre en suspension dans un milieu sans sérum. Compter les cellules avec un hémocytomètre ou un comptoir de diaporama automatique. Ajouter un milieu sans sérum à une concentration finale de 5 x 10 4 cellules / ml. </li><li> Lorsque la matrice de collagène est solidifié (après 30 minutes), ajouter 700 ul de milieu avec soit2% de FBS ou définis charbon-FBS à chaque puits. Sur les 12 inserts par plaque: <ul><li> 3 inserts ont collagène et les puits avec les médias + 2% de FBS </li><li> 3 inserts ont collagène et les puits avec les médias + 2% CS-FBS </li><li> 3 inserts ne ont pas le collagène et les puits avec les médias + 2% de FBS </li><li> 3 inserts ne ont pas le collagène et les puits avec les médias + 2% CS-FBS </li></ul><ol><li> Utilisation des plaques supplémentaires en fonction du nombre de facteurs à tester et d&#39;une commande pas de collagène correspondant à chaque condition. </li></ol></li><li> Ajouter suspension cellulaire aux inserts à 5 x 10 4 cellules / ml, le placage 500 cellules par ul insert dans tous les inserts de migration et d&#39;invasion. </li><li> Incuber les cellules pendant 22 heures à 37 ° C. </li></ol> 6. quantifier le nombre de Migration et invasion des cellules <ol><li> Mettre en place la coloration des puits en utilisant du méthanol fixateur, l&#39;éosine, et hématoxyline, dans des puits séparés. </li><li> Utilisez des cotons-tiges pour éliminer les cellules et la matrice de chaque puits.Rrepeat avec deuxième application de l&#39;écouvillon pour chaque puits. </li><li> Avec une pince, tremper chaque insert 5 fois pendant 1 seconde dans chacune des trois solutions de suite. </li><li> Laisser sécher inserts O / N. </li><li> Soit i) supprimer des filtres avec un scalpel, couper soigneusement autour des bords et monter sur une lame avec lamelle et l&#39;immersion d&#39;huile, ou ii) les inserts permettent de sécher O / N inversé et utilisent les inserts directement pour la microscopie. </li><li> Le lendemain, voir des diapositives ou des inserts sous un microscope avec un objectif 20X et prendre cinq images de différentes régions du filtre. Pour améliorer la cohérence, de prendre quatre champs extérieurs et un centre. </li><li> Compter les cellules pour toutes les conditions en utilisant le logiciel ImageJ et appliquer les formules ci-dessous. </li><li> Déterminer l&#39;invasion de pour cent comme suit: # Moyenne des cellules envahissantes travers collagène je insère = a Moyenne # des cellules migrant à travers insert de commande = b Invasion% = (a / b) * 100 </li><li> Déterminer l&#39;indice Invasion de 2% de FBS comme suit: % Invasion des cellules en cours d&#39;analyse (à 2% de FBS) = c % Invasion de cellules de contrôle non invasifs en (2% de FBS) = d Invasion Index (FBS) = (c / d) </li><li> Déterminer l&#39;indice Invasion 2% CS-FBS comme suit: % Invasion des cellules en cours d&#39;analyse (dans 2% CS-FBS) = e % Invasion des cellules non invasives de contrôle dans (2% CS-FBS) = f Indice Invasion (CS-FBS) = (e / f) </li></ol> 7. Répétez Protocole expérimentale visant à comparer charbon-FBS FBS charbon-dénudés + X n avec de multiples facteurs combinés <ol><li> Répéter la procédure plusieurs fois que nécessaire en utilisant différents composants de &quot;X&quot; ou une combinaison de composants. </li><li> Appliquer les calculs pour déterminer la contribution de chaque facteur &quot;X&quot; pour les effets de la migration et d&#39;invasion. </li></ol>

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The authors have nothing to disclose.

Tumeur métastase est un processus en plusieurs étapes. Les cellules doivent franchir la membrane basale, intravasate soit dans le système lymphatique ou le sang microvaisseaux, dans lesquels ils sont transportés à des sites distants. Les cellules tumorales puis extravasent et colonisent dans un macrométastase 12. La progression à travers la transition épithélio-mésenchymateuse (EMT), l&#39;invasion tumorale et la métastase a été renforcée par des hormones stéroïdes 13,14, les facteu...

