Questo lavoro è stato generosamente sostenuto da fondi provenienti da bambini Cancer Foundation (Baltimore, MD), Brandon Lee Carrington Foundation (Silver Spring, MD), DOD (W81XWH-10-1-0137) e NIH (R01CA108641). Vorremmo ringraziare il Dott. Anton Wellstein e membri del suo laboratorio per condividere la loro esperienza e ci aiuta ad ottimizzare i metodi presentati.

1. Preparazione <ol><li> Tutte le procedure devono essere eseguite in condizioni sterili in una cappa di coltura di tessuti. </li><li> La stazione xCELLigence è posto in un incubatore 37 ° C in presenza del 5% di CO 2. </li><li> Utilizzare cellule HUVEC passaggio basso, preferibilmente non più di passaggio 6. Inoltre, assicurarsi che le cellule non sono più del 75% confluenti al momento della raccolta. </li><li> Cellule HUVEC sono coltivate in EGM-2 supporti ricostituita con kit di EGM-2 proiettile (Lonza Biosciences) contenente i fattori di crescita, integratori e 5% di siero fetale bovino. </li><li> Tutte le tracce di tripsina dovrebbero essere rimossi da sospensioni cellulari da spinning cellule a 200xg, lavare una volta con PBS, e li risospensione di densità cellulari indicati. </li><li> E-coat xCELLigence piastra 16 con 0,1% di gelatina per 1 ora a 37 ° C o per una notte a 4 ° C. Lavare la piastra con PBS e aggiungere 100 microlitri ricostituito EGM-2 media. Eseguire una lettura del bianco sul sistema xCELLigence per misurare sfondoimpedenza in assenza di cellule. </li></ol> 2. Generazione di HUVEC monostrato <ol><li> Raccogliere un pallone sub-confluenti di cellule HUVEC per tripsinizzazione. Risospendere le cellule in ricostituite EGM-2 supporti ad una densità finale di 2,5 x 10 5 cellule / ml. </li><li> A ciascun pozzetto di un fondo calibrato E-piastra contenente 100 microlitri EGM-2 supporti, aggiungere 100 microlitri della sospensione di cellule HUVEC (2,5 x 10 4 cellule HUVEC). </li><li> Installare immediatamente E-Plate nello strumento xCELLigence. </li><li> Software xCELLigence programma per eseguire letture di impedenza ad intervalli di 10 minuti. </li><li> Lasciare che le cellule endoteliali crescere per 18-21 ore fino a formare un monostrato, come evidenziato da un indice di appiattimento cella (Figura 2). </li></ol> 3. Aggiunta di invadere le cellule e la normalizzazione dei dati. <ol><li> Una volta che un monostrato HUVEC stabile è formata, raccolta cellule tumorali da parte tripsinizzazione e risospendere ad un tane finalilità di 1 x 10 5 cellule / ml in mezzi utilizzati per coltivare cellule tumorali (ad esempio RPMI o DMEM contenente il 10% supporti FBS) </li><li> Letture di impedenza Pausa sul software xCELLigence e rimuovere E-Plate dalla stazione. </li><li> Rimuovere EGM-2 supporti per aspirazione. Aggiungere 100 microlitri di sospensione cellulare tumorale contenente 1 x 10 4 cellule. Generalmente, un rapporto di cellule tumorali 1:2,5: cellule endoteliali inseminate, funziona meglio per il dosaggio. Tuttavia, il rapporto può essere ottimizzato per linee cellulari individuali. </li><li> Posizionare