Supported by grants from the National Institute of Health grants CA133085, CA182518 and CA185909 (to KL) and VA Merit Review Award BX001962 (to KL).

Tutte le procedure seguono linee guida e protocolli approvati da parte della Georgia Regents dell&#39;Università degli animali e del Comitato Usa Care. 1. Istituzione di ortotopico tumore cancro al seno <ol><li> Un giorno prima dell&#39;esperimento, la cultura di circa 2 x 10 6 cellule tumorali 4T1-Luc in un piatto di coltura di 10 cm in 10 ml RPMI mezzo contenente 10% FBS. Incubare la piastra di coltura in un incubatore CO 2 a 37 ° C e 5% CO 2. </li><li> Il giorno di iniezione delle cellule tumorali, togliere terreno di coltura dalla piastra di coltura con il vuoto, aggiungere 5 ml di fosfato sterile salina tamponata (PBS), pH 7,4 al piatto cultura, e scuotere delicatamente il piatto per lavare la superficie della cultura, rimuovere il PBS sotto vuoto. </li><li> Aggiungere 2 ml di tripsina-EDTA (0,05% tripsina e 0,53 mM EDTA) al piatto di coltura, incubare nell&#39;incubatore CO 2 a 37 ° C per circa 5 min. Controllare cellule su un microscopio invertito per assicurarsi che tutte le cellule sono staccate dal fondo del piatto cultura. &lt;/ Li&gt; <li> Quench tripsina aggiungendo 8 ml medie siero contenenti celle di trasferimento ad un tubo conico da 15 ml e centrifugare le cellule a circa 450 xg per 3 min a RT. </li><li> Rimuovere il surnatante dalle cellule del vuoto e risospendere in 10 ml di PBS. cellule centrifugare a circa 450 xg per 3 min. Rimuovere il surnatante dal vuoto, e risospendere le cellule in 10 ml di PBS. </li><li> Pipettare 10 ml di sospensione cellulare in un emocitometro sotto il vetro di copertura. Contare le cellule per determinare la concentrazione cellulare. Risospendere le cellule in PBS ad una densità di 2 x 10 5 cellule / ml HBSS. Trasferire le cellule ad un 1,5 ml provetta da microcentrifuga sterile e mantenere le cellule in ghiaccio fino al momento dell&#39;uso. </li><li> Utilizzare femmina BALB / c topi di 6-8 settimane per l&#39;iniezione delle cellule tumorali. <ol><li> Radere capelli vicino circostante capezzolo # 3 utilizzando un trimmer capelli elettronica. L&#39;iniezione di cellule 4T1 nel mammaria cuscinetto adiposo ghiandola 3 genera riproducibile metastasi polmonari. Pulire la zona rasata utilizzando il bastoncino di cotone imbevuto nel 70% ethanol. Risospendere le cellule invertendo la provetta 2 - 3 volte. Delicatamente aspirato 50 ml di sospensione cellulare in un ½ CC 27 G 1/2 tubercolina siringa. </li><li> Iniettare le cellule tumorali nel cuscinetto di grasso mammaria sotto # 3 ghiandola mammaria di un topo BALB / c. Posizionare il mouse in una gabbia pulita e riportare la gabbia per impianto di stabulazione degli animali. Attendere circa 21 - 30 giorni per lo sviluppo del tumore. La crescita del tumore deve essere monitorata in maniera regolare secondo le norme dell&#39;istituto per gli studi tumorali. </li></ol></li></ol> 2. vivo mouse bioluminescenza Imaging di crescita tumorale <ol><li> topi immagine ogni 3 giorni dopo i topi di trasferimento di iniezione del tumore alla struttura di imaging. Iniettare 100 luciferina microlitri (30 mg / ml in PBS) per via intraperitoneale (ip) a topi prima imaging. Dopo circa 10 minuti, anestetizzare topi in una camera di induzione con O 2 / flusso isoflurano 2%. Utilizzare le pinze per toccare la gamba del mouse. Nessuna reazione al tatto conferma that il mouse è completamente privo di sensi. <ol><li> Posizionare i topi in camera di imaging con continuo 2% isoflurano e O 2 ad un flusso di 2 L / min. Posizionare il mouse tale che l&#39;area di interesse (cioè, il pad mammaria dell&#39;iniezione tumore iniziale) di fronte alla telecamera del sistema di imaging. Assicurarsi che il naso e la bocca del mouse sono saldamente fissato all&#39;interno