Nous sommes reconnaissants à Ken Iwata (OSI Pharmaceuticals, New York, Etats-Unis) pour les réactifs et Fred Miller (Barbara Ann Intitute cancer Karmanos, Detroit, Etats-Unis) pour les lignées cellulaires.

1. Préparation de la solution méthocel (500 ml) <ol><li> Autoclave 6 g de méthylcellulose dans une bouteille de 500 ml contenant un agitateur magnétique. </li><li> Dissoudre la méthylcellulose dans 250 ml de DMEM/F12 sans sérum préchauffé (60 ° C) pendant 20 min. </li><li> Ajouter 250 ml de DMEM/F12 (RT) contenant du sérum de cheval à 10%. Mélanger la nuit (4 ° C). </li><li> Effacer la solution par centrifugation (5000g, 2h, 4 ° C). </li><li> Prenez la solution claire très visqueux dans un nouveau tube (environ 90-95% de la solution mère). </li><li> Conserver à 4 ° C jusqu&#39;à utilisation. </li></ol> 2. La culture Sphéroïde <ol><li> Préparer une solution à 20% Methocel (10 ml et 40 méthocel milieu de croissance ml). </li><li> Laver les cellules deux fois avec PBS, ajouter 0,1% de trypsine et les cellules incuber à 37 ° C </li><li> Resuspendre les cellules dans un milieu de croissance et de compter les cellules. </li><li> Préparer une suspension de 10 000 cellules par ml dans méthocel 20%. </li><li> Ajouter la suspension cellulaire à la S 96 puits à fond rondplaques USPENSION (100 pi par puits). </li><li> Le jour suivant, vérifiez la formation sphéroïde dans les plaques de suspension. Sphéroïdes sera prêt à l&#39;emploi après 1-3 jours. </li></ol> 3. Préparation du collagène <ol><li> Préparer sur la glace une solution de collagène 8 ml, 1 ml PBS 10X, 1 ml de NaOH 0,1 N et 3 gouttes de HCl 0,1 N. Vérifiez si le pH est de 7,4 en repérant certains de la solution sur les bandes indicatrices de pH. Ajuster le pH si hors de portée. </li><li> Ajouter 50 ul de solution de collagène neutralisé dans le puits d&#39;une plaque à 96 puits </li><li> Incuber à 37 ° C jusqu&#39;à ce que le collagène est solidifié (90 min). </li><li> Préparer méthocel-collagène-ligand des solutions sur la glace: pour chaque pipette ligand 1 partie de la solution de collagène. Ajouter 20 ng par ml de TGF-β collagène. Après dilution avec méthocel et la diffusion sur les différentes couches, la concentration finale de TGF-β sera de 5 ng / ml Mélanger au vortex. Ajouter une partie de méthocel-solution. Mélanger au vortex. </li></ol> 4. Sphéroïde intégrerDing <ol><li> Placez une extrémité 200 pi, qui a environ 5 mm de la pointe coupée, sur une pipette 200 pi. Aspirez une sphéroïde avec la pipette et soigneusement Répartir le milieu dans une assiette vide. Regardez si le sphéroïde reste à l&#39;intérieur de la pointe. Le sphéroïde est visible comme un point blanc. Sans relâcher le piston, sucer jusqu&#39;à 100 ul collagène méthocel mélange et se dispenser de l&#39;ellipsoïde dans le mélange de collagène dans un revêtus de collagène de 96 puits. Faites cela pour 12 sphéroïdes pour chaque condition. </li><li> Laissez collagène solidifier au moins 30 minutes à 37 ° C </li><li> Retirer les bulles