Un nouveau dosage d&#39;invasion a été développé dans le but d&#39;identifier l&#39;implication de certaines hormones, des cytokines et / ou facteurs de croissance chimiotactiques comme dans l&#39;invasion des cellules cancéreuses. La capacité des cellules tumorales à envahir à travers la matrice extracellulaire (ECM) est une caractéristique du phénotype métastatique 3.1. chambres d&#39;invasion ont été largement utilisés comme outils pour étudier in vitro l&#39;invasion des cellules cancéreuses et la migration et peuvent fournir des connaissances sur les mécanismes de l&#39;invasion et la métastase tumorales in vivo. Les chambres sont constituées d&#39;inserts de culture de cellules cylindriques emboîtés dans les puits de plaques de culture de cellules. Le fond des inserts en polycarbonate ou polystyrène sont des membranes semi-perméables des tailles de pores définies. Dans l&#39;essai d&#39;invasion standard, les cellules sont ensemencées sur la membrane de l&#39;insert avec du milieu sans sérum et on les place dans des puits de culture de cellules qui sont remplis de sérum ou de sérum comme agents chimio-attractifs pour set jusqu&#39;à un chimioattractives vigueur. Cela conduit de force cellules de se déplacer à travers la membrane semi-perméable (migration) ou encore, par un revêtement de la matrice extracellulaire (invasion), qui peut ensuite être teinté avec des colorants classiques pour détecter et quantifier le nombre de cellules. Le nombre de cellules est ensuite normalisée en fonction de l&#39;étendue de la migration et de l&#39;invasion dans une lignée cellulaire non invasive. Cette méthode permet à un chercheur d&#39;évaluer le potentiel invasif de différents types de cellules dans une variété de conditions, y compris les manipulations génétiques. Les hormones, les cytokines et les facteurs de croissance sont des éléments essentiels de sérum et sont représentés plus en plus à être associé à l&#39;entraînement du phénotype invasif 4. Cependant, la nature, le rôle et la spécificité de ces molécules sériques chimioattractives dans la médiation invasion encore demeurent insaisissables. Le défi reste à déterminer quels facteurs spécifiques dans le sérum ou de sérum comme chimioattractifs sont responsables de la conduiteinvasion des cellules cancéreuses ainsi que des molécules qui peuvent inhiber le processus d&#39;invasion. Dans ce rapport, nous décrivons une méthodologie pour évaluer l&#39;implication potentielle des hormones, des cytokines et / ou facteurs de croissance présents dans le sérum, en tant que molécules de conduire l&#39;chimioattraction et l&#39;invasion des cellules cancéreuses. Dans ce protocole modifié proposé, le charbon de décapage est utilisé pour éliminer les hormones, les cytokines et les facteurs de croissance de 2% de sérum bovin fœtal (FBS) pour générer 2% FBS charbon de bois (CS-FBS). Les deux 2% de FBS et 2% CS-FBS sont utilisés comme agents de mettre en place la force chimioattractives à conduire l&#39;invasion des cellules cancéreuses. Utilisation de 2% de CS-FBS comme agent chimio-attractifs par rapport à la normale 2% de FBS offre plusieurs avantages, y compris d&#39;une part, et le plus en évidence, la possibilité d&#39;étudier le phénotype d&#39;invasion du cancer en l&#39;absence réduit / relative des hormones, des cytokines et des facteurs de croissance. Il permet également à l&#39;enquêteur de déterminer si l&#39;élimination collective des hormones, facteurs de croissance, et CYtokines entraîne une augmentation ou diminution de l&#39;indice invasive. Le dosage est alors conçue pour déterminer si l&#39;addition d&#39;un composant individuel, désignée comme «X» peut inhiber, diminuer, augmenter