del tubo di anestetico. Gli animali devono essere anestetizzati per norme veterinarie presso l&#39;istituto. </li></ol></li><li> Acquisire l&#39;imaging bioluminescenza con una serie di vari tempi di esposizione. Ad esempio, impostare l&#39;esposizione a 30, campo visivo (FOV) a 25, altezza dell&#39;oggetto da 1,5 cm, e di ottenere immagini fotografiche luminescenti con i raggi X a bassa potenza. Impostare le unità di misura per &quot;splendore&quot;. <ol><li> Utilizzare una gamma colore automatica lineare con un massimo di circa 2,4 unità x 10 7 radianza. Dopo aver acquisito le immagini iniziali, regolare le impostazioni e ottenere un immagine ottimizzata (Figuras 3 e 4). </li></ol></li><li> topi per pulire le gabbie e assicurano topi riacquistano coscienza con la deambulazione, prima di tornare gli animali alla struttura alloggiativa tornare. </li><li> Analizzare i dati con il software di imaging di vita associata allo strumento di imaging. Utilizzando un programma di imaging, tracciare una linea chiusa intorno alla regione di interesse (ROI). Utilizzare il software di imaging per determinare l&#39;intensità splendore del ROI. Registrare le intensità relative di ogni ROI per ogni mouse. NOTA: elevate intensità di radianza indicano maggiori livelli di cellule luminescenti, e così una maggiore crescita del tumore. </li><li> Esaminare visivamente il mouse ogni 3 giorni per gli effetti avversi. NOTA: I topi saranno pesati una volta alla settimana. Una perdita di peso del peso corporeo del 15% sarà considerato effetto negativo e topi portatori di tumore sarà eutanasia come descritto nella fase saranno sacrificati 5. I topi con tumori ulcerate. </li></ol> 3. La rimozione chirurgica dei tumori primari NOTE: forbici Autoclave, pinze e le clip ferita e ferita clip di applicatore (Figura 1). <ol><li> Eseguire l&#39;intervento in una cappa sterile per mantenere una condizione sterile per ridurre il rischio di infezione. Un giorno prima dell&#39;intervento chirurgico, radere l&#39;area che circonda i tumori dei topi portatori di tumore con un trimmer capelli elettrici. </li><li> Circa 2 - 4 ore prima dell&#39;intervento chirurgico, alimentare i topi compresse masticabili carprofen formulati per topi ad una dose di 5 mg / kg di peso corporeo. </li><li> Mettere i topi in una camera contenente 2% isoflurano in ossigeno per circa 30 sec. Continuamente anestetizzare il mouse utilizzando un&#39;apparecchiatura anestesia durante l&#39;intero periodo chirurgica. </li><li> Usare un bastoncino di cotone sterile per applicare veterinario unguento per gli occhi del mouse per prevenire la secchezza mentre sotto anestesia. Utilizzare le pinze per toccare la gamba del mouse. Nessuna reazione al tatto conferma che il mouse è completamente privo di sensi. </li><li> Disinfettare l&#39;area pelle intorno al sito del tumore con tre alternatasalviette di uno scrub sapone disinfettante. Pulire l&#39;area chirurgica con un alcool al 70% pulire. </li><li> Utilizzare un bisturi sterilizzato (Figura 1) per tagliare una piccola incisione vicino al tumore. </li><li> Inserire la punta di un paio di forbici sterilizzati per l&#39;incisione per tagliare la pelle attorno al tumore per esporre il nodulo tumorale (Figura 2A). Tenere il tumore con pinze sterili e utilizzare le forbici per separare il tumore della pelle (Figura 2B). Salvare il campione del tumore congelando in un -80 ° C congelatore o fissando in formalina al 10% per un uso futuro. </li><li> Chiudere il sito chirurgico con clip ferita da 9 mm utilizzando l&#39;applicatore automatico-clip (Figura 1). Ritorna il mouse in una gabbia pulita con biancheria da letto in autoclave. Gli animali devono essere monitorati secondo le linee guida istituzionali. NOTA: Il mouse di solito riprende conoscenza sufficiente e inizia a muoversi nella gabbia di circa 