This work was conducted with funds to M.B. from the Canadian Institutes of Health Research (CIHR) grant MOP 107972. M.B. is a recipient of a CIHR New Investigator Salary Award. D.C. is a recipient of studentships from the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, London Regional Cancer Program. C.G. is recipient of studentships from the Translational Breast Cancer Research Unit, London Regional Cancer Program and from the CIHR- Strategic Training Program in Cancer Research and Technology Transfer. SGD.

1. Culture tridimensionnelle de cellules de cancer du sein dans la membrane basale Matrix (La Technique Embedment) <ol><li> Manipulation Matrigel matrice de la membrane basale: Décongeler sur la glace pendant une nuit à 4 ° C. Basement membrane-matrice est liquide à basse température mais se solidifie à la température ambiante. Gardez sous-sol matrice de la membrane sur la glace (figures 1A-B). </li><li> Couvrir le plat à fond de verre n ° 1 confocale avec 50 pl de la matrice de la membrane basale par des moyens de diffusion de la matrice en utilisant la pointe d&#39;un P-200 Pipetman dans un motif en spirale (figure 1C). Faites preuve de prudence lors de l&#39;épandage de la matrice de la membrane basale pour éviter la formation de bulles d&#39;air. De même, éviter la propagation de la matrice de fermer les frontières du plat à fond de verre pour empêcher la formation de ménisque. Si la manipulation inexpérimentée sous-sol matrice de la membrane, puis refroidi au préalable le plat, P-200 Pipetman et pipettes peut être sur la glace (alternativement à gauche la nuit dans un réfrigérateur à 4 ° C). Cette étape offresun délai supplémentaire pour se propager de la matrice de membrane basale avant solidification. Placer la capsule (s) dans un incubateur de culture de cellules (à 37 ° C avec 5% de CO2) pendant au moins 30 min pour permettre à la matrice de membrane basale à se solidifier (figure 1D). </li><li> Bien que la matrice est en cours de solidification, trypsiniser une confluence de 100 mm plaque de cellules de 70-80%. Une fois que les cellules ont commencé à soulever hors de la plaque, les remettre en suspension dans 10 ml de milieu RPMI 1640 contenant 10% (v / v) de sérum bovin fœtal (FBS), afin d&#39;inactiver la trypsine (figure 1E). Puis transférer les cellules remises en suspension dans un tube conique de 15 ml (figure 1F). </li><li> Faire tourner les cellules présentes dans le tube (conique) à 100 g pendant 3 min dans une centrifugeuse de culture cellulaire spécifique (figures 1G-H). </li><li> Alors que les cellules sont centrifugées, aliquote de 50 ul de matrice dans un tube à centrifuger de 1 ml (note: chaque plat à fond de verre est attribué un tube à centrifuger; donc, sil&#39;expérience nécessite 3 plats, il s&#39;ensuit que 3 microtubes sont nécessaires aussi bien), puis placer le tube sur de la glace (figure 1B). </li><li> Aspirer le milieu du tube conique de l&#39;étape 1.4, tout en laissant le culot non perturbées (figure 1I). Resuspendre les cellules (qui ont été centrifugées) dans 1 ml de milieu RPMI supplémenté de FBS (figure 1J). </li><li> Une fois que les cellules sont comptées en utilisant un hémocytomètre (figure 1K), ou un compteur aliquote de particules de cellule 2,5 x 10 4 cellules dans le tube à centrifuger et couronner le tout en utilisant les médias appropriés de façon à obtenir le volume total de 50 pi (figure 1L). </li><li> Mélanger les cellules provenant de l&#39;étape 1.7 (25 000 cellules dans 50 ul) avec le tube à centrifuger contenant une matrice à partir de l&#39;étape 1.5 dans un rapport de 1:1; volume final sera de 100 pi (figure 1M). </li><li> La plaque doucement 100 pl de la matrice: mélange de cellules provenant de l&#39;étape 1.8 sur la smembrane basale plat de matrice revêtu olidified de l&#39;étape 1.2 (Figure 1 N). Ceci permet pour les cellules devant être enrobées dans une matrice de membrane