Il finanziamento per questa ricerca è da Cancer Research UK.

1. Etichettatura retrovirali di cellule con le proteine ​​fluorescenti <ol><li> Piastra di cellule imballaggio retrovirali (ad esempio Phoenix) fuori a 0,25 x 10 6 cellule per pozzetto di un 6-bene piatto nel 10% siero fetale bovino (FBS) / DMEM. </li><li> Trasfezione delle cellule con il DNA retrovirale 48 ore più tardi le cellule usando Effectene secondo le istruzioni dei produttori &#39;. </li><li> Sciacquare due volte con pozzi di media 24 ore più tardi, quindi aggiungere 1,5 ml di 10% FBS / DMEM per bene. </li><li> Raccogliere virus confezionato in colture di tessuti di media dopo 48 ore pipettando e trasferendo ai tubi microcentrifuga da 2 ml. </li><li> Centrifugare a 1600 rpm per 5 minuti per far sedimentare tutte le cellule. </li><li> Rimuovere il supernatante in una provetta pulita e conservare a -80 ° C. </li><li> Celle da retrovirally trasdotte sono placcati fuori a 1,5 x 10 5 cellule per pozzetto di un piatto ben 6 e posto in un incubatore umidificato a 37 ° C durante la notte. </li><li> Il giorno successivo, rimuovere il supporto da parte delle cellule e add 1 ml di brodo virus integrato con 4 polibrene microlitri (2 mg / ml) in ciascun pozzetto. Sostituire le piastre in incubatrice. </li><li> In seguito a incubazione per 5-6 ore, aggiungere 2 ml 10% FBS / DMEM in ciascun pozzetto e le piastre sono sostituiti in inbubator 37 ° C durante la notte. </li><li> Il giorno successivo, sostituire i media e consentono alle cellule 24 ore prima di aggiungere appropriati terreni selettivi. </li><li> Quando confluenti (che dipende dal tasso di crescita delle cellule, e l&#39;efficienza della trasduzione retrovirale, ma richiede in genere 4-14 giorni), trypsinize cellule e di ordinamento per le celle a fluorescenza utilizzando non trasdotte come riferimento, raccogliere e cellule con piscina fluorescenza maggiore rispetto ai non-trasdotte aliquote e congelare a -80 ° C per uso futuro. </li></ol> 2. Inversa Matrigel invasione test <ol><li> Lentamente scongelare un&#39;aliquota del Matrigel completa (cioè che contiene fattori di crescita) sul ghiaccio (Figura 1A). </li><li> Una volta scongelato, diluire Matrigel ghiaccio 1:1 in PBS freddo (insieme a qualsiasi altra ulterioretrattamenti a concentrazione 2x nella PBS prima della diluizione; Figura 1B). Per rendere più facile da maneggiare Matrigel prima della polimerizzazione, tutti Plasticware (ad esempio punte, tubi, ecc) dovrebbe essere ghiacciato. </li><li> Inserisci come molti pori di 8 micron 6,5 mm di diametro rivestito Transwells come richiesto nei pozzetti di una piastra ben 24 colture di tessuti, poi accuratamente pipetta 100 microlitri della Matrigel diluito nei pozzetti e lasciare incubare per circa 30 minuti a 37 ° C a solidificare (Figura 1C). </li><li> Durante questo tempo, preparare uno o più fluorescenza marcata con sospensioni cellulari tra 1 a 4 x 10 5 cellule per ml, a seconda della linea cellulare, da ogni pre-trattati condizione (e. g. SiRNA, trattamento farmacologico) nella loro crescita normale medio (Figura 1D). </li><li> Quando il Matrigel si è solidificato, invertire la Transwells e pipettare 100 l di sospensione cellulare sul lato inferiore rivolta verso l&#39;alto del filtro (Figura 1E). </li><li> Coprire attentamente la Transwells con la base di un tessuto ben 24piastra di coltura, prendere contatto con ogni goccia di sospensione cellulare (Figura 1F). </li><li> Incubare la piastra in stato invertito per 4 ore per consentire l&#39;attacco delle cellule (Figura 1G). </li><li> In seguito a questo punto, girare i piatti destra-up e lavare ogni Transwell per immersione sequenziale in 2 x 1 ml di mezzo di siero libero (Figura 2A). </li><li> Lasciare il Transwells in un terzo pozzo contenente farmaci o trattamenti necessari (Figura 2B). </li><li> Delicatamente pipetta 100 l di 10% FBS / DMEM più chemiotattico (e. g. FEG a 25 ng / ml) nel Transwell in cima alla Matrigel solidificato / miscela PBS, sostituire il coperchio e incubare per 3-5 giorni a 37 ° C con il 5% di CO 2 (Figura 2C). </li></ol> 3. Colorazione e la visualizzazione <ol><li> Cellule che esprimono proteine ​​fluorescenti possono saltare i passaggi riportati di seguito prima di microscopia confocale </li><li> Per l&#39;immagine non fluorescenti le cellule invadono Matrigel, Transwells luogo fresco in 24 piatti bene e pipettare 1 ml di 4 mMCalceina AM soluzione macchia sulla parte superiore di ogni spina Matrigel, permettendo così di versare sopra i lati e macchia dalla parte superiore e inferiore. Calceina AM (estere acetossimetil di calceina) è un colorante cellule vive che macchia le cellule intera verde e non richiede