Le financement de cette recherche est de Cancer Research du Royaume-Uni.

1. Étiquetage rétrovirale de cellules avec des protéines fluorescentes <ol><li> Cellules de la plaque d&#39;encapsidation rétrovirale (ex Phoenix) à à 0,25 x 10 6 cellules par puits d&#39;une 6-même plat dans 10% sérum de veau fœtal (FBS) / DMEM. </li><li> Cellules avec un ADN rétroviral Transfecter 48 heures plus tard les cellules en utilisant Effectene conformément aux instructions du fabricant. </li><li> Rincer les puits deux fois avec support 24 heures plus tard, puis ajouter 1,5 ml de 10% de FBS / DMEM par puits. </li><li> Recueillir le virus en culture de tissus conditionnés moyenne 48 heures plus tard par pipetage et le transfert à 2 ml microtubes. </li><li> Centrifuger à 1600 rpm pendant 5 min pour culotter les cellules. </li><li> Enlever le surnageant dans un tube propre et conserver à -80 ° C. </li><li> Les cellules à un retrovirus transduites sont étalées à 1,5 x 10 5 cellules par puits d&#39;un plat à 6 puits et placés dans un incubateur humidifié à 37 ° C pendant la nuit. </li><li> Le lendemain, retirez le support à partir des cellules et des annoncesd 1 ml de stock de virus complété avec 4 polybrène ul (2 mg / ml) dans chaque puits. Remplacer les plaques dans un incubateur. </li><li> Après incubation pendant 5-6 heures, ajouter 2 ml 10% de FBS / DMEM à chaque puits et les plaques sont remplacés par 37 ° C inbubator nuit. </li><li> Le lendemain, remplacez les médias et permettre aux cellules 24 heures avant d&#39;ajouter milieux sélectifs appropriés. </li><li> Lorsque confluentes (qui dépend du taux de croissance des cellules, et l&#39;efficacité de la transduction rétrovirale, mais prend généralement de 4 à 14 jours), trypsiniser cellules et de tri des cellules par fluorescence à l&#39;aide non-transduites comme une référence, la collecte et la piscine avec des cellules fluorescence plus grande que les non-cellules transduites et des aliquots congeler à -80 ° C pour une utilisation future. </li></ol> 2. Inverse test d&#39;invasion de Matrigel <ol><li> Lentement le dégel d&#39;une partie aliquote de Matrigel complète (c&#39;est à dire contenant des facteurs de croissance) sur la glace (figure 1A). </li><li> Une fois décongelé, diluer Matrigel 1:1 dans PBS glacé (avec toute les autres supplémentairestraitements à une concentration 2x dans la PBS avant dilution; figure 1B). Pour le rendre plus facile à manipuler Matrigel avant polymérisation, tous en matière plastique (par exemple des conseils, tuyaux, etc) doit être glacée. </li><li> Insérer autant de pores 8 microns 6,5 mm de diamètre non couchés Transwells tel que requis dans le puits d&#39;une plaque de culture de tissu 24 puits, puis soigneusement la pipette 100 pi de la Matrigel dilué dans les puits et laisser incuber pendant ~ 30 minutes à 37 ° C pour solidifier (figure 1C). </li><li> Pendant ce temps, préparer un ou plusieurs marqués à la fluorescence des suspensions cellulaires entre 1 à 4 x 10 5 cellules par ml, en fonction de la lignée cellulaire, de chaque condition pré-traités (e. g. Traitement médicamenteux siARN,) dans leur croissance normale moyenne (figure 1D). </li><li> Lorsque le Matrigel s&#39;est solidifié, inverser la Transwells et la pipette 100 ul de la suspension cellulaire sur la partie inférieure orientée vers le haut du filtre (figure 1E). </li><li> Soigneusement couvrir les Transwells avec la base d&#39;un tissu à 24 puitsplaque de culture, de prendre contact avec chaque gouttelette de suspension cellulaire (figure 1F). </li><li> Incuber la plaque dans l&#39;état inversé pendant 4 heures pour permettre la fixation des cellules (figure 1G). </li><li> Après ce temps, tournez les plaques droite-up et lavez chaque Transwell par trempage séquentielle en 2 x 1 ml de sérum moyennes libre (figure 2A). </li><li> Laissez le Transwells dans un troisième puits contenant des médicaments ou des traitements nécessaires (figure 2B). </li><li> Délicatement la pipette 100 ul de 10% de FBS / DMEM plus chimiotactique (e. g. EGF à 25 ng / ml) dans le Transwell sur le dessus du Matrigel solidifié / mélange PBS, remettre le couvercle et incuber pendant 3-5 jours à 37 ° C avec 5% de CO 2 (figure 2C). </li></ol> 3. Coloration et la visualisation <ol><li> Les cellules exprimant des protéines fluorescentes peuvent sauter les étapes ci-dessous avant de microscopie confocale </li><li> Pour l&#39;image non fluorescent cellules envahissant Transwells Matrigel place, en eau douce 24 plats bien et pipette de 1 ml de 4 uMCalcéine AM solution de coloration sur le dessus de chaque fiche de Matrigel, lui permettant de déborder sur les côtés et tacher d&#39;en haut et en bas. Calcéine AM (ester acétoxyméthyle de calcéine) est un colorant de cellules vivantes qui colore les cellules ensemble vert et ne nécessite aucune fixation. </li><li> Incuber 