MB est soutenu par des subventions d&#39;une bourse de la MRC. KP et AHM sont pris en charge par des subventions de recherche médicale Wessex et Cancer Research UK / RCS (Angleterre) (C28503/A10013). Nous sommes reconnaissants pour le soutien de l&#39;Université de Southampton Unité de recherche histochimie.

1. Mise en place des cultures de fibroblastes primaires de coliques explants <ol><li> Obtenir des échantillons de tissu de colon humain muqueuse normale directement à partir de la salle d&#39;opération chirurgicale, et suspendre à 7-10 ml de PBS supplémenté avec 100 unités / ml de pénicilline, 100 pg / ml de streptomycine, et 0,25 pg / ml de Fungizone. </li><li> Dans le laboratoire, placer les échantillons au centre d&#39;une boîte de culture de tissu de 10 cm et laver 3 fois avec du PBS / Pen-angine / Fungizone. Ne pas aspirer après le lavage final. </li><li> Avec des pinces stériles et scalpel, couper le tissu en petits morceaux (environ 2 mm) et placer chaque pièce au centre d&#39;une croix tracée avec la lame de scalpel dans une plaque de 12 puits. Basculez le scalpel doucement sur le tissu afin de lui permettre d&#39;adhérer. </li><li> Culture des échantillons dans 750 ul de milieu de croissance des fibroblastes primaires (DMEM supplémenté avec 20% de sérum de veau foetal (SVF), 100 U / ml de pénicilline, 100 pg / ml de streptomycine et 292 ug / ml de L-glutamine), ce qui devrait submerge de tissu, sans lui permettre de flotter. Éviter de perturber la plaque au cours des 5 prochains jours sauf réalimentation tous les 2 jours. </li><li> Au cours des 5 prochains jours fibroblastes vont lentement commencer à se développer à partir du bord du tissu. À environ 7 jours, ce sera plus visible. Au bout d&#39;environ 3 à 4 semaines, et si il ya suffisamment de croissance des fibroblastes, ajoutez 750 ul de trypsine à chaque puits. Après que les cellules ont détaché (qui peut prendre 5-10 min), la piscine et le transfert à un flacon de culture tissulaire T-25. </li></ol> 2. Organotypique Préparation Préparation du gel imprégné fibroblastes organotypique: <ol><li> Préparer une suspension de cellules de fibroblaste contenant 5 x 10 5 cellules par organotypique dans du DMEM supplémenté avec 10% de SVF et 292 ug / ml de L-Glutamine. </li><li> Faites les gels organotypiques sur la glace, dans les rapports suivants: <ul><li> 7 volumes de collagène de queue de rat: Matrigel mélange (1:1) </li><li> 1 volume de DMEM filtré 10x </li><li> 1 volume de FCS </li><li> 1 volume de suspension cellulaire de fibroblastes contenant 5 x 10 5 fibroblastes Pour 9 gels (1 ml par gel) préparer 10 ml et mélanger doucement pour éviter les bulles. </li></ul></li><li> Ajouter 1 ml dans chaque puits d&#39;une plaque à 24 puits et incuber pendant 1 heure à 37 ° C dans une atmosphère humidifiée pour permettre de fixer des gels. Après 1 h ajouter 1 ml de DMEM (10% de FCS) / Glut sur des gels et revenir à l&#39;incubateur pendant la nuit. </li><li> Le jour suivant, le support d&#39;aspiration à partir de la partie supérieure de la plaque de gels et de 1 ml de milieux contenant 5 x 10 5 cellules epitheliales du CRC. </li></ol> Préparation des feuilles de nylon enduit de gel: <ol start="5"><li> Préparer stérile, feuilles de nylon autoclave mesure 1,5 cm x 1,5 cm, à l&#39;avance. </li><li> En utilisant des pinces stériles, placer le nombre requis de feuilles de nylon dans une boîte de culture de 10 cm (généralement 4 feuilles de nylon peuvent être logés par boîte). </li><li> Maquillage gel sur la glace dans l&#39;ordre suivant (250 pi totalevolume est nécessaire par feuille de nylon): <ul><li> 7 volumes de collagène de queue de rat </li><li> 1 volume de DMEM filtré 10x </li><li> 1 volume de FCS </li><li> 1 volume de DMEM 10% SVF / Glut </li></ul></li><li> Si la solution est jaune, neutraliser par addition de NaOH 0,1 M dans des aliquotes de 50 pl jusqu&#39;à ce que la solution vire au rose. </li><li> Ajouter 250 pi de cette solution à chaque feuille de nylon et incuber à 37 ° C dans une atmosphère humidifiée de 5% de CO2 pendant 30 min pour permettre de mettre en gel. </li><li> Maquillage solution de glutaraldéhyde à 1% dans du PBS. Ajouter 10 ml à chaque boîte de culture de 10 cm, une fois le gel est fixé et incuber à 4 ° C pendant 1 heure. </li><li> Les feuilles de nylon Laver 3x dans du PBS 1x et dans du DMEM (10% de FCS) / Glu, et incuber pendant une nuit dans du DMEM / Glu à 4 ° C. </li></ol> Augmenter gels organotypiques sur des grilles en acier pour l&#39;invasion: <ol start="12"><li> Pré-préparer des grilles d&#39;échafaudage par pliage de tôles d&#39;acier inoxydable dans une formation de trépied. Autoclave avant utilisation.</li><li> Utiliser des pinces stérilisées dans de l&#39;éthanol afin de placer une grille d&#39;acier dans chaque puits d&#39;une plaque à six puits. </li><li> Placer une feuille de nylon enduit de gel avec le côté le plus élevé du collagène, sur chaque grille. </li><li> Soigneusement transférer les gels organotypiques de la plaque de 24 puits à la feuille de nylon soulevé à l&#39;aide d&#39;une spatule stérilisé dans de l&#39;éthanol. </li><li> Remplir le puits avec du DMEM 10% SVF / Glut supplémenté avec 10% de FCS jusqu&#39;à ce que la feuille de nylon est en contact avec le milieu, mais non immergée. Assurez-vous que le support ne touche pas le gel organotypique. </li><li> Incuber pendant 14 jours à 37 ° C dans une atmosphère humidifiée de 5% de CO 2, en remplacement de support tous les 2 jours. </li></ol> 3. Organotypique fixation <ol><li> Retirer organotypique ensemble, y compris la feuille de nylon de bien et placer sur une pellicule de plastique. </li><li> Bissecter organotypique et la feuille de nylon à l&#39;aide d&#39;un scalpel jetable propre et fixer les deux moitiés dans du formol pendant 24 heures à température ambiante. </li><li> Remplacer formaldéhyde avec 70% ethanol après 24 h et laisser une nuit avant l&#39;enrobage dans de la paraffine, la coupe et la coloration. </li></ol> 4. Laser microdissection de la marge envahissantes <ol><li> Section monté à 10 um d&#39;épaisseur sur une membrane diapositives. </li><li> Déparaffiner sections en appliquant xylène pendant 1 min, puis retirez le xylène et fixer à 75% d&#39;éthanol pendant 1 min. </li><li> Éliminer l&#39;éthanol et tache avec une solution de 0,125% de crésyl violet pendant 1 min. Le violet de crésyl souligne cellules épithéliales et permet la discrimination facile du stroma. </li><li> Retirer le violet de crésyl et appliquer 100% d&#39;éthanol pendant 1 min avant de rincer à 100% d&#39;éthanol. Laisser les lames sécher à l&#39;air pendant 30 minutes. </li><li> Effectuer microdissection laser en utilisant la plate-forme choisie (par exemple, le système Leica AS). </li><li> Placez la lame de verre avec la section teinté à microdissection face vers le bas sur la platine du microscope. </li><li> Monter un tube de 0,5 ml à centrifuger dans la cassette de collecte et ajouter 50 ul deun tampon de lyse cellulaire (par exemple, ADN, ARN ou un tampon de lyse protéique) à la coiffe en tissu qui permettra de recueillir microdissection. </li><li> Sous vision directe, utilisez le joystick pour identifier un tissu d&#39;intérêt, et l&#39;utilisation de l&#39;interface du logiciel, d&#39;annoter la section organique souillé, soulignant cellules à microdissection à l&#39;avant de l&#39;invasion tumorale. </li><li> Demandez le laser à feu, ce qui devrait réduire à la fois la section en surbrillance et propulser tissu microdissection dans le bouchon du tube à centrifuger. </li><li> Une fois suffisamment de matériel a été recueilli, éjecter la cassette de collecte, fermer le tube à centrifuger et tourner doucement pour l&#39;extraire de la mémoire tampon de lyse au fond du tube. </li><li> Placer les échantillons sur la glace jusqu&#39;à ce que la microdissection est terminée. Traiter les échantillons par une méthode appropriée pour extraire l&#39;analyte d&#39;intérêt. </li></ol>