basale. </li><li> Transférer la capsule (s) dans l&#39;incubateur de culture de cellules (à 37 ° C avec 5% de CO 2) et de permettre la matrice: mélange de la cellule de se solidifier pendant au moins 30 min. </li><li> Une fois la matrice: mélange de cellules est solidifiée, ajouter 2 ml de milieu RPMI FBS complétés à l&#39;antenne (figure 1O) et prendre le plat arrière de l&#39;incubateur où il sera stocké pour le reste de l&#39;expérience (figure 1P). </li><li> Changer médias tous les jours pendant une période de 5 jours (ou selon requis pour une analyse; la durée de l&#39;essai pour les cellules MDA-MB-231 cellules est de 5 jours de longueur). </li><li> L&#39;utilisation d&#39;un microscope optique, prendre interférence différentiel (DIC) des images des cellules MDA-MB-231 colonies en suspension dans la matrice de la membrane basale (figure 3A) contraste. Image 20 zones représentatives à 10X objectif une fois par jourpendant cinq jours afin de déterminer la morphogenèse colonie (Figure 3C). </li><li> Analyser les images aveuglément à déterminer colonie de cellules formation stellaire (figure 3B). Une colonie est réputée être stellaire si une ou plusieurs saillies à partir de la sphéroïde de cellules sont perçus. Pour déterminer le pourcentage de colonies étoilées envahissantes, diviser le nombre de colonies étoilées par le nombre total de colonies de cellules par image acquise, puis la moyenne des colonies étoilées pourcentages de 20 images pour chaque jour. </li></ol><img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig1highres.jpg" width="500" /> Figure 1. Culture tridimensionnelle de cellules MDA-MB-231 cellules de cancer du sein de la matrice de membrane basale (la technique d&#39;implantation). AB) Un schéma du dispositif expérimental (effectué dans la hotte). C) Le bien de la boîte à fond de verre enduitavec 50 ul de sous-sol matrice de la membrane. D) vaisselle (s) placé dans un incubateur de culture de cellules (à 37 ° C avec 5% de CO 2) pour permettre à la matrice de se solidifier pendant au moins 30 min. E) Les cellules ont été traitées à la trypsine. F ) Les cellules ont été remises en suspension dans un tube de 15 ml conique. GH) Cells (présents dans le tube conique) ont été centrifugés à 100 g pendant 3 min dans une culture centrifugeuse de tissu. I) culot cellulaire. J) cellules (qui ont été filés bas) ont été remises en suspension dans 1 ml de milieu RPMI FBS supplémenté. K) Les cellules ont été comptées en utilisant un hématimètre. L) 2,5 x 10 4 cellules aliquotées dans le tube à centrifuger et complété en utilisant les médias appropriés de façon à obtenir le volume total de 50 pi. M ) mélanger les cellules provenant de l&#39;étape 1.7 (25 000 cellules dans 50 ul) avec le tube à centrifuger contenant une matrice à partir de l&#39;étape 1.5 dans un rapport de 1:1; volume final sera de 100 μ.; L N) plaque délicatement 100 pi de la matrice: mélange de cellules de l&#39;étape 8 sur le plat de matrice revêtu solidifié de l&#39;étape 1.2 O) Une fois la matrice: mélange de cellules est solidifiée, ajouter 2 ml de milieu RPMI FBS complétées à. le plat. P) Placez le plat dans l&#39;incubateur où il sera stocké pour le reste de l&#39;expérience. 2. L&#39;examen des caractéristiques des cultures Morphogenic 3D avec immunofluorescence <ol><li> Mettre en place un bac à glaçons, le phosphate froid solution tamponnée (PBS), 20% d&#39;acétone: méthanol à 80% (de la solution de fixation), et 3% d&#39;albumine de sérum bovin (BSA; la solution de blocage) (figure 2A) avant de retirer la coupelle (s) à partir de l&#39;incubateur. </li><li> Placer le plat (s) sur le plateau de glace et aspirer les médias, puis laver 3 fois avec 2 ml de PBS froid (figure 2B). </li><li> Une fois le dernier lavage PBS est aspiré, ajouter 2 ml d&#39;acétone à 20%: solution de méthanol à 80% dans le plat (s) (figure 2C </strong&gt;) pour fixer les cellules pendant 20 min à 