Nous tenons à remercier le Nikon Imaging Center à Harvard Medical School, en particulier des eaux Jennifer, Lara Petrak et Salmon Wendy, de formation et de l&#39;utilisation de leurs microscopes timelapse. Nous aimerions également remercier Rosa Ng et Achim Besser pour des discussions utiles. Ce travail a été soutenu par des subventions du NIH 5695837 (à M. Iwanicki) et GM064346 à ACC; par une subvention de la Dre Miriam et Sheldon Adelson G. Medical Research Foundation (pour ACC).

1. Cancer de l&#39;ovaire Sphéroïde cellule Formation <ol><li> DP-exprimant les cellules cancéreuses de l&#39;ovaire sont cultivées dans un milieu de base de 10% (un milieu de culture de cellule personnalisé contenant un mélange 50:50 de 199 et MCDB105, inactivé à 10% sérum de veau fœtal et 1% stylo-streptococcique). Pour exprimer la demande de propositions non étiquetés cellules cancéreuses de l&#39;ovaire, transfecter les cellules avec un plasmide contenant DP et sélectionnez des cellules exprimant RFP. Alternativement, les vecteurs viraux peuvent être utilisés pour exprimer transitoirement protéines fluorescentes, ou des cellules peuvent être pré-incuber avec un rouge fluorescent cellule traqueur colorant (Invitrogen). </li><li> Avant de former des sphéroïdes cancer de l&#39;ovaire, il est nécessaire de préparer à faible adhérence 96 puits ronds plats culture sur le fond. Pour produire les plaques de culture à faible adhérence, 30μl poly-HEMA (6mg polyhydroxyéthylméthacrylate dans 1 ml à 95% EtOH) solution est ajoutée à chaque puits de 96 puits plat Corning culture cellulaire. Les plaques à 96 puits sont incubés dans une 37 ° C non humidifié incubateur pour évaporer l&#39;éthanol, le fait de laisser un film de poly-HEMA sur chaque puits. Ce film de poly-HEMA empêche les cellules de se fixer à la partie inférieure du puits, forçant les cellules à croître en suspension 18. [Sinon, Ultra-Low des plaques de culture de fixation (Corning) peut être utilisé à la place de la poly-HEMA plats revêtus.] </li><li> Après les plaques à faible adhérence des culture sont préparés, trypsiniser une plaque de cellules cancéreuses de l&#39;ovaire, les cellules à granules dans une centrifugeuse de paillasse (Heraeus) à 900 RCF pendant 3 minutes, aspirer le surnageant et remettre en suspension dans un milieu de base 10%. </li><li> Compter les cellules en utilisant un hémocytomètre. </li><li> Ajuster la concentration de ces cellules qu&#39;il ya 100 cellules par 50 pi de milieu de base de 10%. </li><li> Ajouter 50 pi de la suspension cellulaire diluée suspension uniforme dans chaque puits de la boîte de culture et 96 poly-HEMA couché. </li><li> Incuber la plaque à 96 puits dans une étuve à 37 ° C de culture cellulaire pendant 16 heures (cette quantité de temps doit être augmenté ou diminué en fonction de la quantité de temps nécessaire pour queune lignée cellulaire particulière pour former sphéroïdes multicellulaires ou souhaités conditions expérimentales) pour permettre aux cellules cancéreuses de l&#39;ovaire à se regrouper, former un sphéroïde multicellulaire unique dans chaque puits. Certaines cellules tumorales peuvent subir l&#39;apoptose au cours de cette période, il est donc important de choisir un moment avant l&#39;induction de l&#39;apoptose. </li></ol> 2. Mésothéliales Formation monocouche de cellules <ol><li> Dans une hotte de culture cellulaire, pré-enduire les puits d&#39;une à 6 puits à fond de verre plat MatTek avec de la fibronectine en ajoutant 2 ml d&#39;une fibronectine 5 pg / ml solution de PBS dans chaque puits de la parabole et l&#39;incubation à température ambiante pendant 30 minutes. La qualité optique du verre-fonds dans les plats MatTek permettre l&#39;imagerie haute résolution microscopique. </li><li> GFP-exprimant les cellules mésothéliales sont cultivées dans un milieu de base de 10%. Trypsiniser une plaque de cellules mésothéliales, ralentit dans une centrifugeuse de table (Heraeus) à 900rpm pendant 3 minutes, aspirer le surnageant, et