fissazione. </li><li> Incubare per 1 ora a 37 ° C in 5% CO 2 atmosfera umidificata, in cui le cellule punto sono completamente colorati e pronti per essere ripreso al microscopio confocale. </li><li> In alternativa, non fluorescenti le cellule invadono Matrigel possono essere fissate e colorate come descritto di seguito in 3,5-3,9. </li><li> Trasferimento di ogni Transwell a un nuovo 24-pozzetti. Sovrapposizione 1 ml del 4% para-formaldehyde/0.2% Triton X-100. </li><li> Incubare a temperatura ambiente per 0,5 ore. </li><li> Rimuovere il fissativo e lavare 3 volte con 1 ml di PBS. </li><li> Rimuovere RNA citoplasmatici con 30 minuti di trattamento RNasi con 100 mcg / ml RNasi. Lavare 2 volte con PBS. </li><li> Per la visualizzazione aggiungere 0,01 mg / ml di ioduro di propidio (PI) diluito in PBS e lasciare a temperatura ambiente in dark per 0,5 ore. Lavare 3 volte con PBS. In questa fase, PI macchiato transwell possono essere conservati a temperatura ambiente al buio per almeno 1 mese. </li><li> Metodi di imaging preciso mediante microscopia confocale sono dipendenti dalle risorse disponibili. Utilizzando un microscopio invertito confocale, Transwell luogo con una piccola quantità di residuo PBS su largo coprioggetti (garantire l&#39;assenza di bolle sono presenti) per il mancato obiettivo immersione 20 X e catturare sezioni ottiche ogni 10-15 micron dalla parte inferiore della spina matrigel. Singoli fette ottico può essere utilizzato per quantificare l&#39;entità di invasione (Figura 3A) o di costruire in 3-oggetti tridimensionali utilizzando un software appropriato, come Volocity (Figura 3B). </li></ol> 4. Rappresentante Risultati Un esempio di serie Z di fette ottica è mostrata in figura 3a. In questo caso, le cellule sono state colorate con calceina AM e il numero di cellule invasori fino dal filtro può essere visto a diminuire con la distanza. Quantificazione dellevasion può essere fatto analizzando il rapporto di calceina AM pixel per pixel positivo negativo ad ogni intervallo, oppure utilizzando la fissazione / colorazione metodo sopra descritto e il conteggio dei nuclei PI positivo in ogni posizione. Un vantaggio di calceina AM colorazione è che il 3-dimensionale ricostruzioni di invasione delle cellule possono essere assemblati utilizzando software come Volocity, dando una descrizione visiva del modo di invasione (Figura 3B). Se le cellule sono etichettati con l&#39;espressione di proteine ​​fluorescenti, poi le posizioni di ogni cella del colore possono essere visualizzati in 3-dimensionale ricostruzioni, sia visti di lato (Figura 3C) o facendo fette attraverso la ricostruzione (Figura 3D). <img alt="Figura 1" src="/files/ftp_upload/3525/3525fig1.jpg" /> Figura 1. Schema delle tappe per la creazione di test inverso invasione. A) Matrigel ECM scongelati sul ghiaccio. B) Matrigel viene diluito 1:1 con PBS contenente eventuali trattamenti farmaco ad una concentrazione finale due volte. C)<html> Inserti Transwell sono posti in piastre a pozzetti multipli, e in ogni Matrigel pipettati. D) le sospensioni cellulare fatto a concentrazione desiderata. E) Una volta che il Matrigel ha fissato, il piatto è invertito e rimosso, le cellule sono placcati sul filtro inferiore del inserti Transwell. F) in posizione capovolta, la lastra alveolare viene accuratamente posizionato sopra inserti Transwell, prendere contatto con la sospensione cellulare. G) Le cellule possono aderire al filtro per 4 ore. <img alt="Figura 2" src="/files/ftp_upload/3525/3525fig2.jpg" /> Figura 2. Proseguimento dello schema per il saggio invasione inverso. A) Una volta che le cellule hanno aderito, ciascuno Transwell tuffo in serum-free dei media due volte per rimuovere le cellule sciolto. B) Luogo lavati Transwell in un media finale contenente ben più trattamenti come richiesto. C) Media contenente chemiotattico (es. 10% di siero fetale bovino) con trattamenti come richiesto viene accuratamente a strati su Matrigel. ENT &quot;&gt; <img alt="Figura 3" src="/files/ftp_upload/3525/3525fig3.jpg" /> Figura 3. Immagini rappresentative dei risultati del test invasione inversa. A) sezioni ottiche di cellule colorate con calceina AM invasori Matrigel, prese a intervalli di 15 micron al microscopio confocale. B) Ricostruzione di una 3-dimensionale ricostruzione di invasione delle cellule da una pila di confocale serie Z immagini, viste dal lato. Ristampato da riferimento 10. C) Ricostruzione tridimensionale di cellule GFP e RFP-etichettati invasori Matrigel visto di lato. D) fetta ottico attraverso la 3-dimensionale ricostruzione delle cellule GFP e RFP-etichettati. Ristampato da riferimento 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>