4 ° C (soit sur ​​la glace ou dans un réfrigérateur) (figure 2D). </li><li> Une fois les 20 min de fixation est terminée, apporter les plats de retour à la température ambiante, aspirer le fixateur, puis laver 3 fois avec PBS. Une fois que le dernier lavage PBS est aspiré, ajouter 2 ml de BSA à 3% à l&#39;antenne (s) pour bloquer pendant au moins 30 min à température ambiante (figure 2E). </li><li> Au cours de la période de blocage préparer des dilutions de primaire (laminine V 1:100) et des anticorps secondaires (à une dilution appropriée) dissous dans 3% d&#39;albumine de sérum bovin (BSA), le tampon de blocage. S&#39;il vous plaît noter que 400-500 ul de la solution «BSA + anticorps primaire» devrait être ajouté directement sur la matrice: mélange de cellules. </li><li> Une fois que le blocage de 30 minutes sont écoulées, ajouter les anticorps primaires et incuber pendant au moins 1 heure à la température ambiante (Figure 2F). S&#39;il vous plaît noter que aucun lavage PBS doivent être réalisés après 30 min blockinpériode de g. </li><li> À la fin de l&#39;étape 2.6, éliminer la solution &#39;BSA + anticorps primaire », et laver 3 fois avec PBS. </li><li> Ajouter l&#39;anticorps secondaire dissous dans 3% de BSA (à une dilution appropriée) directement sur ​​la matrice: mélange de cellules, couvrir les plats et incuber pendant 1 heure à température ambiante (figure 2G). </li><li> Retirez le «BSA + anticorps secondaire» solution, et laver 3 fois avec PBS. </li><li> Ajouter 2 ml de Hoechst 33258 (1:10000) dissous dans du PBS pour la coloration des noyaux, et incuber pendant 5 min dans une feuille d&#39;aluminium (ou recouvrir d&#39;un récipient opaque pour limiter photoblanchiment) (figure 2H). </li><li> Une fois la 5 min d&#39;incubation avec Hoechst 33258t ont expiré, laver la vaisselle (es) 5x avec du PBS. </li><li> Ajouter milieu de montage directement sur la matrice: mélange de cellules dans le plat confocale et couvrir avec lamelle de verre avec précaution pour éviter toute perturbation induisant à l&#39;intégrité des colonies dans la matri de la membrane basalex: mélange de cellules (figure 2I). </li><li> Laisser le plat (s) à sécher pendant la nuit (ou 24 h) à température ambiante (figure 2I). Une fois sec, le plat (s) peut être conservé à -20 ° C, mais il est fortement recommandé qu&#39;ils soient imagés aussi rapidement que possible. </li><li> Acquérir des images en utilisant un microscope à fluorescence avec des longueurs d&#39;onde de laser appropriés d&#39;anticorps utilisés (figures 3D-E). </li></ol><img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig2highres.jpg" width="500" /> Figure 2. Examen des cultures 3D par immunofluorescence. . A) Un schéma du dispositif expérimental B) Plat (s) placé sur le plateau de glace et médias aspiré; ensuite les cellules ont été lavées trois fois avec 2 ml de PBS froid C) 2 ml d&#39;acétone à 20%:. solution méthanolique à 80% ajouté dans le récipient métallique (s) D) pour réparer les cellules.20 min à 4 ° C (soit sur ​​la glace ou dans un réfrigérateur). E) 2 ml de BSA à 3% ajouté à la boîte (s) pour bloquer pendant au moins 30 min à température ambiante. F) Une fois que la 30 min d&#39; blocage ont expiré, ajouter les anticorps primaires et incuber pendant au moins 1 heure à température ambiante G) Les anticorps secondaires dissous dans 3% de BSA (à une dilution appropriée) ont été ajoutés directement sur ​​la matrice de membrane basale. mélange de cellules; . ensuite plats recouverts et incubés pendant 1 heure à température ambiante H) 2 ml de Hoechst (1:10.000; dissous dans du PBS) a été ajouté à la dishe (s) et les cellules incubées pendant 5 min dans du papier d&#39;aluminium I) Milieu de montage ajouté directement. sur la matrice: mélange de cellules dans le plat confocal et le plat est recouvert de lamelle de verre. Antenne (s) mis à sécher pendant une nuit (ou 24 h) à température ambiante.