ou rétablir l&#39;indice invasive à sa valeur initiale (voir les figures 2 et 3). Bien que cette méthodologie est spécifique pour atteindre les niveaux physiologiques de l&#39;élément qui est éliminée à partir du sérum au cours de charbon décapage, il convient également de noter que le charbon de décapage peut aussi affecter les voies de transduction du signal. Par exemple, il a été rapporté que le charbon de décapage peut diminuer l&#39;activité de la phosphatase alcaline de cellules ostéoprogénitrices et induire l&#39;adipogenèse par la réduction des activateurs de 5 MAPK. Charcoal médias dénudées sont commercialement disponibles et sont basés sur le protocole décrit qui combine charbon dextran et incubées avec du sérum fœtal bovin O / N 6. Cet essai a été développé en Response à un résultat rapporté par Zucker et al. 7 Les auteurs ont démontré que le phénotype mutant affectant une communication écart de jonction fait preuve d&#39;une augmentation de l&#39;indice d&#39;invasion lors de la migration vers la normale médias avec 2% de FBS. Cette augmentation a été éliminée avec le remplacement de sérum de charbon-dépouillé. De ce fait, les auteurs ont conclu que ce mutant affecté migration vers une molécule lipophile (plus spécifiquement, une hormone, un facteur de croissance ou cytokines) 7. Il est bien connu que les hormones, facteurs de croissance, cytokines et médiateurs voies impliquées dans la promotion de la tumeur et invasion quatre signalisation. Ainsi, il est important pour les chercheurs scientifiques pour déterminer quel facteur (s) sont le moteur de l&#39;invasion tumorale des cellules cancéreuses ou des mutants particulier à l&#39;étude. L&#39;essai est conçu pour examiner le rôle des composants individuels lors de leur concentration physiologique telle que déterminée en fonction de la différence entre la concentration du composant et# 8220; X &quot;dans FBS normale et le CS-FBS. Grâce au développement de ce test, les enquêteurs peuvent gagner plus de perspicacité dans la voie invasive spécifique régissant leur système. Des hormones, des facteurs de croissance, cytokines et ont souvent été considérés comme des promoteurs de tumeurs 8. Certains de ces facteurs tels que EGF sont utilisés comme sources directes pour chimioattractifs dans des chambres d&#39;invasion neuf. Ainsi, il semble probable que ceux-ci représentent les principales composantes du sérum qui invasion tumorale directe. Ce protocole propose un moyen simple, mais significative, de la modification de l&#39;essai in vitro classique dans d&#39;invasion qui permet à un chercheur pour évaluer l&#39;implication des hormones, des facteurs de croissance, des cytokines dans la médiation et le potentiel invasif des cellules cancéreuses. Cependant, le test est conçu pour fournir à l&#39;enquêteur une réponse quant à savoir si la procédure sera efficace pour leur étude à une étape précoce dans l&#39;analyse, afin de ne pas utiliser un temps précieux et ressources si jugées inutiles. Cette méthode utilise CS-FBS et se appuie sur la discrétion des enquêteurs à laquelle les molécules de poursuivre en tant que candidats de plomb qui interviennent potentiellement invasion des cellules cancéreuses. Les résultats de ces analyses devraient se avérer utiles dans l&#39;identification des composants du sérum qui servent d&#39;agents chimio-attractifs ou des inhibiteurs de la lignée cellulaire particulière ou un mutant de l&#39;étude. En outre, cette approche peut aider l&#39;enquêteur d&#39;identifier les voies de signalisation clés soit favoriser ou inhiber l&#39;invasion des cellules cancéreuses; dirigeant ainsi l&#39;avenir la conception de médicaments.