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Saggi di invasione Matrigel sono tradizionalmente stati istituiti con le cellule posto su uno strato di proteine ​​della matrice extracellulare con chemoattractant indotta motilità verso e attraverso un filtro in basso. L&#39;invasività è stata valutata in funzione del numero di cellule potevano contare sulla parte inferiore del filtro. Anche se in pratica vi è poca differenza con il saggio &quot;inversa&quot; invasione di cui sopra, ci sono informazioni molto più sul processo di invasione che può essere deter...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> DMEM (Medium Dulbecco Modified Eagle) </td><td> GIBCO </td><td> 21969 </td></tr><tr><td> Siero bovino fetale </td><td> PAA </td><td> A15-101 </td></tr><tr><td> Penicillina Streptomicina </td><td> GIBCO </td><td> 15140 </td></tr><tr><td> 200 mM L-glutammina (100x) </td><td> GIBCO </td><td> 25050-032 </td></tr><tr><td> Puromicina </td><td> Sigma-Aldrich </td><td> P8833 </td></tr><tr><td> 0,05% tripsina EDTA </td><td> GIBCO </td><td> 25300 </td></tr><tr><td> Polibrene </td><td> Sigma-Aldrich </td><td> AL-118 </td></tr><tr><td> Lipofectamine 2000 reagente </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> Transwells 6.5mm, 8,0 micron diametro dei pori </td><td> Corning </td><td> 3422 </td></tr><tr><td> Completa Matrigel </td><td> BD Biosciences </td><td> 354234 </td></tr> &lt;tr&gt; <td> Calceina AM </td><td> Invitrogen </td><td> C1430 </td></tr><tr><td> RNasi </td><td> Qiagen </td><td> 19101 </td></tr><tr><td> Propidio ioduro </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