la E-piastra sul sistema xCELLigence in incubatrice e continuare letture di impedenza ad intervalli di 10 minuti. </li><li> Invasione può essere monitorato in tempo reale nei prossimi 6-12 ore come una goccia in indice cella a causa della retrazione delle giunzioni endoteliali e penetrazione di invadere le cellule tumorali. </li><li> Normalizzare i risultati al momento dell&#39;aggiunta di invadere le cellule tumorali. Il software permette la normalizzazione xCELLigence a qualsiasi punto e risultati tempopuò essere visualizzato direttamente nella finestra del software. </li></ol> 4. Rappresentante dei risultati: Un esempio di HUVEC monostrato invasione di cellule metastatiche e non metastatico è mostrato in Figura 3. K7M2 è una linea cellulare di osteosarcoma metastatico. Queste cellule esprimono alti livelli della proteina del citoscheletro linker ezrin, che rappresenta le proprietà invasive di questa linea cellulare 13,14. Quando le cellule HUVEC sono sfidati con K7M2 cellule, vi è un calo di resistenza elettrica entro 6 ore. Questa diminuzione della resistenza elettrica rappresenta l&#39;invasione di monostrato HUVEC dalle cellule tumorali invasori. La cella K12-line, invece, esprime livelli molto bassi di ezrin ed è di conseguenza meno metastatica 13. Sfidando il monostrato HUVEC con K12 risultati cellule in un meno ripido calo nella resistenza come le cellule sono in grado di aderire o invadere attraverso il monostrato HUVEC (Figura 3). <imgsrc = &quot;/ files/ftp_upload/2792/2792fig1.jpg&quot; alt = &quot;Figura 1&quot; /&gt; Figura 1. Rappresentazione schematica del processo di flusso di lavoro per l&#39;invasione delle cellule endoteliali da parte delle cellule tumorali. Cellule HUVEC sono placcati su E-piastre rivestite di gelatina e ha permesso di formare un monostrato confluente. Cellule maligne sono aggiunti una volta un monostrato è formata e invasione viene monitorata in tempo reale come indice cella cambia a causa invasione del monostrato endoteliale. <img src="/files/ftp_upload/2792/2792fig2.jpg" alt="Figura 2"/> Figura 2. Formazione monostrato HUVEC. Le variazioni dell&#39;indice delle cellule come le cellule HUVEC attaccano e si diffondono su elettrodi d&#39;oro gelatina rivestite. La formazione di un monostrato confluente è rappresentato da un indice di stabilizzazione cellule dopo 18 ore. <img src="/files/ftp_upload/2792/2792fig3.jpg" alt="Figura 3"/> Figura 3. Invasione di cellule HUVEC da K7M2 e K12 cellule di osteosarcoma. Una diminuzione netta delle cell-index avviene entro poche ore dalla introduzione di K7M2 cellule altamente metastatiche. K12 celle, d&#39;altro canto, sono meno metastatica e sono in grado di penetrare il monostrato endoteliale nel modo più efficiente K7M2 cellule. Questo è rappresentato da una meno ripida diminuzione dell&#39;indice cella quando il monostrato HUVEC è sfidato con K12 celle. Controllo rappresenta impedenza del monostrato HUVEC in assenza di cellule tumorali. Esperimenti sono stati eseguiti in triplicato.