remettre en suspension dans un milieu de base 10%. Lacellules mésothéliales utilisés ici ont été déjà exprimant la GFP quand ils ont été obtenus, mais non marqués cellules mésothéliales peut être produite par transfection avec un plasmide contenant la GFP ADNc, ou pré-incubation des cellules dans une cellule à fluorescence verte suiveur de colorant (Invitrogen). </li><li> Après l&#39;incubation fibronectine de 30 minutes (à l&#39;étape 2.1), laver les puits de la boîte de MatTek avec 2 ml de PBS. </li><li> Aspirer le PBS et la plaque 6 x10 5 cellules mésothéliales par puits dans chaque puits de la boîte de 6 puits MatTek. Incuber le plat MatTek dans un incubateur à 37 ° C durant la nuit de culture cellulaire pour permettre aux cellules mésothéliales de joindre à l&#39;antenne et de former une monocouche. </li></ol> 3. Mésothéliales Assay élimination des cellules <ol><li> Utiliser une pipette pour recueillir les sphéroïdes cancer de l&#39;ovaire de la 96 puits poly-HEMA plaque revêtue. </li><li> Aspirer le milieu d&#39;un puits de la boîte de 6 puits contenant MatTek une monocouche de cellules mésothéliales. Laver une fois avec 2 ml de PBS. Ajouter tous les sphéroïdes de la 96 puits ptard pour un puits de la vaisselle MatTek (~ 3x le nombre de sphéroïdes qui vont être imagée pour tenir compte de l&#39;atterrissage sphéroïdes de la part de l&#39;antenne qui ne peuvent pas être imagée). </li><li> Placer le plat MatTek sur la scène d&#39;un microscope inversé à fluorescence à champ large capable d&#39;effectuer imagerie time-lapse pour la durée d&#39;au moins 8 heures. Utilisez une platine motorisée à des postes d&#39;images multiples dans le plat, avec de multiples événements intercalation sphéroïde, en une seule expérience. Nous utilisons un Nikon Ti-E inversé motorisé Widefield fluorescence time-lapse microscope avec système de mise au point parfaite et intégrée à faible [20x-0.75 ouverture numérique (NA)] agrandissement / NA différentiels de contraste d&#39;interférence (DIC) optique, un transilluminateur halogène Nikon à 0,52 NA longue distance de travail (GDL) condenseur, Nikon rapide (&lt;100 millisecondes le temps de commutation) filtres d&#39;excitation et d&#39;émission (GFP Ex 480/40, Em 525/50, DP-mCherry Ex 575/50 Em 640/50), Sutter rapide transmissibles question à deux volets à épifluorescence trajet de la lumière intelligentetres, un Nikon linéaire codé platine motorisée, un Hamamatsu ORCA-AG refroidi charge-coupled device (CCD), une chambre sur mesure microscope d&#39;incubation avec la température et le CO 2 de contrôle, Nikon NIS-Elements AR logiciel version 3, et un Table TMC isolation contre les vibrations. </li><li> Les sphéroïdes cellulaires de cancer ovarien se déposent au fond du plat et le joindre à la monocouche de cellules mésothéliales. Recueillir la GFP, RFP et les images de phase de 20 + sphéroïde / monocouche interactions, toutes les 10 minutes, pendant 8 heures. </li><li> Les sphéroïdes DP-exprimant cellulaires de cancer ovarien envahira dans la monocouche GFP cellule exprimant mésothéliale créant un trou dans la monocouche. Au bout de 8 heures, de mesurer la dimension des trous en traçant les trous noirs dans les images en utilisant un logiciel GFP éléments (ou un autre logiciel approprié tel que l&#39;image J). Normaliser la taille du trou de la taille initiale sphéroïde en divisant la dimension du trou à 8 heures par la taille de l&#39;ellipsoïde de l&#39;image correspondant au temps zéro DP. Dans cet exexemple, la taille du trou a été mesurée seulement une fois, mais elle peut être mesurée à plusieurs reprises tout au long de l&#39;expérience huit heures afin de mieux comprendre la dynamique de l&#39;intercalation. </li></ol> 4. Les résultats représentatifs Dans cet exemple, nous avons comparé la capacité de clairance mésothéliales OVCA433 sphéroïdes cellulaires de cancer ovarien qui ont atténué l&#39;expression de la taline-1 pour contrôler OVCA433 sphéroïdes. OVCA433 sphéroïdes de chaque groupe