d&#39;air avec une pointe de 200 pi. </li><li> Ajouter 50 ul de milieu avec du sérum de 1,6% et les inhibiteurs dissous dans du DMSO. Pour le SB-431542 ajouter 4 ul de la solution mère (10 mm) à 1 ml de milieu. Après la diffusion, la concentration finale de l&#39;inhibiteur sera de 10 uM. Le TGF-β et les inhibiteurs maintiendra le cap de l&#39;expérimentation. Prenez des photos de sphéroïdes sous le microscope en utilisant un grossissement de 40X. </li><li> Après 48h de stimulation par le TGF-β et / ou inhibiteurs, prendre des photos des sphéroïdes sous le microscope en utilisant un grossissement de 40X. </li><li> Mesurer la surface des sphéroïdes envahir après 2 jours et soustraire la zone de départ. Mesure de la surface d&#39;un sphéroïde: Ouvrir l&#39;image à partir du sphéroïde dans Adobe Photoshop Extended (figure 2A) et sélectionnez l&#39;outil Sélection rapide (figure 2B). Le pointeur de la souris se transforme en un cercle avec un «plus» (+) signe (figure 2C). Tout en faisant glisser le curseur sur le sphéroïde, Photoshop reconnaître les frontières de la sphéroïde. Si une zone à l&#39;extérieur de l&#39;ellipsoïde est sélectionné, cette région ne peut être exclu de la sélection en appuyant sur la touche Alt ou en utilisant l&#39;outil de sélection négative qui est indiqué par un signe - dans la barre d&#39;outils (). Le curseur se transforme en un cercle avec un «moins» (-) signe et en faisant glisser le curseur sur la zone sélectionnée à tort, cette zone est retiré de la sélection (figure 2D). Si nécessaire, plusieurs régions peuvent être sélectionnées en appuyant sur le ShBouton ift. Pour travailler avec plus de précision, de la taille du pointeur de la souris peut être ajustée en changeant le diamètre pinceau dans la barre d&#39;outils (figure 2e). Lorsque le sphéroïde entier est sélectionné, la zone de la sélection est calculée par l&#39;analyse-mesure dans la barre de menu ou en appuyant sur Ctrl + Maj + M (figure 2F). La fenêtre d&#39;enregistrement de mesure apparaîtra indiquant le nom du fichier et le domaine de la sélection en pixels, ainsi que d&#39;autres données (figure 2G). Si plusieurs sélections sont mesurées, la première ligne indique la somme de tous les domaines. Les mesures des sélections individuelles sont présentées dans les lignes ci-dessous. Les données de toutes les mesures peuvent être exportées dans un fichier texte et ouvert dans Excel. </li></ol> 5. Les résultats représentatifs: Un exemple de l&#39;essai sphéroïde MCF10A1 (M1) cellules mammaires normales épithéliales, H-Ras transformée MCF10AneoT (M2) et des cellules dérivées de M2 ​​MCF10CA1a (M4) est donnée dans la Figure 3. Cellules M1 a montré que l&#39;invasion faible sansstimulation, mais envahie significativement meilleure lors de la stimulation par le TGF-β. En revanche, le RAS-transformé M1-M2 dérivés ont envahi déjà efficacement sans plus de stimuli, et ce fut encore augmenté fois 4-5 lors du TGF-β traitement. Cellules M4 a montré la plus forte invasion, à la fois