Questo lavoro è stato finanziato, in parte, dal National Institutes of Health R01 CA131990 (BFS e RRM). Live-cell di imaging è stata effettuata nel Nucleo Microscopia, Imaging e Citometria Risorse sostenuta, in parte, NIH Centro concessione P30 CA22453 il Karmanos Cancer Institute, Wayne State University e dal Perinatologia Research Branch del National Institute of Child Health e Sviluppo , Wayne State University.

1. Preparare DQ-substrati <ol><li> Consentire liofilizzato DQ-substrati a temperatura ambiente prima di fiale di apertura, preparare la soluzione stock di 1 mg / ml di DQ-substrato in acqua deionizzata, dividere in aliquote di 50 pl e conservare a 4 ° C. </li></ol> Può essere necessario agitare DQ-substrato in un bagno d&#39;acqua ultrasonico per ~ 5 minuti e si riscalda a 50 ° C per facilitare la dispersione. <ol start="2"><li> Scongelare RBM su ghiaccio notte a 4 ° C; meccanismi devono essere manipolati in ghiaccio in ogni momento. </li><li> Per rendere fosfato 10X soluzione salina tamponata (PBS), sciogliere 80 g di NaCl (1,37 M), KCl 2 g (0,027 M), 14,4 g di Na 2 HPO 4 (1 M), e 2,4 g di KH 2 PO 4 (0,02 M) in 800 ml di acqua ultrapura. Regolare il pH della soluzione tampone PBS a 7,4 con 1,0 M HCl. Portare il volume ad 1 litro, sterilizzare mediante filtrazione. </li><li> Preparare una soluzione di collagene I su ghiaccio: 8 parti di collagene refrigerato I 1 parte di 10X PBS freddo. Regolare il pHdi miscela a 7,2-7,6 usando sterile 0,1 M NaOH. Controllare il pH con le strisce pH e regolare il volume finale a 10 parti con acqua sterile. </li><li> Preparare DQ-collagene I: collagene I matrice diluendo DQ-collagene I collagene I in soluzione in un tubo prechilled ad una concentrazione finale di 25 pg / ml e 2,4 mg / ml, rispettivamente. </li><li> Preparare DQ-collagene IV: membrana basale ricostituita (MLF) matrice diluendo DQ-collagene IV in RBM (Cultrex o Matrigel) in un tubo prechilled ad una concentrazione finale di 25 pg / ml e 12-15 mg / ml, rispettivamente. Miscelare sul ghiaccio usando pipettaggio delicato per evitare di creare bolle. </li></ol> 2. Preparare co-colture MAME Vedi Figura 1 per un diagramma schematico di co-colture. <ol><li> Inserire due coprioggetti rettangolari di plastica (sterilizzato in alcool al 70% per 10 minuti e lasciarlo asciugare all&#39;aria) in un 35 mm piatto di coltura o di utilizzare un ibiTreat 35 mm μ-Dish. Indicazioni qui, sono per entrambi coprioggetto e u-Piatti, che usiamo per la STUmuore su un microscopio verticale. Per gli studi su un microscopio invertito, usiamo solo u-Piatti. </li><li> Mescolare il numero desiderato di fibroblasti con DQ-collagene I: I matrice di collagene. Per lungo termine co-colture illustrate nelle figure 2 e 3, si usa 500 fibroblasti in 10 pl di mezzi più 60 pl di DQ-collagene I: I matrice di collagene. </li><li> Utilizzando un micropipettatore 100-pl, accuratamente pipetta e diffondere 70 pl di fibroblasti: DQ-collagene I: collagene I matrice su tutta la superficie di ciascun coprioggetto e lasciare a 37 ° C in un incubatore umidificato senza CO 2 per 30 minuti per solidificare. </li><li> Transfer da 35 mm piatti contenenti coprioggetti a 37 ° C incubatore con 5% di CO 2 per 10 min per equilibrare. </li><li> Togliere dal incubatore e lasciare i piatti da 35 mm sotto il cofano fino a che non raggiunga la temperatura ambiente. </li><li> Aggiungere 60 pl di DQ-collagene IV: meccanismi matrice sopra l&#39;solidificato DQ-collagene I: collagene I matrice con fibroblasti incorporati. Con la punta di pipetta, carefully diffondere DQ-collagene IV: RBM matrice in modo uniforme, evitando di graffiare il fibroblasti: DQ-collagene I: I matrice di collagene. </li><li> Transfer da 35 mm piatti contenenti coprioggetti a 37 ° C incubatore con 5% di CO 2 per 10 min a solidificarsi. Mentre il DQ-collagene IV: RBM matrice solidificazione, Tripsinizzare e contare le cellule