Una caratteristica distintiva della malignità del cancro è l&#39;invasione e metastasi 1. In alcuni tipi di cancro (e. g. Glioma 2), invasione locale nel circostante tessuto sano è la causa principale di malattia e morte. Per altri tipi di tumore (e. g. Mammella, polmone, ecc.), È il processo di metastasi, in cui le cellule tumorali passare da una massa tumorale primaria, colonizzare siti distale e in ultima analisi, contribuire alla insufficienza d&#39;organo, che alla fine porta a morbilità e mortalità 3. E &#39;stato stimato che l&#39;invasione e le metastasi sono responsabili del 90% delle morti per cancro 4. Di conseguenza, vi è stato intenso interesse per individuare i processi molecolari e mediatori proteina critica di invasione e metastasi ai fini del miglioramento della diagnosi e del trattamento 5. Una sfida per gli scienziati è quello di sviluppare il cancro test invasione che sufficientemente simile alla situazione in vivoper permettere la modellazione accurata della malattia 6. Bidimensionale saggi di motilità cellulare sono solo informativi circa un aspetto della invasione e non tengono conto della matrice extracellulare (ECM) rimodellamento proteina che è anche un elemento critico. Recentemente, la ricerca ha raffinato la nostra comprensione di invasione delle cellule tumorali e ha rivelato che le singole cellule possono spostare da modalità allungate o arrotondate 7. Inoltre, vi è stata maggiore apprezzamento per il contributo di invasione collettiva, in cui le cellule invadono in fili, fogli e dei cluster, in particolare nei tumori altamente differenziate che mantengono le caratteristiche epiteliali, per la diffusione del cancro 8. Vi presentiamo un metodo raffinato 9 per esaminare il contributo delle proteine ​​candidate per invasione collettiva 10. In particolare, le piscine di ingegneria separati di cellule di esprimere diverse proteine ​​fluorescenti, è possibile sezionare molecolarmente le attività e PROTEIns richiesto nelle cellule principali rispetto a quelli richiesti nelle cellule seguito. L&#39;uso di RNAi fornisce lo strumento molecolare di smontare sperimentalmente i processi coinvolti nell&#39;invasione delle cellule individuali così come in diverse posizioni di invasione collettiva. In questa procedura, le miscele di cellule fluorescenza marcata sono placcati sul fondo di un inserto Transwell precedentemente riempiti con proteine ​​Matrigel ECM, poi il permesso di invadere &quot;verso l&#39;alto&quot; attraverso il filtro e nel Matrigel. Ricostruzione delle serie Z stack immagine, ottenuta da imaging confocale, in rappresentazioni tridimensionali permette la visualizzazione di collettivamente fili invadere e analisi della rappresentazione di cellule fluorescente-etichettato leader contro seguenti posizioni.

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

Modelli di invasione delle cellule tumorali in tre dimensioni della matrice extracellulare meglio riflettere la<em&gt; In vivo</emSituazione&gt; quelli a due dimensioni saggi di motilità. Usando saggi invasione matrice combinata con l&#39;imaging confocale di cellule fluorescenza-etichettato, informazioni dettagliate sulle modalità di invasione e il contributo distinto di leader rispetto ai seguenti cellule possono essere ottenuti.

Modellazione e imaging 3-Dimensional Invasion cella Collettiva

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 Views.  University of Strathclyde. Modelli di invasione delle cellule tumorali in tre dimensioni della matrice extracellulare meglio riflettere la In vivo quelli a due dimensioni saggi di motilità. Usando saggi invasione matrice combinata con l'imaging confocale di cellule fluorescenza-etichettato, informazioni dettagliate sulle modalità di invasione e il contributo distinto di leader rispetto ai seguenti cellule possono essere ottenuti.

Video: 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-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

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

Ricerca

Medicina

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.

Saggio sferoide Misura TGF-β-indotta 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.

3-Dimensional Casting resina e imaging di Vena mouse portale o sistema biliare intraepatica Duct

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.

Imaging molecolare a Target trapiantate cellule progenitrici muscolari

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.

Generazione e quantificazione 3-Dimensional di arteriosa lesioni nei topi mediante tomografia ottica di proiezione

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;

Molecular Profiling del microambiente tumorale invasiva in un modello 3-Dimensional delle cellule del cancro colorettale e<em&gt; Ex vivo</em&gt; fibroblasti

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.

A Modified<em&gt; In vitro</em&gt; Invasion test per determinare il ruolo potenziale di ormoni, citochine e / o fattori di crescita nella mediazione Cancer Invasion cellulare

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.

Modellazione di Carcinoma renale metastatico spontanea (MRCC) in topi dopo nefrectomia

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.

Isolamento e Immortalizzazione di paziente derivati ​​da linee cellulari provenienti da Muscle Biopsia per la malattia Modeling

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.

Un Cancer Cell Saggio sferoide valutare Invasion in un Ambiente 3D

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