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Le développement de la 3D des techniques de culture cellulaire a permis aux chercheurs d&#39;étudier la transformation des cellules épithéliales mammaires, ce qui nous permet de visualiser les changements morphologiques spectaculaires. Outre l&#39;analyse de l&#39;invasion des cellules, les sphéroïdes épithéliales mammaires ou multicellulaires simples peuvent être utilisés pour évaluer les changements dans l&#39;adhésion cellulaire, la prolifération, la taille et basal-apical polarité. Contrairement aux m?...

La migration et l&#39;invasion des cellules individuelles ou collectives sont deux caractéristiques du cancer, et requis pour la propagation métastatique des cellules cancéreuses 4.1. La capacité des cellules cancéreuses à initier une métastase dépend de leur capacité à migrer et à envahir les tissus voisins utilisant invadopodia de dégrader la membrane basale des cellules. Invadopodia sont des saillies de dégradation de la matrice riche en actine dynamiques qui permettent la dégradation de la matrice extracellulaire par l&#39;intermédiaire de la libération de proteases dégradant la matrice 5. l&#39;invasion des cellules du cancer implique la dégradation de la matrice, suivi par la migration des cellules cancéreuses, ce qui s&#39;accompagne d&#39;une réorganisation de la tridimensionnel (3D) de l&#39;environnement de la matrice 2. Ainsi, pour pénétrer à travers la matrice, une cellule doit transformer sa forme et à interagir avec la matrice extracellulaire (ECM) 2. Le maintien de l&#39;intégrité du tissu mammaire dépend étroitement controlled architecture tissulaire depuis les jonctions cellule-ECM et l&#39;adhérence cellule-cellule influencent l&#39;expression génique et la perturbation de la polarité épithéliale peut conduire à l&#39;apparition du cancer 10.6. Cependant, la plupart des essais in vitro de migration et d&#39;invasion dans tels que transwell tests de chambre ou des essais plaie-rayures sont à deux dimensions (2D) et donc les négligent les interactions complexes entre les cellules et leur environnement adjacent 3,6,8,11-14. Diversités morphologiques et fonctionnelles importantes, y compris les variations de la morphologie cellulaire, la différenciation cellulaire, des adhérences cellule-matrice et des profils d&#39;expression génique ont été détectés par mise en culture des cellules dans des cultures 3D qui sont couramment défaut dans 2D dosages 2,6,8,11. Ainsi, l&#39;utilisation de tests 3D sont nettement bénéfique en récapitulant un plus physiologique en état ​​vivo, conduisant à une meilleure application des conclusions révolutionnaires de la recherche fondamentale à la clinique 6-10. Toutefois, il convient de noter, En dépit des nombreux avantages obtenus avec l&#39;utilisation de cultures 3D, ce modèle ne peut pas capturer toutes les complexités du microenvironnement tumoral in vivo en incluant différents types cellulaires. Cependant, il est possible d&#39;incorporer des cellules stromales dans les modèles 3D (par exemple, les fibroblastes, les leucocytes et les macrophages) pour étudier l&#39;effet des interactions tumeur-stroma sur l&#39;adhérence des cellules cancéreuses et l&#39;invasion de 15 à 17. Cellules épithéliales mammaires en culture se développent le plus efficacement lorsque les protéines de la MEC comme la laminine et le collagène sont présents. Avec cette connue, un mélange de matrice disponible dans le commerce a été dérivé de Engelbreth-Holm-Swarm (EHS) tumeur murine et est connu comme matrice de la membrane Matrigel sous-sol 2,8. Un certain nombre de techniques ont été mis en place pour cultiver des cellules épithéliales que colonies 3D dans la matrice de membrane basale 2,8. La membrane basale modèle de matrice 3D est efficace pour établir cellule à la fois malin et non malignes du seincroissance ressemblant à ce qui se produit dans l&#39;environnement in vivo 18,19. MCF10A cellules sont des cellules épithéliales mammaires non malignes. Lorsqu&#39;il est cultivé en sous-sol matrice de la membrane, ces cellules présentent en traits in vivo de cellules mammaires normales et subir une prolifération cellulaire contrôlée, de la polarisation cellulaire, l&#39;apoptose et pour établir l&#39;espace de lumière 8,12,20. Par ailleurs, l&#39;apparition de noyaux cellulaires de cellules formant des acini MCF10A dans des cultures 3D ressemblent plus étroitement à celles des cellules épithéliales mammaires en tissu que ceux cultivés dans monocouche 21. Des études menées par Bissell et ses collègues ont été les premiers à révéler que les cellules malignes du sein peuvent être différenciées à partir de cellules non malignes du sein lorsqu&#39;il est cultivé dans un laminine riche environnement, puisque les cellules malignes présentent un phénotype très désorganisé, une prolifération accrue, une diminution de cellule-à- l&#39;adhésion cellulaire, l&#39;augmentation de l&#39;expression de marqueurs mésenchymateuses et une augmentation du nombre de structures formées 3,6,2 invasive2. Des anomalies de l&#39;environnement cellulaire peuvent influencer la formation de tumeur 20. Le procédé de culture en 3D peut être utilisée pour étudier efficacement la communication qui se produit entre les cellules tumorales et leur environnement et à déterminer comment les influences d&#39;expression de protéines telles 14,20,21,23 de communication. Cet article fournit une méthodologie détaillée de croître MDA-MB-231 cellules de cancer du sein dans les cultures 3D pour analyser envahissant, et d&#39;étudier la perte de la morphologie épithéliale en utilisant un laminine marqueur épithélial, un composant de la membrane basale de la cellule 18,19,24, 25. Les procédures détaillées offrent la possibilité de quantifier avec précision et de façon reproductible stellaire (invasive) formation de la structure par une cellule de cancer invasif et n&#39;est pas limitatif pour les lignées cellulaires de cancer du sein commun (telles que des cellules MDA-MB-231, Hs578T, MCF-7, ou T47D ). Ainsi, ce test peut servir de plate-forme pour évaluer la façon dont l&#39;expression protéique dans les cellules ou le traitement avec pro-ou anti-composés invasives régulent dégradation de la matrice extracellulaire, par des cellules uniques ou multiples.