epiteliali. </li><li> Luogo 50 microlitri di una sospensione di cellule epiteliali / tumore sul coprioggetto rivestito con DQ-collagene IV: RBM matrice. Luogo da 35 mm piatti contenenti i coprioggetti in un incubatore a 37 ° C. Lasciare 40 a 60 minuti per le cellule da allegare al DQ-collagene IV: RBM matrice. Per lungo termine co-colture illustrate nelle figure 2 e 3, si usa 2500 cellule epiteliali o tumore. </li><li> Aggiungere 2 ml di terreno di coltura contenente 2% meccanismi per ogni 35 mm piatto. Se fare qualsiasi trattamenti farmacologici, i farmaci dovrebbero essere aggiunti in questo momento. </li><li> Incubare per periodo desiderato di tempo prima di imaging. Per le colture a lungo termine, i media dovrebbe essere cambiato ogni 3-4 giorni. Le cellule possono essere mantenutenella loro mezzo di coltura normale. Per MAME co-colture di linee cellulari mammarie, usiamo epiteliale medio basale (MEBM-PRF) integrato con SingleQuots MEGM. </li></ol> 3. Eseguire 4D (3D + tempo), l&#39;imaging di co-colture MAME dal vivo mediante microscopia confocale <ol><li> Per i proof-of-principio studi illustrati, co-colture MAME sono state marcate con un colorante fluorescente di monitoraggio delle cellule in base alla nostra procedura di pubblicazione 1. Dopo lavaggio con PBS, co-colture sono state incubate con 5 arancione CellTracker pM in mezzo MEGM per 45 min a 37 ° C incubatore, nuovamente lavate con PBS e incubate con MEGMmedium preriscaldato per 30 min a 37 ° C con 5% CO 2 incubatore prima di imaging . </li><li> Immagine co-colture MAME vivere in un basso ingrandimento (10X, obiettivo acqua immersione) su un LSM-510 Zeiss META microscopio confocale. </li><li> Sezioni ottici vengono acquisiti a intervalli durante l&#39;intera profondità delle strutture. Per i nostri studi, abbiamo catturare sezioni ottiche a 10intervalli micron. </li><li> Utilizzare sezioni ottiche di ricostruire le immagini in 3D. Usiamo software Volocity per generare ricostruzioni 3D. </li><li> Realizzare filmati delle ricostruzioni 3D, in modo che le strutture, l&#39;interazione fra cellule e le interazioni con le matrici circostanti possono essere visualizzati. Abbiamo fatto i film con QuikTime Player 7. </li></ol> 4. Risultati rappresentativi La co-colture MAME che abbiamo sviluppato è un modello sperimentale per trattabile live-cella immagini in tempo reale delle interazioni tra i vari costituenti cellulari che compongono un tumore del seno ed il suo microambiente. Qui si dimostra la capacità dei modelli di MAME per ricapitolare l&#39;interazione fra cellule durante la progressione del cancro della mammella e del live-cell imaging per seguire queste interazioni nel corso del tempo. Illustriamo la possibilità di interazioni tra una immagine preinvasive linea cellulare umana MCF10.DCIS al seno e un seno umano cancro-associata linea di fibroblasti, WS-12TI, oltreun periodo di più di tre settimane in coltura. Il co-colture sono stati ripresi a 3, 16 e 23 giorni. Sezioni ottiche attraverso l&#39;intero volume della co-colture sono state ricostruite in 3D e esempi rappresentativi sono mostrati per ciascuno dei tre punti temporali in figura 2. La fluorescenza rossa in figura 2 rappresenta il MCF10.DCIS e WS-12TI cellule. Per discriminare tra i due tipi di cellule e di lungo termine co-colture, si può transfettare o trasdurre tipi cellulari individuali con proteine ​​fluorescenti prima di stabilire le culture. Una co-coltura di cellule MAME MCF10.DCIS e WS-12TI che sono differenzialmente etichettati con proteine ​​fluorescenti per l&#39;identificazione è illustrato in Figura 3. Per substrati contenenti, in questo caso DQ-collageni, nella co-colture MAME, si può valutare e quantificare i cambiamenti nel tempo in proteolisi associato transizione ad un fenotipo invasivo. Abbiamo stabilito metodi per quantificare i prodotti di scissione fluorescenti che risultatoda DQ-collagene degradazione 2,3. Abbiamo