ont été ajoutées à un plat contenant MatTek ZT monocouches de cellules mésothéliales. Six sphéroïdes de chaque groupe ont été imagées toutes les 10 minutes pour huit heures (Figure 4, Film 3, Film 4). Les trous réalisés dans la monocouche par les sphéroïdes d&#39;épandage ont été mesurés et six positions de chaque groupe ont été en moyenne. La figure 4 montre que la zone de dégagement moyenne créée par sphéroïdes Tallinn knockdown 1 était significativement plus petite que la superficie moyenne créée par sphéroïdes de contrôle, ce qui suggère que taline est nécessaire pour le dédouanement mésothéliales par OVCA433 sphéroïdes cancer de l&#39;ovaire. <img alt="Figure 1" src="/files/ftp_upload/3888/3888fig1.jpg" /> Figure 1. Métastases du cancer de l&#39;ovaire. Tumeurs ovariennes primaires se développent soit à partir de l&#39;épithélium de surface de l&#39;ovaire ou des trompes de Fallope. Les cellules tumorales ou groupes se détachent de la tumeur primaire et de recueillir dans la cavité péritonéale. Les cellules tumorales peuvent alors se regroupent pour former des sphéroïdes multicellulaires. Sphéroïdes puis fixez les monocouches de cellules mésothéliales qui tapissent la cavité péritonéale. Les cellules mésothéliales sont exclus de dessous le sphéroïde cancer de l&#39;ovaire joint, permettant les sphéroïdes pour accéder à la membrane sous-sol sous-jacent. Film 1. Métastases du cancer de l&#39;ovaire. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">Cliquez ici pour voir le film</a> . <img alt="Figure 2" src="/files/ftp_upload/3888/3888fig2.jpg" /> Figure 2. </strong&gt; ligne mésothéliales cellules de la surface de tissu péritonéal humaine et sont exclus de la sous implants cellulaires de cancer ovarien. <img alt="Figure 3" src="/files/ftp_upload/3888/3888fig3.jpg" /> Figure 3. Essai Dégagement mésothéliales. Cancer de l&#39;ovaire sphéroïdes sont formés par incubation 100 DP-exprimant cellules de cancer ovarien par puits dans une poly-HEMA revêtu 96 coupelle inférieure puits de culture ronde à 37 ° C pendant 16 heures. Poly-HEMA empêche les cellules de se fixer à la boîte de culture, les cellules permettant de rester en suspension et d&#39;adhérer les uns aux autres pour former une grappe unique par puits. Monocouches de cellules mésothéliales sont préparés en étalant 6x10 5 cellules mésothéliales par puits dans une fibronectine revêtu 6 puits plat MatTek et l&#39;incubation de la plaque à 37 ° C pendant 16 heures. Les sphéroïdes sont ensuite transférés à la boîte de MatTek avec la monocouche mésothéliales et les deux populations cellulaires sont imagées toutes les 10 minutes pour 8 heures en utilisant un Nikon Ti-E Inverted motorisé Widefield fluorescence time-lapse microscope et logiciels Elements. Movie 2. Essai Dégagement mésothéliales. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">Cliquez ici pour voir le film</a> . <img alt="Figure 4" src="/files/ftp_upload/3888/3888fig4.jpg" /> Atténuation Figure 4. De Talin une expression dans OVCA433 sphéroïdes diminue la capacité de clairance mésothéliales. OVCA433 sphéroïdes (rouge) avec et sans expression atténuée de taline 1 ont été autorisés à se fixer et envahir une monocouche ZT mésothéliales (vert). Les deux populations de cellules ont été imagées toutes les 10 minutes pour 8 heures en utilisant un Nikon Ti-E inversé motorisé Widefield fluorescence time-lapse microscope et un logiciel éléments. Le graphique montre que taline une atténuationdiminue significativement la clairance des cellules mésothéliales (parcelle quantile avec des barres vertes au moyen). Movie 3. Contrôle OVCA433 sphéroïdes (rouge) qui envahissent en une monocouche mésothéliales (vert). <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">Cliquez ici pour voir le film</a> . Atténuation Movie 4. De Talin une expression dans OVCA433 sphéroïdes (rouge) diminue mésothéliales (vert) la capacité de clairance. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">Cliquez ici pour voir le film</a> .