10 - 30 min dopo l&#39;intervento chirurgico. </li><li> Somministrare Carprofen atopi nuovamente 24 ore dopo l&#39;intervento chirurgico. Carprofen è per post-operatorio analgesia e la prescrizione di post-operatorio analgesia vanno verificate mediante consultazione diretta con il personale veterinario dell&#39;istituzione. Rimuovere clip ferita dopo la ferita guarisce (di solito entro 5 - 7 giorni dopo l&#39;intervento). Rimuovere clip ferita utilizzando il dispositivo di rimozione auto-clip. </li></ol> 4. vivo mouse bioluminescenza Imaging di tumore metastasi <ol><li> topi Immagine ogni 3 giorni dopo l&#39;intervento, come descritto al punto 2. </li></ol> 5. La convalida del polmone metastasi Utilizzando inflazione India inchiostro di polmoni tumore-cuscinetto NOTA: Per convalidare i luciferasi in tempo reale risultati di imaging del tumore, eseguire l&#39;India l&#39;inflazione inchiostro dei polmoni del mouse tumore-cuscinetto per quantificare noduli tumorali. Cellule 4T1 metastatizzano anche ad altri organi e tessuti (Figura 5) e che, quindi, l&#39;esame istologico di tali organi per convalidare metastasi tumorali dovrebbe essere eseguita se necessario. Questo uso del protocollo lung metastasi come un esempio. <ol><li> Mantenere i topi nella loro gabbia casa esistente per l&#39;eutanasia. Quando ospitato con altri animali, spostare quei topi non sottoposti a eutanasia di una gabbia diversa. </li><li> Togliere la parte superiore del filtro gabbia e sostituirlo con la parte superiore del filtro di CO 2. Accendere cilindro compresso gas CO 2 (100%) che è collegato alla parte superiore del filtro CO 2. Impostare la velocità di flusso a 2 L / min e mantenere CO 2 per almeno 10 min. Se i topi sono respiravano ancora alla fine di 10 min, lasciare la CO 2 fino almeno 1 min dopo topi interrompere la respirazione. Spegnere CO 2 di controllo del cilindro e il misuratore di portata. Attendere altri 3 minuti e rimuovere la parte superiore del filtro. </li><li> Posizionare il mouse sacrificato sulla sua schiena su una tavola di polistirolo. Pin le gambe per garantire l&#39;accesso senza ostacoli alla trachea. Spruzzare i topi con il 70% di etanolo. </li><li> Utilizzando un paio di forbici, tagliare lungo la linea mediana della metà addome del mouse attraverso la gabbia toracica e verso il salivariare ghiandole. Osservare la trachea. Usare pinze per rimuovere tessuti circostanti la trachea per esporre la trachea. </li><li> Infilare un puntale sotto la trachea. Tenendo la punta con una mano, sollevare delicatamente la trachea alto e lontano dal corpo. </li><li> Ruotare la piattaforma tenendo premuto il mouse 180 °. Utilizzare un ½ CC 27 G 1/2 tubercolina siringa per iniettare l&#39;India inchiostro (10% inchiostro di china e lo 0,1% di idrossido di ammonio) nei polmoni attraverso la trachea. Completamente gonfiare i polmoni con inchiostro finché non si avverte una forte resistenza. </li><li> Utilizzare un paio di forbici per tagliare la trachea. Utilizzare pinze per tenere il mouse e inserire un altro set di pinze sotto i polmoni e tirare i lobi polmonari fuori dal mouse. Risciacquare brevemente i polmoni in un bicchiere contenente acqua. </li><li> In cappa, trasferire i polmoni in una fiala di vetro scintillazione contenente 3 ml di soluzione di Fekete (50% etanolo, 6% formaldeide e 3% di acido acetico glaciale). Chiudere la fiala per evitare che la soluzione evaporazione. NOTA: I tessuti possono essere conservati in soluzione di Fekete indefinitamente. </li><li> Dopo alcuni minuti, osservare noduli tumorali come punti bianchi sui polmoni neri (Figura 5). Il nodulo tumorale bianco è visibile agli occhi. Trasferire i polmoni di un piatto in una cappa aspirante e contare il numero di punti bianchi. Ogni macchia bianca rappresenta un singolo nodulo tumorale metastatico. </li></ol>