avec et sans le TGF-β plus. Ensuite, nous avons examiné si nous pouvions interférer avec le TGF-β induite par l&#39;invasion de ce modèle sphéroïde en utilisant des inhibiteurs chimiques. À cette fin, nous avons utilisé le SB-431 542, un analogue de l&#39;ATP et inhibiteur sélectif de l&#39;activité kinase de TβRI, ainsi que l&#39;activine de type IB récepteur (ActRIB) et l&#39;activine receptor-like kinase (ALK) 7 12. Comme prévu, le SB-431542 inhibe puissamment le TGF-β induit l&#39;invasion de M1, M2 et M4 cellules (Fig. 4), indiquant que le TGF-β invasion induite est TβRI-kinase dépendante. En outre, l&#39;invasion basale des sphéroïdes a également été fortement inhibée, ce qui suggère que l&#39;invasion basale est également deindépendante sur le TGF-β. <img alt="Figure 1" src="/files/ftp_upload/3337/3337fig1.jpg" /> Organigramme Figure 1. De l&#39;analyse 3D sphéroïde. Tout d&#39;abord, les cellules sont plaquées sous conditions non-adhérentes en plaques suspensions en forme de U. Cellules attestation globale en sphéroïdes multcellular et peuvent être intégrés dans une matrice de collagène. Dans cette matrice de collagène, ils sont capables d&#39;envahir. <img alt="Figure 2" src="/files/ftp_upload/3337/3337fig2.jpg" /> Figure 2. Quantification de la zone des sphéroïdes en utilisant Adobe Photoshop Extended. (A) L&#39;image du sphéroïde est ouvert dans Adobe Photoshop élargie du (B) Choix de l&#39;outil de sélection rapide (C) Glisser le curseur sur le sphéroïde pour sélectionner la zone du sphéroïde (D) Suppression d&#39;une zone incluse wongly utilisant le négative outil Sélection rapide (E) Ajustement de la taille du pinceau de l&#39;outil Sélection rapide pour sélectionner plus précisément la zone (F) Sélection de laMesure commande à enregistrer (G) Exemple d&#39;un enregistrement de la mesure. <img alt="Figure 3" src="/files/ftp_upload/3337/3337fig3.jpg" /> Figure 3. TGF-β induite par l&#39;invasion de sphéroïdes spontanément immortalisé MCF10A1 (M1) (A), RAS-transformé MCF10AneoT (M2 (B) et son dérivé métastatique MCF10CA1a (M4) (C). M1, M2 et M4 sphéroïdes embarqué en collagène ont été traités avec 5ng/ml du TGF-β pour les 48h comme indiqué. AC images représentant prises à la journée d&#39;intégration et deux jours plus tard. D invasion relative a été quantifiée comme différence région le jour de moins 2 jours 0. Les résultats sont exprimés en moyenne ± SD Adaptée à partir de 13. <img alt="La figure 4a" src="/files/ftp_upload/3337/3337fig4a.jpg" /> <img alt="Figure 4b" src="/files/ftp_upload/3337/3337fig4b.jpg" /> Invasion de la figure 4. Sphéroïde basocellulaire et le TGF-β-induite est inhibée bSB-431542 Y. Sphéroïdes M1, M2 et M4 ont été intégrés dans le collagène et traités avec le TGF-β 5ng/ml et / ou 10 uM SB-431 542 comme indiqué. Images représentant prises après 2 jours (A). L&#39;invasion relative a été quantifiée comme différence région le jour de moins 2 jours 0 (B). Les résultats sont exprimés en moyenne ± SD Adaptée à partir de 13.