normalizzare prodotti di degradazione nell&#39;intero volume 3D della co-colture MAME su una base per cellula di etichettatura e contando nuclei delle cellule. Inoltre, siamo in grado di localizzare il degrado a compartimenti extracellulari e intracellulari e quantificare i totali, prodotti di degradazione intracellulari ed extracellulari, come abbiamo descritto in precedenza 2. La fluorescenza verde nelle figure 2 e 3 rappresenta prodotti di scissione delle due DQ-collagene substrati. In questo momento, collageni differenzialmente marcati non sono disponibili che permetta di distinguere tra degradazione del collagene di tipo IV e degradazione del collagene di tipo I. Le interazioni in co-colture MAME sono dinamici e possono cambiare nel tempo. Per ottenere informazioni temporali e dinamica, lo stesso campo di vista può essere sezioni immaginati e ottiche adottate attraverso l&#39;intero campione del volume più volte in un periodo di tempo da alcuni minuti a settimana,quindi raccogliendo dati 4-D. Nella figura 2, si illustrano i cambiamenti piuttosto drammatici che si verificano più di 23 giorni: cambiamenti nel numero di cellule, la migrazione cellulare e le interazioni cellulari, volumi, forme struttura la struttura, le deviazioni delle strutture di sfericità, escrescenze invasive e proteolisi. Inoltre, abbiamo anche dimostrato un cambiamento generale di volume a causa della co-colture MAME comprometterne la matrice extracellulare che circonda in questo periodo di tempo. A tre giorni come illustrato in Figura 2A, il volume è 110.000 um 3. Al contrario, il volume del co-colture MAME a 16 e 23 giorni è solo 46.250 um 3, come illustrato nelle figure 2B e C. <img alt="Figura 1" src="/files/ftp_upload/3661/3661fig1.jpg" /> Figura 1. Schema di co-colture MAME. Vetrino in plastica è rivestita con il collagene I, contenente DQ-collagene I e fibroblasti (magenta). Un 2 ° strato della membrana basale ricostituita (RBM) contenente DQ-collagen IV viene aggiunto. Le cellule tumorali (rosso) sono rivestiti in alto e il 2% RBM nella cultura media è sovrapposto (puntini bianchi) ad ogni cambio di media. Il verde rappresenta i prodotti di scissione fluorescenti di DQ-collagene I e IV. <img alt="Figura 2" src="/files/ftp_upload/3661/3661fig2.jpg" /> Figura 2. MAME co-colture di cellule del seno MCF10.DCIS umane e WS-12TI fibroblasti materno. I fibroblasti sono stati incorporati in collagene I / DQ-collagene I e sovrapposti, con DQ-RBM contenente collagene IV. Le cellule di carcinoma duttale in situ sono state poi seminate sul RBM e ricoperto con supporti contenenti 2% RBM. Nel corso dei 23 giorni in co-coltura qui illustrati, c&#39;era la proliferazione cellulare, la degradazione del collagene IV-DQ e I (verde) e matrici di collagene, come indicato dalla riduzione in volume del co-colture. Le cellule sono state localizzate, etichettando con Orange CellTracker in situ prima della creazione dell&#39;immagine (rosso). Le co-colture stesse dal vivo sono state osservate nel corso dei 23 giorni con le immagini catturate da cmicroscopia onfocal a 3, 16 e 23 giorni di co-coltura. Le fette risultanti ottici sono stati ricostruiti in 3D con il software Volocity. Ingrandimento, 10X. <img alt="Figura 3" src="/files/ftp_upload/3661/3661fig3.jpg" /> Figura 3. MAME 16 al giorno co-colture di cellule del seno MCF10.DCIS umane e WS-12TI fibroblasti umani della mammella che esprimono RFP (rosso) e YFP (bianco), rispettivamente. Co-colture sono state stabilite e analizzate come in Figura 2. I due tipi di cellule era mostrato qui, tuttavia, sono differenzialmente etichettati in modo che possano essere distinti l&#39;uno dall&#39;altro. Le immagini ingrandimento maggiore in questa figura, rispetto a quelli in figura 2, illustrano DQ-proteolisi di collagene IV sulla superficie delle cellule DCIS e proteolisi diffusa di DQ-collagene I in zona dei fibroblasti. Ingrandimento, 20X.