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Mice were purchased from the Jackson Laboratory and housed in the Georgia Regents University animal facility. Experiments and care/welfare were in agreement with federal regulations and an approved protocol by the Georgia Regents University IACUC committee. The authors have nothing to disclose.

Sono stati sviluppati molti tipi di modelli di topi transgenici di metastasi del cancro al seno. 1 Questi topi transgenici hanno un&#39;alta incidenza di tumore che vanno da 60 a 100%. Tuttavia, l&#39;incidenza di metastasi di questi topi transgenici è molto inferiore rispetto all&#39;incidenza del tumore (14 - 100%). Le cellule tumorali metastatizzano a LN e polmoni nella maggior parte di questi modelli di topi transgenici di metastasi del cancro al seno. La latenza metastasi varia da modello a modello e va...

Secondo l&#39;American Cancer Society, il cancro al seno è la forma più frequentemente diagnosticata di cancro nelle donne negli Stati Uniti. La diagnosi precoce in combinazione con terapie mirate recentemente sviluppati ha ridotto significativamente la mortalità di cancro al seno negli ultimi due decenni. Tuttavia, il cancro al seno è ancora la seconda causa di morte per cancro nelle donne negli Stati Uniti 1. La maggior parte dei decessi di pazienti affetti da cancro al seno sono dovute a metastasi delle cellule tumorali. Purtroppo, la maggior cancro al seno è invasivo e spesso metastatizza al linfonodo e successivamente in organi distanti, tra cui ossa, polmoni, fegato e cervello. 1,2 al momento non c&#39;è una terapia efficace per il carcinoma mammario metastatico. Pertanto, lo sviluppo di agenti chemioterapici e immunoterapia per sopprimere carcinoma mammario metastatico è di grande importanza. modelli di metastasi spontanea del mouse Vari cancro al seno abbiano ieen sviluppato per studiare i meccanismi molecolari alla base la progressione delle cellule tumorali della mammella e metastasi e da utilizzare come modelli per lo sviluppo di agenti terapeutici. 1,3-5 Tuttavia, la maggior parte di questi modelli di topo sono modelli tumorali genetiche che, mentre gli ottimi modelli per meccanicistica studi, non sono adatti per testare agenti terapeutici in quanto la metastasi vogliono mesi per sviluppare in questi modelli genetici, richiedendo in tal modo l&#39;amministrazione costosi a lungo termine degli agenti anti-cancro. 6,7 di monitoraggio progressione del tumore nei topi dal vivo è anche tecnicamente impegnativo. Al contrario, un seno modello di metastasi del cancro trapiantato ha i vantaggi della progressione tumorale termine breve e facile il monitoraggio della progressione del tumore nei topi vivi. Il modello murino di metastasi spontanea 4T1 ortotopico tumore al seno è un tale modello di tumore trapiantato. 8 In questo modello, le cellule del tumore al seno sono trapiantate nel cuscinetto di grasso mammaria per stabilire noduli tumorali primarie. Il tumore primariopossono poi essere rimosso chirurgicamente come nei pazienti con cancro mammario umano. 4T1 cellule tumorali sono altamente invasiva. 8,9, 3,10 Quasi tutti i tumori-cuscinetto topi sviluppano metastasi in 30 giorni dopo il trapianto del tumore nel cuscinetto adiposo mammario. Tuttavia, i tumori 4T1 crescono in modo aggressivo nei siti primari e le dimensioni del tumore spesso superano i limiti a cui è consentito nella maggior parte dei protocolli di animali. In questa fase, le metastasi sono spesso micrometastasi. Pertanto, è essenziale per rimuovere i tumori primari di consentire il progresso metastasi per testare agenti terapeutici. Qui, si segnala l&#39;istituzione di una procedura semplice e tuttavia sensibile del trapianto di tumore primario, la rimozione chirurgica del tumore primario e bioluminescenza quantificazione di imaging-based della crescita tumorale 4T1 e la progressione in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="973">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Ami-X Imaging System&#160;</td>
			<td style="width:437px;">Spectral Instruments Imaging Inc. Tucson, AZ</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">IsoTec SurgiVet</td>
			<td style="width:437px;">Anesthesia Service &#38; Equipment , Inc., Atlanta, GA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">AutoClip Physicians Kit</td>
			<td style="width:437px;">Becton Dickinson Primary Care Diagnostics. Sparks, MD</td>
			<td style="width:119px;">427638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">9 MM AutoClip Applier&#160;</td>
			<td style="width:437px;">Becton Dickinson Primary Care Diagnostics. Sparks, MD</td>
			<td style="width:119px;">427630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">9 MM AutoClip Remover</td>
			<td style="width:437px;">Becton Dickinson Primary Care Diagnostics. Sparks, MD</td>
			<td style="width:119px;">427637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">9 MM AutoClip Wound Clip</td>
			<td style="width:437px;">Becton Dickinson Primary Care Diagnostics. Sparks, MD</td>
			<td style="width:119px;">427631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:437px;">Fisher</td>
			<td style="width:119px;">8940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Fine-Pointed Forceps</td>
			<td style="width:437px;">Fisher</td>
			<td style="width:119px;">8875</td>
			<td></td>
		</tr>
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an orthotopic mouse model of spontaneous breast cancer metastasis