<ol>
	<li>Nystrom, M. L., Thomas, G. J., Stone, M., Mackenzie, I. C., Hart, I. R., Marshall, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J Pathol. 205 (4), 468-475 (2005).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-153+supports+colorectal+cancer+progression+via+pleiotropic+effects+that+enhance+invasion+and+chemotherapeutic+resistance.">miR-153 supports colorectal cancer progression via pleiotropic effects that enhance invasion and chemotherapeutic resistance.</a> Cancer Res. , (2013).</li><li>Bullock, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+actions+of+miR-21+highlight+the+critical+role+of+deregulated+stromal+microRNAs+during+colorectal+cancer+progression.">Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.</a> Cell Death Dis. 20 (4), (2013).</li><li>Jenei, V., Nystrom, M. L., Thomas, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+invasion+in+an+organotypic+model.">Measuring invasion in an organotypic model.</a> Methods Mol Biol. 769, 223-232 (2011).</li><li>De Wever, O., Demetter, P., Mareel, M., Bracke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+myofibroblasts+are+drivers+of+invasive+cancer+growth.">Stromal myofibroblasts are drivers of invasive cancer growth.</a> Int J Cancer. 123 (10), 2229-2238 (2008).</li><li>Fries, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+fibroblast+heterogeneity+and+the+role+of+fibroblast+subpopulations+in+fibrosis.">Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.</a> Clin Immunol Immunopathol. 72 (3), 283-292 (1994).</li><li>Moutasim, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betel-derived+alkaloid+up-regulates+keratinocyte+alphavbeta6+integrin+expression+and+promotes+oral+submucous+fibrosis.">Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.</a> J Pathol. 223 (3), 366-377 (2011).</li><li>Winter, J., Jung, S., Keller, S., Gregory, R. I., Diederichs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+roads+to+maturity:+microRNA+biogenesis+pathways+and+their+regulation.">Many roads to maturity: microRNA biogenesis pathways and their regulation.</a> Nat Cell Biol. 11 (3), 228-234 (2009).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+miRNA+expression+patterns+using+total+RNA+extracted+from+matched+samples+of+formalin-fixed+paraffin-embedded+(FFPE)+cells+and+snap+frozen+cells.">Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells.</a> BMC Biotechnol. 7 (36), (2007).</li><li>Szafranska, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+molecular+characterization+of+formalin-fixed,+paraffin-embedded+tissues+by+microRNA+expression+profiling.">Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.</a> J Mol Diagn. 10 (5), 415-423 (2008).</li></ol>