<ol>
	<li>Sameni, M., Dosescu, J., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol. Imaging. 2, 159-175 (2003).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr. Protoc. Cell Biol. 39, (2008).</li><li>Jedeszko, C., Victor, B. C., Podgorski, I., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+hepatocyte+growth+factor+promotes+invasion+of+human+mammary+ductal+carcinoma+in+situ.">Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.</a> Cancer Res. 69, 9148-9155 (2009).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+live-cell+imaging+demonstrates+that+beta1-integrin+promotes+type+IV+collagen+degradation+by+breast+and+prostate+cancer+cells.">Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.</a> Mol. Imaging. 7, 199-213 (2008).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> Neoplasia. 10, 314-328 (2008).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantifying+the+dynamics+of+tumor+associated+proteolysis.">Imaging and quantifying the dynamics of tumor associated proteolysis.</a> Clin. Exp. Metastasis. 26, 299-309 (2009).</li><li>Cavallo-Medved, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+demonstrates+that+proteases+in+caveolae+of+endothelial+cells+degrade+extracellular+matrix.">Live-cell imaging demonstrates that proteases in caveolae of endothelial cells degrade extracellular matrix.</a> Exp. Cell. Res. 315, 1234-1246 (2009).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li></ol>

Non abbiamo nulla da rivelare.

Come dimostrato, la co-colture MAME può essere utilizzato per live-cella di imaging in tempo reale delle interazioni tra i vari costituenti cellulari che compongono un tumore del seno ed il suo microambiente. In studi in corso nel nostro laboratorio, abbiamo usato co-colture MAME quello di individuare percorsi proteolitici associati con la transizione da carcinoma duttale in situ di carcinoma duttale invasivo, nonché le interazioni tra i sentieri e percorsi proteolitici coinvolti in questa transizione, come chemochine...

<table border="1"><tbody><tr><td> Reagente / Attrezzature </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> Membrana basale ricostituita (Cultrex) </td><td> Trevigen </td><td> 3445-005-01 </td><td> Paragonabile ad Matrigel (BD Biosciences) </td></tr><tr><td> Collagene I </td><td> Laboratori di coesione </td><td> 5005-B </td><td></td></tr><tr><td> DQ-substrati (Collagene I, IV) </td><td> Invitrogen </td><td> DQ-Col I-D12060 DQ-col IV D12052 </td><td></td></tr><tr><td> Coprioggetti plastica 22-mm </td><td> Fisher Scientific Co. </td><td> 12-547 </td><td> Tagliare a metà, lasciare in 70% di etanolo per 10 minuti e asciugare all&#39;aria prima dell&#39;uso. </td></tr><tr><td> Coltura cellulare medio </td><td> Lonza </td><td> MEBM-PRF CC-3153 MEGM CC-4136 </td><td> Rosso fenolo-free </td></tr><tr><td> ibiTreat μ-Dish </td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Volocity software </td><td> Perkin-Elmer </td><td> Versione 5.5 </td><td> Altre marche di software di imaging possono essere utilizzati per generare ricostruzioni in 3D e filmati. </td></tr></tbody></table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