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Le &quot;test Dégagement mésothéliales&quot; présentée ici utilise la microscopie time-lapse pour surveiller les interactions de sphéroïdes multicellulaires cancer de l&#39;ovaire et les monocouches de cellules mésothéliales, dans le détail spatial et temporel grande. Auparavant, plusieurs groupes ont utilisé des dosages 8-14 points de terminaison pour montrer que les cellules cancéreuses de l&#39;ovaire et de joindre à envahir des monocouches de cellules mésothéliales. Ce test est unique en ce...

<table border="1"><tbody><tr><td> Réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> OVCA433 cellules ovariennes cancéreuses </td><td></td><td></td><td> Don de Dr. Dennis Slamon </td></tr><tr><td> ZT cellules mésothéliales </td><td></td><td></td><td> Cadeau du Dr Tan Ince </td></tr><tr><td> Milieu 199 </td><td> Gibco </td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td> Cellule Applications Inc </td><td> 117-500 </td><td></td></tr><tr><td> FBS-inactivé par la chaleur </td><td> Gibco </td><td> 10082 </td><td></td></tr><tr><td> Pen-Strep </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Plaques à 96 puits </td><td> Corning Costar </td><td> 3799 </td><td></td></tr><tr&gt; <td> Polyhydroxyéthylméthacrylate (poly-HEMA) </td><td> Sigma-Aldrich </td><td> 192066-25G </td><td> Pour poly-HEMA solution dissoudre 6mg poly-HEMA poudre dans 1 ml de EtOH 95% </td></tr><tr><td> EtOH </td><td> Pharmco-AAPER </td><td> 111ACS200 </td><td> Diluer à 95% en 2 0 dH </td></tr><tr><td> Hotte de culture cellulaire </td><td> Nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td> De culture de tissu incubateur </td><td> Thermo Scientific </td><td> 3110 </td><td></td></tr><tr><td> incubateur pour le poly-HEMA plaques </td><td> Instruments Labline </td><td> Imperial III 305 </td><td></td></tr><tr><td> Centrifugeuse de table </td><td> Heraeus </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 puits à fond de verre plat </td><td> MatTek Corp. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> La fibronectine </td><td> Sigma </td><td> F1141-1MG </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-CV </td><td></td></tr><tr><td> Microscope Timelapse: </td><td></td><td></td><td></td></tr><tr><td> Microscope </td><td> Nikon </td><td></td><td> Ti-E inversé motorisé fluorescence time-lapse microscope avec système de mise au point intégré Parfait </td></tr><tr><td> Lentille </td><td> Nikon </td><td></td><td> 20X-0.75 apeture numérique </td></tr><tr><td> Transilluminateur halogène </td><td> Nikon </td><td></td><td> 0,52 NA condenseur longue distance de travail </td></tr><tr><td> Filtres d&#39;excitation et d&#39;émission </td><td> Chroma filtres passe uniques dans le logement Nikon </td><td></td><td> GFP Ex 480/40, Em 525/50 DP-mCherry Ex 575/50 Em 640/50 </td></tr><tr><td> Transmissibles et Epifluoresce trajet de la lumière </td><td> Sutter </td&gt; <td></td><td> Volets Smart </td></tr><tr><td> Linéaire codé platine motorisée </td><td> Nikon </td><td></td><td></td></tr><tr><td> Refroidi caméra à dispositif à couplage de charge </td><td> Hamamatsu </td><td> ORCA-AG </td><td></td></tr><tr><td> Microscope chambre d&#39;incubation avec la température et le CO 2 de commande </td><td> custom-built </td><td></td><td></td></tr><tr><td> Tableau isolation des vibrations </td><td> TMC </td><td></td><td></td></tr><tr><td> NIS-Elements </td><td> Nikon </td><td></td><td> Version 3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