Les auteurs décrivent pas de conflits d&#39;intérêts potentiels.

Nous décrivons ici un procédé pour isoler et caractériser spécifiquement les cellules tumorales qui ont acquis la capacité d&#39;envahir le stroma associé au cancer au cours des premiers stades de la progression métastatique dans un produit d&#39;assemblage en 3 dimensions de co-culture de cellules épithéliales et stromales. Modèles de co-culture comprenant des cellules épithéliales CRC juxtaposés avec un stroma synthétique contenant ex vivo des fibroblastes du côlon...

Co-cultures organotypiques sont des modèles à partir de tissus qui aide à la reconstruction de la micro-vivo dans la tumeur en trois dimensions en juxtaposant les cellules épithéliales malignes et les cellules stromales dans un gel de collagène contenant des composants de la matrice extracellulaire essentiels 3.1. Principalement conçu comme une méthode de mesure de l&#39;invasion tumorale, organotypics réduire la dépendance in vivo dans des modèles animaux, et d&#39;éviter les défauts des autres techniques in vitro tels que des dosages Transwell, dans lequel les cellules sont forcées artificiellement d&#39;invasion dans un état ​​monodispersé 2-4. L&#39;inclusion de cellules stromales dans ces modèles, telles que les fibroblastes, reflète le rôle fondamental du micro-environnement de la tumeur maligne dans la régulation de l&#39;invasion et la métastase 4.3, cependant l&#39;hétérogénéité phénotypique du stroma est telle que, pour optimiser la pertinence physiologique de organotypique modèles, pour certains organes, anatomiquement exactes ex vivofibroblastes doivent être inclus autant que possible de 3,6. Organotypics sont une plate-forme polyvalente pour étudier les interactions entre la tumeur et les cellules stromales et sont de plus en plus utilisés dans de nouvelles façons d&#39;examiner l&#39;impact sur ​​l&#39;invasion tumorale des inhibiteurs chimiques et des altérations de gènes ciblés 7,8. L&#39;étude de la marge de la tumeur invasive est une perspective particulièrement intéressante. Dans les modèles organotypiques, le cancer colorectal mis en place (CRC) des lignées cellulaires produisent typiquement une couche épithéliale bien stratifié, et en coupe, les cellules qui ont acquis la capacité d&#39;envahir la matrice extracellulaire (MEC) sont facilement repérables. Compte tenu de l&#39;hétérogénéité des micro-topographique établi de tumeur et associés à une tumeur des cellules stromales in vivo, l&#39;extraction de ces cellules à l&#39;aide de laser microdissection et de les étudier dans l&#39;isolement, peut révéler des indications biologiques importantes concernant l&#39;origine des métastases du cancer colorectal et la façon dont il peut être plus efficace, ciblée. Jen la méthodologie suivante, tous les patients qui ont donné des échantillons de tissus fournis le consentement éclairé, et l&#39;étude a été approuvée par les institutions du comité d&#39;éthique de la recherche au niveau régional.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colorectal cancer cell lines - example shown SW480</td>
			<td style="width:132px;">ATCC</td>
			<td style="width:119px;">ATCC CCL-228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagen</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matrigel</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nylon membrane</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">VVLP01300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Metal grid</td>
			<td style="width:132px;">The Mesh Company</td>
			<td>WSS20-A4</td>
			<td>themeshcompany.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser microdissection platform</td>
			<td style="width:132px;">Leica Microsystems</td>
			<td style="width:119px;">Leica AS LMD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Membrane mounted slides</td>
			<td style="width:132px;">Molecular devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cresyl Violet</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">1052350025</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts

Envahir le cancer colorectal (CRC), les cellules ont acquis la capacité de se libérer de leurs cellules sœurs, s&#39;infiltrer dans le stroma, et remodeler la matrice extracellulaire (MEC). La caractérisation de la biologie de ce groupe phénotypique distincte de cellules pourrait améliorer considérablement notre compréhension des événements précoces lors de la cascade métastatique. invasion tumorale est un processus dynamique facilitée par des interactions bidirectionnelles entre épithélium malin et le stroma de cancer associé. Afin d&#39;examiner les réponses spécifiques des cellules au stroma tumoral interface, nous avons combiné organotypique co-culture et des techniques de micro-dissection laser. Modèles organotypiques, dans lesquels les mandants stromales clés telles que les fibroblastes sont 3 dimentioanally co-cultivées avec des cellules épithéliales cancéreuses, sont des outils expérimentaux très manipulables qui permettent invasion et interactions stroma-cancer à étudier dans un proche-phconditions ysiological. Microdissection laser (LMD) est une technique qui implique la dissection chirurgicale et l&#39;extraction des différentes couches au sein du tissu tumoral, avec une précision de l&#39;ordre du micron. En combinant ces techniques avec le profilage génomique, transcriptomique et épigénétique nous visons à développer une compréhension plus profonde des caractéristiques moléculaires des cellules tumorales envahissantes et tissu stromal environnante, et en faisant éventuellement révéler de nouveaux biomarqueurs et des opportunités pour le développement de médicaments dans CRC.

Nous avons appliqué le procédé ci-dessus pour de multiples combinaisons de lignées cellulaires de CRC et des cellules stromales. Un exemple présenté ici est des lignées de cellules épithéliales SW480 CRC ex vivo avec des fibroblastes humains primaires du côlon. Co-cultures en 3 dimensions sont construits (figure 1), soumis à la microscopie et la microdissection par capture laser (figure 2), et analysés par microARN (miARN) profilage (figure 3) pour ...

Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Profilage moléculaire des cellules laser microdissection et le stroma des modèles synthétiques, de l&#39;ingénierie tissulaire du cancer colorectal représente une approche nouvelle et manipulable à caractériser et à disséquer la biologie particulière à l&#39;interface entre les cellules tumorales à la cellules stromales associées au cancer invasif et avant.

Profil moléculaire du microenvironnement de la tumeur invasive dans un modèle 3-dimensionnelle des cellules du cancer colorectal et<em&gt; Ex vivo</em&gt; fibroblastes

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

molecular-profiling-invasive-tumor-microenvironment-3-dimensional

Research

JoVE Journal

Medicine

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

11.0K vues.  University of Southampton School of Medicine.  Profilage moléculaire des cellules laser microdissection et le stroma des modèles synthétiques, de l'ingénierie tissulaire du cancer colorectal représente une approche nouvelle et manipulable à caractériser et à disséquer la biologie particulière à l'interface entre les cellules tumorales à la cellules stromales associées au cancer invasif et avant. 

Vidéo: Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

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

Modeling and Imaging 3-Dimensional Collective Cell Invasion

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

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

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

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

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Dépistage du mélanome Modificateurs l&#39;aide d&#39;un modèle de tumeur Zebrafish Autochtone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Vivez l&#39;imagerie des réponses de drogues dans le microenvironnement des tumeurs dans les modèles murins de cancer du sein

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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 ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Une<em&gt; Ex vivo</em&gt; Modèle pour étudier action hormone dans le sein humain

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

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 ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Etudier le cancer du pancréas Stem Cell Caractéristiques pour le développement de nouvelles stratégies de traitement

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

L&#39;isolement et la propagation des cellules tumorales circulantes d&#39;un cancer Modèle Souris

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Une tumeur au cerveau / organotypiques Slice système de co-culture pour l&#39;étude des tumeurs Microenvironnement et ciblée Thérapies de drogue

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...