<ol>
	<li>Klein, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer.+The+metastasis+cascade.">Cancer. The metastasis cascade.</a> Science. 321, 1785-1787 (2008).</li><li>Chiang, A. C., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+metastasis.">Molecular basis of metastasis.</a> N Engl J Med. 359, 2814-2823 (2008).</li><li>Orr, F. W., Wang, H. H., Lafrenie, R. M., Scherbarth, S., Nance, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+cancer+cells+and+the+endothelium+in+metastasis.">Interactions between cancer cells and the endothelium in metastasis.</a> J Pathol. 190, 310-329 (2000).</li><li>Bacac, M., Stamenkovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+cancer+cell.">Metastatic cancer cell.</a> Annu Rev Pathol. 3, 221-247 (2008).</li><li>Christofori, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+signals+from+the+invasive+front.">New signals from the invasive front.</a> Nature. 441, 444-450 (2006).</li><li>Kramer, R. H., Nicolson, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+tumor+cells+with+vascular+endothelial+cell+monolayers:+a+model+for+metastatic+invasion.">Interactions of tumor cells with vascular endothelial cell monolayers: a model for metastatic invasion.</a> Proc Natl Acad Sci U S A. 76, 5704-5708 (1979).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+fibroblast+behavior+in+tissue+culture+with+an+applied+electric+field.">Monitoring fibroblast behavior in tissue culture with an applied electric field.</a> Proc Natl Acad Sci U S A. 81, 3761-3764 (1984).</li><li>Giaever, I., Keese, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromotion+of+mammalian+cells+measured+electrically.">Micromotion of mammalian cells measured electrically.</a> Proc Natl Acad Sci U S A. 88, 7896-7900 (1991).</li><li>Tiruppathi, C., Malik, A. B., Del Vecchio, P. J., Keese, C. R., Giaever, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+method+for+detection+of+endothelial+cell+shape+change+in+real+time:+assessment+of+endothelial+barrier+function.">Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.</a> Proc Natl Acad Sci U S A. 89, 7919-7923 (1992).</li><li>Whipple, 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=Epithelial-to-mesenchymal+transition+promotes+tubulin+Detyrosination+and+Microtentacles+that+enhance+endothelial+engagement.">Epithelial-to-mesenchymal transition promotes tubulin Detyrosination and Microtentacles that enhance endothelial engagement.</a> Cancer Res. 70, 8127-8137 (2010).</li><li>Boyd, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell-microelectronic+sensing+technique+for+profiling+cytotoxicity+of+chemicals.">A cell-microelectronic sensing technique for profiling cytotoxicity of chemicals.</a> Anal Chim Acta. 615, 80-87 (2008).</li><li>Khanna, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane-cytoskeleton+linker+ezrin+is+necessary+for+osteosarcoma+metastasis.">The membrane-cytoskeleton linker ezrin is necessary for osteosarcoma metastasis.</a> Nat Med. 10, 182-186 (2004).</li><li>Ren, L. <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+linker+protein+ezrin+is+regulated+during+osteosarcoma+metastasis+by+PKC.">The actin-cytoskeleton linker protein ezrin is regulated during osteosarcoma metastasis by PKC.</a> Oncogene. 28, 792-802 (2009).</li></ol>