1 heure à 37 ° C dans 5% de CO 2 atmosphère humidifiée, à laquelle les cellules sont entièrement teinté points et prêt à être visualisé par microscopie confocale. </li><li> Alternativement, les cellules non fluorescentes invasion de Matrigel peuvent être fixées et colorées comme décrit ci-dessous en 3.5 à 3.9. </li><li> Transfert de chaque Transwell à une nouvelle plaque de 24 puits. Superposition de 1 ml de 4% para-formaldehyde/0.2% de Triton-X 100. </li><li> Incuber à température ambiante pendant 0,5 heure. </li><li> Retirer fixateur et laver 3 fois avec 1 ml de PBS. </li><li> Retirer ARN cytoplasmique avec 30 min de traitement utilisant la RNase 100 pg / ml de RNase. Laver 2 fois avec du PBS. </li><li> Pour la visualisation ajoutez 0,01 iodure de propidium mg / ml (PI) dilué dans du PBS et laisser à température ambiante dans la daRK pendant 0,5 heure. Laver 3 fois avec du PBS. A ce stade, PI teinté transwell peut être conservé à température ambiante dans l&#39;obscurité pendant au moins 1 mois. </li><li> Méthodes d&#39;imagerie par microscopie confocale précis dépendent des ressources disponibles. En utilisant un microscope inversé confocal, Transwell place avec une petite quantité de résidus de PBS sur la lamelle de grande taille (s&#39;assurer qu&#39;aucun des bulles sont présentes) sur les non-objectif 20 X d&#39;immersion et de capture des sections optiques toutes les 10-15 um à partir du bas de la fiche Matrigel. Individuels tranches optiques peuvent être utilisés pour quantifier l&#39;ampleur de l&#39;invasion (figure 3A) ou à construire en 3 dimensions des objets en utilisant un logiciel approprié tel que Volocity (figure 3B). </li></ol> 4. Les résultats représentatifs Un exemple d&#39;une série Z des tranches optiques est montré dans la figure 3A. Dans ce cas, les cellules ont été colorées avec calcéine AM et le nombre de cellules jusqu&#39;à l&#39;invasion du filtre peut être vu à diminuer avec la distance. Quantification devasion peut être fait en analysant le rapport de calcéine AM pixels positive à négative pixels à chaque intervalle, ou en utilisant la méthode de fixation / coloration détaillées ci-dessus et le comptage PI noyaux positifs à chaque position. Un avantage de calcéine AM coloration est que les reconstructions en 3 dimensions de l&#39;invasion des cellules peuvent être assemblés en utilisant des logiciels tels que Volocity, donnant une représentation visuelle du mode d&#39;invasion (figure 3B). Si les cellules sont marquées par l&#39;expression de protéines fluorescentes, les positions de chacune des cellules de couleur peuvent être visualisées en trois dimensions des reconstructions, soit vu de côté (figure 3C) ou en faisant des tranches grâce à la reconstruction (figure 3D). <img alt="Figure 1" src="/files/ftp_upload/3525/3525fig1.jpg" /> Figure 1. Schéma de principe d&#39;étapes impliquées dans la mise en place de dosage d&#39;invasion inverse. A) Matrigel ECM décongelées sur glace. B) Matrigel est dilué 1:1 avec PBS contenant aucun des traitements médicamenteux à une concentration deux fois plus définitive. C)<html> Insère Transwell sont placés dans des plaques multipuits, et Matrigel pipetés dans chacune. D) Les suspensions cellulaires faites à la concentration désirée. E) Une fois le Matrigel a mis, la plaque est inversé et enlevé, les cellules sont étalées sur le filtre sous-face des inserts Transwell. F) en position inversée, la plaque alvéolaire est soigneusement placée sur inserts Transwell, prise de contact avec la suspension cellulaire. G) Les cellules sont autorisés à adhérer au filtre pendant 4 heures. <img alt="Figure 2" src="/files/ftp_upload/3525/3525fig2.jpg" /> Figure 2. Poursuite du schéma pour le dosage de l&#39;invasion inverse. A) Une fois les cellules ont adhéré, tremper chaque Transwell en milieu sans sérum à deux reprises pour enlever les cellules lâches. B) Lieu lavés dans un média Transwell finale puits contenant plus de traitements nécessaires. C) Les médias contenant chimiotactique (par exemple 10% de sérum de veau fœtal) avec des traitements comme l&#39;exige est soigneusement déposé sur Matrigel. ent &quot;&gt; <img alt="Figure 3" src="/files/ftp_upload/3525/3525fig3.jpg" /> Figure 3. Images représentant des résultats de dosage de l&#39;invasion inverse. A) des sections optiques des cellules colorées avec calcéine AM invasion de Matrigel, prises à intervalles de 15 um par microscopie confocale. B) la reconstruction d&#39;une reconstruction en 3 dimensions de l&#39;invasion des cellules d&#39;une pile de confocale série Z des images, des vues de côté. Reproduit de la référence 10. C) Reconstruction tridimensionnelle de cellules GFP et RFP marqué l&#39;invasion de Matrigel vu de côté. D) slice optique grâce à la reconstruction en 3 dimensions de cellules GFP et RFP-étiquetés. Reproduit de la référence 10.