Abbiamo sviluppato modelli di co-coltura 3D, che noi chiamiamo MAME (m ammary uno rchitecture e m icroenvironment MBIENTALE e), e li hanno usati per la live-cell imaging in tempo reale di interazione fra cellule. Il nostro obiettivo generale era quello di sviluppare modelli che ricapitolano l&#39;architettura delle lesioni mammarie preinvasive di studiare la loro progressione verso un fenotipo invasivo. Specificamente, abbiamo sviluppato modelli per analizzare interazioni tra pre-maligne varianti cellule epiteliali mammarie e altri tipi di cellule del microambiente tumorale che sono stati implicati nel migliorare o ridurre la progressione delle cellule epiteliali mammarie preinvasive a invasive carcinomi duttali. Altri tipi cellulari studiati fino ad oggi sono cellule mioepiteliali, fibroblasti, macrofagi e sangue e le cellule endoteliali microvascolari linfatici. Oltre ai modelli MAME, che sono progettati per ricapitolare le interazioni cellulari all&#39;interno del seno duranteprogressione tumorale, abbiamo sviluppato modelli paragonabili per la progressione dei tumori della prostata. Qui si illustrano le procedure per la definizione delle co-colture 3D insieme con l&#39;uso di live-cellula imaging e un saggio funzionale proteolisi per seguire la transizione di co-colture di carcinoma mammario duttale in situ (DCIS) cellule e fibroblasti ad un fenotipo invasivo nel tempo, in questo caso più di venti-tre giorni di coltura. Il co-colture MAME costituiti da strati multipli. I fibroblasti sono incorporati nello strato di fondo di collagene di tipo I. Su che si trova uno strato di membrana basale ricostituita (meccanismi) su cui sono seminate cellule DCIS. Uno strato finale del 2% MLF è incluso e rifornito ad ogni cambio di media. Per proteolisi immagine associata con la progressione di un fenotipo invasivo, usiamo proteine ​​fluorescenti colorante-temprate (DQ) della matrice (DQ-collagene I mescolato con lo strato di collagene I e DQ-collagene IV miscelato con lo strato intermedio di rBM) e osservare colture vive usando la microscopia confocale. Sezioni ottiche vengono acquisiti, elaborati e ricostruita in 3D con il software di visualizzazione Volocity. Nel corso di 23 giorni in co-colture MAME, le cellule proliferano DCIS e fondersi in grandi strutture invasive. I fibroblasti migrano e venire incorporato in queste strutture invasive. Fluorescenti frammenti proteolitici dei collageni si trovano in associazione con la superficie di strutture DCIS, intracellulare, e anche disperse in tutta la matrice circostante. Farmaci che percorsi proteolitica bersaglio, chemochine / citochine e chinasi o modifiche nella composizione del co-colture cellulari può ridurre l&#39;invasività, suggerendo che i modelli MAME possono essere utilizzati come schermi preclinico per nuovi approcci terapeutici.

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

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

Abbiamo sviluppato modelli di co-coltura 3D per live-cell imaging in tempo reale delle interazioni tra cellule tumorali del seno e altre cellule nel loro microambiente che hanno un impatto progressione verso un fenotipo invasivo. Questi modelli possono servire come schermi preclinici di farmaci per colpire paracrino indotte proteolitici, percorsi chemochine / citochine e chinasi implicati nella invasività.

Modelli per MAME 4D Live-Imaging cellule di tumore: Interazioni microambiente che Impact progressione maligna

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

mame-models-for-4d-live-cell-imaging-tumor-microenvironment

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Medicine

14.3K Views.  Wayne State University. Abbiamo sviluppato modelli di co-coltura 3D per live-cell imaging in tempo reale delle interazioni tra cellule tumorali del seno e altre cellule nel loro microambiente che hanno un impatto progressione verso un fenotipo invasivo. Questi modelli possono servire come schermi preclinici di farmaci per colpire paracrino indotte proteolitici, percorsi chemochine / citochine e chinasi implicati nella invasività.

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

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

Xenotrapianto ortotopico di Human-luciferasi con tag cellule maligne del tumore della guaina dei nervi periferici per In vivo Test di agenti terapeutici candidati

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

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Medicina

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Valutazione della migrazione delle cellule staminali del cancro mediante compartimentazione dispositivi microfluidici e Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

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

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

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

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro test Clearance mesoteliali che modella i primi passi di metastasi del cancro ovarico

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Eterotipica tridimensionale<em&gt; In Vitro</emModellazione&gt; di stromale-epiteliali interazioni Durante iniziazione e la progressione del cancro ovarico

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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.

Vivo Imaging di risposte droga nel microambiente tumorale in modelli murini di cancro al seno

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

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Un romanzo ad alta risoluzione<em&gt; In vivo</em&gt; Tecnica di imaging per studiare la risposta dinamica di strutture intracraniche alla crescita del tumore e Terapeutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

Ad alta frequenza di Ecografia del mouse cervicali Linfonodi

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

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;

Un tumore cerebrale / organotipica fetta sistema di co-coltura per lo Studio microambiente tumorale e mirata terapie farmacologiche

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

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Bioluminescenza-Based Metodo Tumore quantificazione per il monitoraggio del tumore Progressione e gli effetti del trattamento in modelli murini linfoma

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Ex vivo dal vivo Imaging di polmone metastasi e il loro micro

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...