Il costo della pubblicazione di questo manoscritto è stato gentilmente fornito da Roche Applied Science.

Il tempo reale saggio di invasione HUVEC è un metodo semplice, ma potente per il monitoraggio invasione. Esso fornisce risultati in tempo reale, che è un vantaggio significativo rispetto ad altre tecniche tradizionali che si basano su analisi end-point. Il dosaggio può essere modificato per testare farmaci, anticorpi, ligandi e proteine ​​come inibitori o stimolatori di metastasi. L&#39;effetto di agenti diversi su endoteliale / carcinoma diafonia può essere valutata come bene. Nell&#39;introdurre agenti esogeni, quali fattori di crescita e medicinali nei terreni di coltura, è importante garantire che non influenzano l&#39;integrità del monostrato HUVEC. Perturbazione del monostrato HUVEC può essere testato introducendo l&#39;agente esogeno in assenza di invadere le cellule, e garantendo che non vi è alcun cambiamento di indice cella. Così, gli opportuni controlli dovrebbero essere inclusi come parte del disegno sperimentale. Diverse linee cellulari differenti curve dimostrano cell-index quanto formano un confmonostrato luent. La forma della curva, così come il massimo cellula-index raggiunto, è tipo cellulare specifico. Cellule HUVEC mostrano una caratteristica transitoria appiattimento indice cella 4-6 ore dopo la semina, seguito da un altro stabilizzazione ad un indice cella superiore dopo 16-18 ore. Quindi, è importante permettere alle cellule formano un monostrato confluente per almeno 18 ore prima dell&#39;aggiunta di cellule tumorali. Poiché l&#39;indice cella dipende l&#39;ambiente ionico in corrispondenza dell&#39;elettrodo / cellula interfase, vari fattori come cellula-morfologia e qualità delle adesioni cellula-cellula influenzano la cellula-index, oltre alla zona del fondo ben coperto. Così, la diminuzione dell&#39;indice cella, che si verifica su di invasione delle cellule tumorali, è funzione di diversi fattori, che includono la retrazione delle giunzioni endoteliali e successiva invasione delle cellule tumorali. Lo strumento xCELLigence è prodotto in tre diverse configurazioni: analizzatore cellula tempo reale(RTCA) SP, RTCA MP e RTCA DP. Lo strumento RTCA SP detiene un 96-ben E-piastra che lo strumento RTCA MP può contenere fino a sei a 96 pozzetti e-piastre contemporaneamente. Questo permette di high-throughput screening di farmaci e composti. Gli esperimenti in questo articolo sono eseguite utilizzando lo strumento RTCA DP, che può contenere tre a 16 e E-piastre. Ogni stazione di partecipazione E-piastra può essere utilizzato in modo indipendente, che consente a più utenti di eseguire esperimenti contemporaneamente. Lo strumento RTCA DP può essere utilizzato anche per studiare la migrazione utilizzando CIM-piastre. Queste sono le piastre a 16 pozzetti con camere superiori e inferiori separati da un polietilene tereftalato (PET) membrana con dimensione dei pori media di 8um. I sensori elettronici sono situati sul lato inferiore della membrana porosa della camera superiore. La camera inferiore funge da serbatoio per chemiotattico per cellule nella camera superiore. Tuttavia, uno dei principali vantaggi del saggio di invasione HUVEC negli CIM-piastre è che l&#39;invasione delle cellule tumorali nel primo is non è limitata dalla dimensione dei pori, l&#39;invasione delle cellule endoteliali dipende diafonia tra le cellule tumorali ed endoteliali. Il saggio di invasione HUVEC può anche essere eseguita su strumento ECIS Z, prodotto da Applied Biophysics (Troy, NY). Lo strumento ECIS Z utilizza una tecnologia simile allo strumento xCELLigence, con differenze nelle configurazioni di array elettrodo. Abbiamo effettuato con successo i test di invasione HUVEC utilizzando gli array ECIS 8 pozzetti. È importante notare che la superficie di fondo è ben maggiore nelle matrici ECIS, che richiede un numero maggiore di cellule endoteliali e tumorali di essere placcato: 2,5 x 10 5 e 1 x 10 5 cellule rispettivamente.

<table border="1">
	<tbody>
 <tr>
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr> 
 <tr>
 	<td>xCELLigence RTCA DP station</td>
 <td>Roche	</td>
 <td>05469759001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>RTCA control unit</td>
 <td>Roche</td>
 <td>05454417001</td>
 <td>Notebook with preinstalled RTCA software</td>
 </tr>
 <tr>
 	<td>E-Plate 16</td>
 <td>Roche</td>
 <td>05469830001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>HUVEC cells</td>
 <td>Lonza</td>
 <td>CC-2517</td>
 <td></td>
 </tr>
 <tr>
 	<td>EGM-2 media</td>
 <td>Lonza</td>
 <td>CC-3156</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>EGM-2 bullet kit</td>
 <td>Lonza</td>
 <td>CC-4176</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>0.1% Gelatin in 
sterile H2O
</td>
 <td>Millipore</td>
 <td>MCPROTO045</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells

Diffusione metastatica delle cellule maligne richiede degradazione della membrana basale, dell&#39;adesione delle cellule tumorali all&#39;endotelio vascolare, retrazione delle giunzioni endoteliali ed infine invasione e la migrazione delle cellule tumorali attraverso lo strato endoteliale per entrare nel flusso sanguigno come mezzo di trasporto in sedi lontane l&#39;host 1-3. Una volta nel sistema circolatorio, le cellule tumorali aderiscono per capillarità pareti e stravaso al tessuto circostante per formare tumori metastatici 4,5. I vari componenti di interazione cellula-cellula endoteliale tumorale possono essere replicate in vitro sfidando un monostrato di cellule endoteliali della vena ombelicale umana (HUVEC) con cellule tumorali. Studi eseguiti con elettroni e microscopia in contrasto di fase suggeriscono che la sequenza in vitro di eventi rappresenta in maniera corretta il processo metastatico in vivo 6. Qui, descriviamo una tecnica basata elettrico-impedenza che controlla e quantifica in tempo reale le Invasione delle cellule endoteliali mediante cellule tumorali maligne. Giaever e Keese prima descritto una tecnica per misurare fluttuazioni di impedenza quando una popolazione di cellule crescono sulla superficie degli elettrodi 7,8. Lo strumento xCELLigence, prodotto da Roche, utilizza una tecnica simile a misurare le variazioni di impedenza elettrica come cellule attaccano e dispersi in un piatto di coltura coperto con una matrice microelettrodo oro che copre circa l&#39;80% della zona sul fondo di un pozzo. Come cellule aderiscono e diffondono sulla superficie dell&#39;elettrodo, porta ad un aumento di impedenza elettrica 9-12. L&#39;impedenza viene visualizzata come parametro adimensionale definito cell-index, che è direttamente proporzionale alla superficie totale del tessuto-coltura bene che è coperto da cellule. Quindi, la cellula-indice può essere utilizzato per monitorare l&#39;adesione cellulare, diffusione, morfologia e densità cellulare. Il saggio di invasione descritto in questo articolo si basa su cambiamentiin impedenza elettrica all&#39;elettrodo / cellula interfase, come una popolazione di cellule maligne invadono attraverso un monostrato HUVEC (Figura 1). La distruzione delle giunzioni endoteliali, retrazione del monostrato endoteliale e la sostituzione da parte delle cellule tumorali portare a grandi cambiamenti di impedenza. Questi cambiamenti correlano direttamente con la capacità invasiva delle cellule tumorali, cioè, invasione di cellule altamente aggressivi portare a grandi cambiamenti di impedenza della cella e viceversa. Questa tecnica fornisce un duplice vantaggio rispetto ai metodi esistenti di misurazione invasione, come camera di Boyden e dosaggi Matrigel: 1) l&#39;interazione cellula-tumorale delle cellule endoteliali imita più da vicino il processo in vivo, e 2) i dati sono ottenuti in real- tempo ed è più facilmente quantificabile, al contrario di analisi end-point per altri metodi.

Watch this Scientific Journal Video about A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells at JoVE.com

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

Questo articolo descrive una<em&gt; In vitro</em&gt; Tecnica per le cellule tumorali che invadono monitoraggio mediante un monostrato di cellule endoteliali. I dati vengono acquisiti in tempo reale in funzione delle variazioni dell&#39;impedenza sulla superficie degli elettrodi sul fondo del pozzo.

Un tempo reale impedenza elettrica tecnica basata su Misura Invasione di cellule endoteliali monostrato dalle cellule tumorali

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

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JoVE Journal

Medicine

27.2K Views.  Georgetown University. Questo articolo descrive una In vitro Tecnica per le cellule tumorali che invadono monitoraggio mediante un monostrato di cellule endoteliali. I dati vengono acquisiti in tempo reale in funzione delle variazioni dell'impedenza sulla superficie degli elettrodi sul fondo del pozzo.

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

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Ricerca

Medicina

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 ...

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 ...

Modeling and Imaging 3-Dimensional Collective Cell Invasion

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

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

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 ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Una piattaforma di screening Luciferase basata multiplex per interrogare il cancro-associata trasduzione del segnale in cellule in coltura

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

Liquido cerebrospinale MicroRNA profiling Utilizzando quantitativa Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

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 ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Valutazione del cancro ovarico Spheroid attaccamento e l&#39;invasione delle cellule mesoteliali in tempo reale

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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

Three-Dimensional 3D Tumor Spheroid Invasion Assay

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

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Isolamento di cellule umane endoteliali linfatico da Multi-parametro fluorescenza-attivato cell sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Methods Article

Un tempo reale impedenza elettrica tecnica basata su Misura Invasione di cellule endoteliali monostrato dalle cellule tumorali