<table border="0" cellpadding="0" cellspacing="0" width="1062">
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			<td height="21" style="height:21px;width:323px;">Name of Material/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL tubes</td>
			<td>VWR</td>
			<td>CA10011-700</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100-mm culture dish BD353003</td>
			<td>VWR</td>
			<td>CABD353003</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Falcon tube</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">1 mL filtered tips</td>
			<td>VWR</td>
			<td>10011-350</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">200 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-016</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-008</td>
			<td>Sterile, disposable</td>
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		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed Confocal No.1 culture dishes&#160;</td>
			<td style="width:265px;">MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Precooled before use</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>BioShop</td>
			<td>ALB003.100</td>
			<td>Used at 3% for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Life Technologies</td>
			<td>&#160;A11029</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>&#160;A11011</td>
			<td>1:1200 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-Beta-Catenin&#160;</td>
			<td>BD Transduction Laboratories</td>
			<td>610153</td>
			<td>Mouse-monoclonal; Used at 1:100 for IF</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>Used at 10% (v/v)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg&#8260;mL Solution in Water</td>
			<td>Life Technologies</td>
			<td>H3569</td>
			<td>Used at 0.1% (1:10000 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InVivo Analyzer Suite&#160;</td>
			<td style="width:265px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kisspeptin-10 (KP-10)</td>
			<td style="width:265px;">EZ Biolabs</td>
			<td style="width:119px;">PT0512100601</td>
			<td>Used at 100nM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Laminin&#160;</td>
			<td>Cedarlane</td>
			<td>AB19012(CH)</td>
			<td>Rabbit-polyclonal full length human; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM-510 META laser scanning microscope&#160;</td>
			<td style="width:265px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Used at 63X objective; oil immersion lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel phenol red free (BD356237)</td>
			<td style="width:265px;">VWR</td>
			<td>CACB356237</td>
			<td>&#160;Lot No.2180819; 10.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus IX-81 microscope&#160;</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Antibiotic (added to media; used at 0.01%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipet&#160;</td>
			<td>VWR</td>
			<td>CA53300-523</td>
			<td>Sterile, disposeable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium with Glutamine</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>Used for culturing of MDA-MB-231 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>25200-072</td>
			<td>Used to trypsinize MDA-MB-231 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)</td>
			<td style="width:265px;">Lonza</td>
			<td style="width:119px;">CC-3150</td>
			<td>Used for culturing of MCF10A cells</td>
		</tr>
	</tbody>
</table>

quantification of breast cancer cell invasiveness using a three-dimensional (3d) model