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Aucun conflit d&#39;intérêts.

Nous avons établi un modèle dans lequel sphéroïde MCF10A1 cellules malignes et de ses dérivés envahissent dans une matrice de collagène de manière TGF-β-dépendante. Tout d&#39;abord, sphéroïdes sont formés dans les plaques rondes de 96 puits de suspension en bas, en présence de méthylcellulose. Bien sûr, aussi en forme de U en poly-HEMA plaques revêtues peuvent être utilisés à cette fin. La méthylcellulose est absolument nécessaire dans cette étape, puisque les cellules vont mourir en l&#39;absenc...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires (optionnel) </td></tr><tr><td> Méthyl-cellulose </td><td> Sigma-Aldrich </td><td> M0512 </td><td></td></tr><tr><td> PureCol </td><td> Avancé Biomatrix </td><td> 5005-B </td><td></td></tr><tr><td> bandes indicatrices de pH </td><td> Merck </td><td> 9533 </td><td></td></tr><tr><td> 96 puits de suspension plaques </td><td> Greiner Bio-One </td><td> 650 185 </td><td></td></tr><tr><td> 96 puits d&#39;adhésion TC plaques </td><td> Greiner Bio-One </td><td> 655 180 </td><td></td></tr></tbody></table>

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

Le TGF-β a opposés rôles dans la progression du cancer du sein en agissant comme un suppresseur de tumeur dans la phase initiale, mais stimulant l&#39;invasion et les métastases à 1,2 ultérieurement. Par ailleurs, le TGF-β est fréquemment surexprimé dans le cancer du sein et son expression est corrélée à un mauvais pronostic et les métastases 3,4. Les mécanismes par lesquels le TGF-β induit l&#39;invasion ne sont pas bien comprises. Le TGF-β provoque ses réponses cellulaires via le TGF-β de type II (TβRII) et de type I (TβRI) récepteurs. Sur le TGF-β induit la formation de complexes hétéromériques, TβRII phosphoryle la TβRI. Le TβRI activé initie sa voie de signalisation intracellulaire canonique par Smad récepteurs phosphorylant (R-Smad), c&#39;est à dire Smad2 et Smad3. Ces activé R-Smad former des complexes avec les hétéromériques Smad4, qui s&#39;accumulent dans le noyau et régulent la transcription des 5 gènes cibles. En plus des déjà described voie Smad, les résultats activation du récepteur dans l&#39;activation de plusieurs autres non-Smad voies de signalisation, par exemple Mitogen protéine kinase activée (MAPK) voies 6. Pour étudier le rôle du TGF-β dans les différents stades du cancer du sein, nous avons fait usage du système de pile à MCF10A. Ce système est constitué de cellules MCF10A1 spontanément immortalisé sein (M1) épithéliales 7, le H-Ras transformée M1 dérivés MCF10AneoT (M2), qui produit des lésions précancéreuses chez les souris 8, et le M2 MCF10CA1a dérivés (M4), qui a été établi à partir xénogreffes M2 et les formes de haute qualité avec des carcinomes de la capacité à métastaser au poumon 9. Cette série offre MCF10A la possibilité d&#39;étudier les réponses des cellules avec différents grades de malignité qui ne sont pas biaisées par un fond génétique différent. Pour l&#39;analyse du TGF-β induite par l&#39;invasion, nous avons généré homotypique MCF10A cellules sphéroïdes embe culturesdded dans une matrice de collagène 3D in vitro (figure 1). Ces modèles ressemblent tumeurs humaines in vivo par l&#39;établissement d&#39;un gradient d&#39;oxygène et de nutriments, résultant en cellules actives et invasives sur les cellules à l&#39;extérieur et de repos ou même nécrotiques dans l&#39;intérieur du 10 sphéroïde. Sphéroïde tests basés ont également été montré pour une meilleure résistance aux médicaments que récapituler cultures en monocouche 11. Ce système MCF10 modèle 3D nous a permis d&#39;étudier l&#39;impact du TGF-β de signalisation sur les propriétés invasives des cellules mammaires à différents stades de malignité.

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

Un test pour mesurer quantitativement Transforming Growth Factor (TGF)-β-induite invasion dans des gels de collagène en 3 dimensions est décrite. Ce test prend avantage de la série MCF10A de lignées cellulaires, qui représentent les différentes étapes du développement du cancer du sein. Cette méthode peut être adoptée pour être utilisé avec d&#39;autres lignées cellulaires et pourraient être utilisées pour étudier d&#39;autres activateurs ou des inhibiteurs potentiels de l&#39;invasion.

Assay Sphéroïde pour mesurer le TGF-β induit Invasion

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

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

21.7K vues.  Leiden University Medical Centre. Un test pour mesurer quantitativement Transforming Growth Factor (TGF)-β-induite invasion dans des gels de collagène en 3 dimensions est décrite. Ce test prend avantage de la série MCF10A de lignées cellulaires, qui représentent les différentes étapes du développement du cancer du sein. Cette méthode peut être adoptée pour être utilisé avec d'autres lignées cellulaires et pourraient être utilisées pour étudier d'autres activateurs ou des inhibite...

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

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

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

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

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

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

Recherche

Médecine

Method to Measure Tone of Axial and Proximal Muscle

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

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

Méthode pour mesurer tonicité des muscles axiaux et proximaux

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

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

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

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

Précision en médecine dentaire, une nouvelle façon de mesurer la justesse et fidélité

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

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

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

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

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

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

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

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 ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

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

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

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

Télémétrie intra-utérine mesurer souris contractile pression<em&gt; In Vivo</em

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

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

Three-Dimensional 3D Tumor Spheroid Invasion Assay

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

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 ...

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

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

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

Chick Coeur Invasion Assay pour tester les techniques invasives des cellules cancéreuses et l&#39;activité des composés potentiellement anti-invasives

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

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

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Une cellule cancéreuse Spheroid test pour évaluer Invasion dans un Cadre 3D

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

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

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

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

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

Le modèle Chick Chorioallantoic Membrane In Vivo pour évaluer l'invasion périnéale dans le cancer de la tête et du cou

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