Metastasi è la prima causa di mortalità dei pazienti affetti da cancro al seno. Il meccanismo alla base di metastasi delle cellule tumorali, tra cui metastasi del cancro al seno, è in gran parte sconosciuto e è un focus nella ricerca sul cancro. Vari modelli di cancro al seno spontanei metastasi di topo sono state stabilite. Qui, riportiamo una procedura semplificata per stabilire ortotopico trapiantato tumore primario cancro al seno e conseguente metastasi spontanea che imitano metastasi del cancro al seno umano. In combinazione con l&#39;immagine del tumore dal vivo bioluminescenza, questo modello di topo permette la crescita del tumore e la cinetica di progressione di essere monitorati e quantificati. In questo modello, una dose bassa (1 x 10 4 cellule) delle cellule del cancro al seno 4T1-Luc è stato iniettato in / c cuscinetto di grasso del mouse mammaria BALB utilizzando una siringa da tubercolina. I topi sono stati iniettati con luciferina e ripreso in vari momenti che utilizzano un sistema di imaging bioluminescente. Quando i tumori primari sono cresciuti per il limite di dimensione, come nel protocollo IACUC approvato (approximately 30 giorni), i topi sono stati anestetizzati sotto costante flusso di 2% isoflurano e ossigeno. L&#39;area del tumore è stata sterilizzata con il 70% di etanolo. La pelle del mouse attorno al tumore è stato asportato per esporre il tumore che è stato rimosso con un paio di forbici sterili. La rimozione del tumore primario prolunga la sopravvivenza dei topi 4T-1 tumorale portante per un mese. I topi sono stati poi più volte ripreso per tumore metastatico diffusione agli organi distanti. agenti terapeutici possono essere somministrati per sopprimere la metastasi tumorale a questo punto. Questo modello è semplice e tuttavia sensibile per quantificare la crescita delle cellule del cancro al seno nel sito primario e la cinetica di progressione in organi distanti, e quindi è un ottimo modello per studiare la crescita del cancro al seno e la progressione, e per testare agenti anti-metastasi terapeutici e di immunoterapia in vivo .

Istituzione di ortotopico seno modello di cancro del mouse 4T1 è una linea di cellule di carcinoma mammario aggressivo. L&#39;iniezione di un minimo di 1 x 10 4 cellule nel cuscinetto di grasso mammaria può portare alla creazione di un singolo nodulo tumorale nel sito di iniezione (Figura 2A). Pertanto, il tumore imita carcinoma mammario primario umana. Quasi 100 i topi% sviluppano il tumore ortotopico. La dimensione del tumore può e...

Watch this Scientific Journal Video about An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis at JoVE.com

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Un modello di tumore primario del cancro al seno ortotopica e la rimozione chirurgica del tumore primario di estendere la vita del mouse per generare metastasi spontanee sono descritti. La crescita del tumore e la progressione sono monitorati e quantificati mediante imaging di fluorescenza luciferasi.

Un modello murino ortotopico di spontanea Breast Cancer metastasi

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

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30.5K Views.  Augusta University.  Un modello di tumore primario del cancro al seno ortotopica e la rimozione chirurgica del tumore primario di estendere la vita del mouse per generare metastasi spontanee sono descritti. La crescita del tumore e la progressione sono monitorati e quantificati mediante imaging di fluorescenza luciferasi. 

Video: An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

Un modello ortotopico di cancro alla vescica Murine

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

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Medicina

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

L&#39;analisi quantitativa delle metastasi del cancro utilizzando un modello di aviaria Embryo

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

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 ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

In Imaging bioluminescenza in vivo di Dinamica Tumore ipossia di metastasi cerebrali cancro al seno in un modello murino

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

La radioterapia combinata in un modello di tumore ortotopico cervello di topo

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Un modello di mouse ortotopico di anaplastico della tiroide Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

Un ortotopico cancro della vescica Modello di Gene Studi di consegna

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Bioluminescente modello ortotopico di cancro al pancreas Progressione

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un ortotopico Modello murino di Human Prostate Cancer Metastasi

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Avvio del carcinoma metastatico della mammella dalla Targeting del duttale con Adenovirus-Cre: A Novel modello di topo transgenico di cancro al seno

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...