<ol>
	<li>Olson, M. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

Les auteurs n&#39;ont rien à révéler.

Dosages invasion de Matrigel ont traditionnellement été mis en place avec des cellules placées sur une couche de protéine de la matrice extracellulaire avec chimiotactique induite par la motilité vers et à travers un filtre à la base. Envahissement a été marqué en fonction du nombre de cellules peut être compté sur la partie inférieure du filtre. Bien que pratiquement il ya peu de différence avec le dosage de l&#39;invasion &quot;inverse&quot; décrit ci-dessus, il ya beaucoup plus d&#39;informations sur l...

<table border="1"><tbody><tr><th> Nom du réactif </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> DMEM (milieu Dulbecco modifié Eagle) </td><td> GIBCO </td><td> 21969 </td></tr><tr><td> Sérum de veau fœtal </td><td> AAP </td><td> A15-101 </td></tr><tr><td> La streptomycine Pénicilline </td><td> GIBCO </td><td> 15140 </td></tr><tr><td> 200 mM de L-glutamine (100x) </td><td> GIBCO </td><td> 25050-032 </td></tr><tr><td> Puromycine </td><td> Sigma-Aldrich </td><td> P8833 </td></tr><tr><td> 0,05% de trypsine EDTA </td><td> GIBCO </td><td> 25300 </td></tr><tr><td> Polybrène </td><td> Sigma-Aldrich </td><td> AL-118 </td></tr><tr><td> Lipofectamine 2000 Réactif </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> Transwells 6.5mm, taille des pores 8,0 um </td><td> Corning </td><td> 3422 </td></tr><tr><td> Remplissez Matrigel </td><td> BD Biosciences </td><td> 354234 </td></tr> &lt;tr&gt; <td> Calcéine AM </td><td> Invitrogen </td><td> C1430 </td></tr><tr><td> RNase </td><td> Qiagen </td><td> 19101 </td></tr><tr><td> L&#39;iodure de propidium </td><td> Sigma-Aldrich </td><td> P4864 </td></tr><tr><td> Confocale microcope </td><td> Leica </td><td> SP2MP </td></tr></tbody></table>