<table border="0" cellpadding="0" cellspacing="0" width="987">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:367px;">Item</td>
			<td style="width:195px;">Vendor</td>
			<td style="width:136px;">Catalog #</td>
			<td style="width:289px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td>BioCoat Control Inserts with 8.0&#181;m PET Membrane 
			(12 in each of two 24-Well Plates)</td>
			<td>Corning</td>
			<td>354578</td>
			<td>polyethylene teraphthalate (PET) membranes&#160;&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Collagen I, rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td>Source = rat tail tendon, purity = 90%</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fixative; Diff-Quick</td>
			<td>Thermo Fisher Scientific</td>
			<td>NC9844047</td>
			<td>Methanol-based fixative, Hmatoxylin/Eosin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>12676</td>
			<td>Designated as CS-FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Fetal bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30070</td>
			<td>Designated as FBS</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Charcoal Stripped Fetal Bovine Serum</td>
			<td>Thermo Scientific Hyclone</td>
			<td>SH30068</td>
			<td>Designated as CS-FBS</td>
		</tr>
	</tbody>
</table>

a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion

Le sérum sanguin sert de chimioattractant vers laquelle les cellules cancéreuses migrent et envahissent, ce qui facilite leur intravasation en microvaisseaux. Cependant, les molécules réelles vers lequel les cellules migrent demeurent insaisissables. Ce test d&#39;invasion modifié a été mis au point pour identifier les cibles qui entraînent la migration et l&#39;invasion cellulaire. Cette technique compare l&#39;indice d&#39;invasion dans trois conditions pour déterminer si une hormone spécifique, le facteur de croissance ou cytokine joue un rôle dans la médiation le potentiel invasif d&#39;une cellule cancéreuse. Ces conditions comprennent i) de sérum fœtal bovin normal (FBS), ii) charbon-FBS (CS-FBS), qui supprime les hormones, les facteurs de croissance et des cytokines et iii) molécule CS-FBS + (notée &quot;X&quot;). Un changement important dans l&#39;invasion des cellules CS-FBS avec par rapport à FBS, indique l&#39;implication des hormones, des cytokines ou des facteurs de croissance dans la médiation de la modification. Les molécules individuelles peuvent ensuite être ajoutés revenir à CS-FBS pour doser leur capacité à reverset ou sauver le phénotype d&#39;invasion. En outre, deux ou plusieurs éléments peuvent être combinés pour évaluer l&#39;effets additifs ou synergiques de plusieurs molécules dans la conduite ou l&#39;inhibition de l&#39;invasion. Dans l&#39;ensemble, ce procédé permet au chercheur de déterminer si les hormones, les cytokines et les facteurs de croissance / ou jouent un rôle dans l&#39;invasion cellulaire en agissant comme agents chimio-attractifs ou des inhibiteurs de l&#39;invasion d&#39;un type particulier de cellule cancéreuse ou un mutant spécifique. En identifiant chimiotactiques et des inhibiteurs spécifiques, ce dosage d&#39;invasion modifié peut aider à élucider les voies qui dirigent l&#39;invasion des cellules du cancer de signalisation.

L&#39;indice d&#39;invasion est calculé pour chaque condition de normalisation selon l&#39;une lignée cellulaire non invasive. Pour les expériences, on utilise la ligne 1205Lu cellulaire de mélanome et les lignées cellulaires stables variantes établi comme nos lignes envahissantes ainsi que la variante non invasive prémalignes, WM793 partir duquel les cellules ont été dérivées 1205Lu 10 qui sert de contrôle logique. Nous utilisons également collagène I que la matrice d&#39;invasion parce que ce ...

Watch this Scientific Journal Video about A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion at JoVE.com

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.

Une modification<em&gt; In vitro</em&gt; Invasion Test pour déterminer le rôle potentiel des hormones, des cytokines et / ou facteurs de croissance dans le cancer Médiation invasion cellulaire

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

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Research

JoVE Journal

Medicine

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

10.6K vues.  Roswell Park Cancer Institute. 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.

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

Une technique basée Impédance électrique en temps réel pour mesurer Invasion de monocouche de cellules endothéliales par les cellules cancéreuses

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

Recherche

Médecine

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Une In Vitro Système d&#39;étude dormance tumorale et le commutateur à la croissance métastatique

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

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-photon imagerie de l&#39;invasion des cellules tumorales dans un modèle de souris orthotopique du carcinome épidermoïde oral

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.

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

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

Assay Sphéroïde pour mesurer le TGF-β induit 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.

Modélisation et Imagerie en 3 dimensions l&#39;invasion des cellules collectives

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.

Test in vitro avec dégagement mésothéliales que les modèles les étapes précoces de métastases du cancer de l&#39;ovaire

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 ...

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.

Évaluation de Cancer de l&#39;ovaire Spheroid attachement et l&#39;invasion des cellules mésothéliales en temps réel

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.

Concentrez Formation: Un test cellulaire pour déterminer le potentiel oncogène d&#39;un gène

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

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Mise en place d&#39;un modèle de xénogreffe humain de myélome multiple dans le poulet pour étudier la croissance tumorale, l&#39;invasion et l&#39;angiogenèse

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

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.

Tridimensionnelle (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 ...