Cancer de l&#39;ovaire est le cinquième cause de décès liés au cancer aux États-Unis 1. Malgré une réponse positive initiale à des thérapies, de 70 à 90 pour cent des femmes atteintes d&#39;un cancer de l&#39;ovaire développent des métastases nouvelles, et la récidive est souvent mortelle 2. Il est donc nécessaire de comprendre comment des métastases secondaires surviennent dans le but de développer de meilleurs traitements pour le cancer de l&#39;ovaire stade intermédiaire et tardive. Métastases cancer de l&#39;ovaire se produit lorsque les cellules malignes se détacher de la tumeur primaire et de diffuser dans toute la cavité péritonéale. Les cellules disséminées peuvent former des amas multicellulaires, ou sphéroïdes, qui soit rester seules, ou d&#39;implants sur les organes dans la cavité péritonéale 3 (Figure 1, Film 1). Tous les organes à l&#39;intérieur de la cavité péritonéale sont revêtues d&#39;un unique, continue, la couche de cellules mésothéliales 4-6 (figure 2). Cependant, les cellules mésothéliales sont absents de dessousmasses tumorales péritonéales, tels que révélés par les études d&#39;un microscope électronique excisées de l&#39;homme des coupes de tissus tumoraux 3,5-7 (Figure 2). Ceci suggère que les cellules mésothéliales sont exclus du dessous de la masse tumorale par un processus inconnu. Précédent dans des expériences in vitro ont démontré que des cellules primaires de cancer de l&#39;ovaire joindre plus efficacement à la matrice extracellulaire que de cellules mésothéliales 8, et des études plus récentes ont montré que les cellules mésothéliales péritonéales primaires fournissent effectivement un obstacle à l&#39;adhésion cellulaire et l&#39;invasion de l&#39;ovaire (par rapport à l&#39;adhérence et l&#39;invasion sur des substrats qui n&#39;ont pas été couverts avec des cellules mésothéliales) 9,10. Cela suggère que les cellules mésothéliales agir comme une barrière contre les métastases du cancer de l&#39;ovaire. Les mécanismes cellulaires et moléculaires par lesquels les cellules cancéreuses de l&#39;ovaire abus de cette barrière, et d&#39;exclure le mésothélium ont, jusqu&#39;à récemment, est resté inconnu. Nous décrivons ici ela méthodologie e pour un test in vitro que les modèles de l&#39;interaction entre les sphéroïdes cellulaires de cancer ovarien et les cellules mésothéliales in vivo (figure 3, Film 2). Notre protocole a été adapté à partir des méthodes décrites précédemment pour l&#39;analyse des interactions de cellules tumorales ovariennes avec des monocouches mésothéliales 8-16, et a d&#39;abord été décrite dans un rapport montrant que les cellules tumorales ovariennes d&#39;utiliser une activation de l&#39;intégrine-dépendante de la myosine et la force de traction afin de promouvoir l&#39;exclusion de la à partir de cellules mésothéliales en vertu d&#39;une tumeur 17 sphéroïde. Ce modèle tire parti de time-lapse microscopie à fluorescence pour surveiller les populations de cellules deux en temps réel, fournissant des informations spatiales et temporelles de l&#39;interaction. Les cellules cancéreuses ovariennes exprimer protéine fluorescente rouge (RFP), tandis que les cellules mésothéliales exprimer la protéine fluorescente verte (GFP). DP-exprimant sphéroïdes cellulaires de cancer ovarien joindre à la monocouche GFP-exprimer mésothéliales. La propagation sphéroïdes, envahir, etforcer les cellules mésothéliales côté créant un trou dans la monocouche. Ce trou est visualisée comme l&#39;espace négatif (noir) dans l&#39;image GFP. La superficie du trou peut alors être mesurée pour l&#39;analyse quantitative des différences dans l&#39;activité de contrôle et de jeu entre les populations expérimentales de cancer de l&#39;ovaire et / ou des cellules mésothéliales. Ce test ne nécessite qu&#39;un petit nombre de cellules cancéreuses de l&#39;ovaire (100 cellules par sphéroïdes X 20-30 sphéroïdes par condition), il est donc possible d&#39;effectuer ce test en utilisant des échantillons précieux primaires de cellules tumorales. En outre, ce test peut être facilement adapté pour le criblage haut débit.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

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

Le dosage jeu mésothéliales décrite ici prend avantage des cellules marquées par fluorescence et la microscopie vidéo time-lapse de visualiser et de mesurer quantitativement les interactions de sphéroïdes multicellulaires cancer de l&#39;ovaire et les monocouches de cellules mésothéliales. Ce test modélise les étapes précoces de métastases du cancer de l&#39;ovaire.

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 ...

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Medicine

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

14.4K vues.  Harvard Medical School. Le dosage jeu mésothéliales décrite ici prend avantage des cellules marquées par fluorescence et la microscopie vidéo time-lapse de visualiser et de mesurer quantitativement les interactions de sphéroïdes multicellulaires cancer de l'ovaire et les monocouches de cellules mésothéliales. Ce test modélise les étapes précoces de métastases du cancer de l'ovaire.

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

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Assay Métastases expérimentales

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Recherche

Médecine

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Modèles de MAME pour 4D imagerie des cellules vivantes de la tumeur: Interactions microenvironnement Progression impact malin

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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 ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Modèles de métastases osseuses

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un modèle murin orthotopique de cancer de la prostate humain métastases

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

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 ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Dans l&#39;Enrichissement vitro du cancer des ovaires cellules initiatrices de tumeurs

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Quantification du commerce intra-péritonéale Cancer de l&#39;ovaire Métastase

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modélisation des étapes précoces de cancer de l&#39;ovaire Diffusion dans une culture organotypique de la cavité péritonéale humain

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...