modeling and imaging 3-dimensional collective cell invasion

Une caractéristique de la malignité du cancer est une invasion et la métastase. Dans certains cancers (e. g. Gliome 2), invasion locale dans les tissus environnants sains est à l&#39;origine de la maladie et la mort. Pour d&#39;autres cancers (e. g. Sein, poumon, etc.), C&#39;est le processus de métastase, dans lequel les cellules tumorales de passer d&#39;une masse tumorale primaire, colonisent les sites distale et finalement contribuer à une défaillance organique, qui mène éventuellement à la morbidité et la de mortalité 3. Il a été estimé que l&#39;invasion et les métastases sont responsables de 90% des décès par cancer 4. En conséquence, il a été un vif intérêt dans l&#39;identification des processus moléculaires et les médiateurs protéiques critique de l&#39;invasion et les métastases à des fins d&#39;améliorer le diagnostic et le traitement 5. Un défi pour les scientifiques est de développer un cancer des dosages invasion qui ressemble plus suffisamment à la situation in vivopour permettre la modélisation des maladies précises 6. Deux dimensions tests de motilité des cellules sont seulement informatifs sur un aspect de l&#39;invasion et ne prennent pas en compte remodelage matriciel protéine extracellulaire (ECM) qui est aussi un élément critique. Récemment, la recherche a affiné notre compréhension de l&#39;invasion des cellules tumorales et des cellules individuelles a révélé que peuvent se déplacer par des modes allongés ou arrondis 7. En outre, il a été plus grande appréciation de la contribution de l&#39;invasion collective, dans laquelle les cellules envahir en brins, les feuilles et les grappes, en particulier dans les tumeurs hautement différenciées qui maintiennent des caractéristiques épithéliales, à la propagation du cancer 8. Nous présentons une méthode raffinée 9 pour examiner les contributions des protéines candidates à l&#39;invasion collective 10. En particulier, par des pools d&#39;ingénierie séparées des cellules d&#39;exprimer différentes protéines fluorescentes, il est possible de disséquer moléculairement les activités et Proteins nécessaires dans les cellules de leader par rapport à ceux requis dans les cellules suivantes. L&#39;utilisation de l&#39;ARNi constitue l&#39;outil moléculaire pour démonter expérimentalement les processus impliqués dans l&#39;invasion des cellules individuelles ainsi que dans différentes positions d&#39;invasion collective. Dans cette procédure, des mélanges de cellules marqué par fluorescence sont plaquées sur le fond d&#39;un insert Transwell préalablement rempli par Matrigel ECM protéines, a ensuite permis d&#39;envahir &quot;par le haut» à travers le filtre et dans le Matrigel. Reconstruction des piles d&#39;images de la série Z, obtenue par imagerie confocale, en trois dimensions des représentations permet de visualiser collectivement brins d&#39;invasion et d&#39;analyse de la représentation de cellules marqué par fluorescence dans les principaux postes suivants par rapport.

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

Modèles de l&#39;invasion des cellules tumorales en trois dimensions de la matrice extracellulaire de mieux refléter la<em&gt; In vivo</em&gt; La situation de tests de motilité en deux dimensions. En utilisant des tests d&#39;invasion matrice combinée avec l&#39;imagerie confocale de cellules marqué par fluorescence, des informations détaillées sur les modes d&#39;invasion et les contributions distinctes de leader par rapport suivants cellules peuvent être obtenues.

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 ...

modeling-and-imaging-3-dimensional-collective-cell-invasion

Research

JoVE Journal

Medicine

16.6K vues.  University of Strathclyde. Modèles de l'invasion des cellules tumorales en trois dimensions de la matrice extracellulaire de mieux refléter la In vivo La situation de tests de motilité en deux dimensions. En utilisant des tests d'invasion matrice combinée avec l'imagerie confocale de cellules marqué par fluorescence, des informations détaillées sur les modes d'invasion et les contributions distinctes de leader par rapport suivants cellules peuvent être obtenue...

Vidéo: Modeling and Imaging 3-Dimensional Collective Cell Invasion

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

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

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Recherche

Médecine

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

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

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

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

Assay Sphéroïde pour mesurer le TGF-β induit Invasion

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

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.
A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure1,2, alterations in brain vasculature of a Alzheimer's disease mouse model3, portal vein abnormalities in portal hypertensive and cirrhotic mice4, developmental steps in rat lymphatic maturation between immature and adult lungs5, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury6.
Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.

A method of visualizing and quantifying the 3-dimensional structure of mouse hepatic portal vein or intrahepatic bile duct is described. This resin cast technique can also be applied to other ductal or vascular systems and allows for in situ visualization or quantification of a system's intact communicating architecture.

Résine de coulée 3-Dimensional Imaging et de la veine porte ou de la souris Système biliaires intrahépatiques

In  organs, the correct architecture of vascular and ductal structures is  indispensable for proper physiological function, and the formation and  ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

L&#39;imagerie moléculaire à la cible transplanté des cellules progénitrices musculaires

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Génération et quantification 3-Dimensional des lésions artérielles chez la souris utilisant la tomographie optique de projection

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

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

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

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

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

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 ...

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 ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Modélisation spontanée carcinome rénal métastatique (MRCC) chez des souris après néphrectomie

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolement et immortalisation de lignées cellulaires de patients dérivé de la biopsie musculaire pour la modélisation des maladies

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

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 ...