Il est maintenant bien connu que le microenvironnement cellulaire et tissulaire sont des régulateurs critiques qui influencent l&#39;initiation et la progression tumorale. En outre, la matrice extracellulaire (ECM) a été démontrée comme étant un régulateur critique du comportement des cellules en culture et in vivo de l&#39;homéostasie. L&#39;approche actuelle de la culture de cellules sur deux dimensions (2D), plastiques résultats surfaces de la perturbation et la perte d&#39;interactions complexes entre les cellules et leur microenvironnement. Grâce à l&#39;utilisation de trois dimensions (3D) des analyses de culture, les conditions d&#39;interaction cellule-microenvironnement sont établies ressemblant le micro-environnement in vivo. Cet article propose une méthodologie détaillée pour cultiver des cellules du cancer du sein dans une matrice 3D sous-sol de la protéine de membrane, ce qui illustre le potentiel de la culture en 3D dans l&#39;évaluation de l&#39;invasion de cellules dans le milieu environnant. En outre, nous discuter de la façon dont ces essais 3D ont le potentiel pour examiner la perte de molec signalisationdules qui régulent la morphologie épithéliale par immunomarquage procédures. Ces études aident à identifier les détails mécanistiques importantes dans les processus de régulation invasion, nécessaires à la propagation du cancer du sein.

Un exemple des cellules MDA-MB-231 dans des cellules qui envahissent la matrice 3D est illustré sur la figure 3C. Les cellules sont enrobées dans une matrice (Jour 1), et commencent à se former (étoilées) structures envahissantes par jour 3, et envahissent complètement dans la matrice par Jour 5 (figure 3C). Le nombre de colonies formées étoilées sont comptés, et exprimée en pourcentage du nombre total de colonies par boîte (invasives et non invasives). En outre, puisque les...

Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

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

Cet article fournit des méthodologies détaillées pour l&#39;utilisation de trois dimensions (3D) des essais pour quantifier l&#39;invasion des cellules du cancer du sein. Plus précisément, nous discutons les procédures nécessaires pour mettre en place de tels essais, la quantification et l&#39;analyse des données, ainsi que des méthodes pour examiner la perte de l&#39;intégrité de la membrane qui se produit lorsque les cellules envahissent.

Quantification du cancer du sein cellulaire envahissement aide d&#39;un (3D) du modèle en trois dimensions

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

quantification-breast-cancer-cell-invasiveness-using-three

Research

JoVE Journal

Medicine

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

15.7K vues.  University of Western Ontario.  Cet article fournit des méthodologies détaillées pour l'utilisation de trois dimensions (3D) des essais pour quantifier l'invasion des cellules du cancer du sein. Plus précisément, nous discutons les procédures nécessaires pour mettre en place de tels essais, la quantification et l'analyse des données, ainsi que des méthodes pour examiner la perte de l'intégrité de la membrane qui se produit lorsque les cellules envahissent. 

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

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Dans l&#39;imagerie de bioluminescence in vivo de la tumeur Dynamics hypoxie des métastases cérébrales du cancer du sein dans un modèle murin

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Recherche

Médecine

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modale d&#39;imagerie de l&#39;angiogenèse dans un modèle de rat nu des métastases osseuses du cancer du sein par imagerie par résonance magnétique, la tomodensitométrie et volumétrique ultrasons

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Hétérotypique tridimensionnelle<em&gt; In Vitro</em&gt; Modélisation des interactions stroma-épithéliales lors de l&#39;initiation et la progression du cancer des ovaires

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

L&#39;homme neuroendocriniennes lignées cellulaires tumorales comme un modèle tridimensionnel pour l&#39;étude de la thérapie des tumeurs neuro-endocrines humaines

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Tissue Culture Modèle tridimensionnel pour étudier primaire moelle osseuse humaine et de ses affections malignes

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Initiation de carcinome du sein métastatique en ciblant de l&#39;épithélium canalaire avec adénovirus Cre: un modèle de souris transgénique roman du cancer du sein

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

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

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

Profilage de microARN œstrogène réglementés dans les cellules du cancer du sein

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

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Trois Cultures dimensionnelles: un outil pour étudier l&#39;architecture normale Acinaire vs transformation maligne des cellules mammaires

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

En trois dimensions modèle de co-culture tumeur stromale-Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

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 ...