The authors would like to acknowledge the Netherlands Organization for Scientific Research (NWO, grant 17.106.329)

Gli esperimenti sugli animali sono stati approvati dal comitato il benessere degli animali del Leiden University Medical Center (LUMC). 1. Preparazione di cellule, Strumenti e Mice <ol><li> Un giorno prima del funzionamento, la barba NOD / SCID gamma (NSG) topi dal quarto capezzolo alla linea mediana e pesare i topi per verificare che tutti i topi hanno pesi all&#39;incirca comparabili. </li><li> Autoclave una forbice, due pinzette e due pinze zanzara rette (Figura 1A). </li><li> Il giorno di funzionamento, lavare l&#39;adenocarcinoma mammario umano (MDA-MB-231) cellule che sarà iniettato una volta con tampone fosfato (PBS), pH 7,4 e trypsinize cellule. Quench la tripsina aggiungendo 10 ml di siero contenenti DMEM supporti sulla parte superiore delle cellule. Centrifugare le cellule a 1200 rpm per 7 min a RT per rimuovere il siero risospendendo cellule sia in PBS o nei mezzi senza siero. Centrifugare di nuovo per rimuovere le tracce di siero completamente. Risospendere le cellule in PBS omedia. </li><li> Contare le cellule con un emocitometro e calcolare la quantità di cellule. Utilizzare 500.000 cellule / topo in non più di 150 ml. Opzionale per PBS o supporti, risospendere le cellule in matrigel. NOTA: Determinare la quantità di cellule a seconda del tipo cellulare utilizzato. </li><li> Mettere le cellule in provette da microcentrifuga sterili e tenerli su ghiaccio. </li><li> Riempire uno 50 ml provetta da centrifuga conica con 35 ml di PBS pH 7.4 e un altro 50ml conica provetta da centrifuga con 70% di etanolo. Immergere tamponi di cotone in ogni provetta. </li></ol> 2. iniezione ortotopico <ol><li> Eseguire l&#39;intervento in una cappa sterile per mantenere un&#39;atmosfera sterile. Anestetizzare il mouse sottocutanea iniettando mix Xylazin / ketamina alla dose di 10 mg / kg, rispettivamente 100 mg / kg di peso corporeo. Fissare il mouse su una piastra elettrica. Confermare il successo di anestesia per la mancanza di reazione al pizzico punta. NOTA: Se uno non è molto familiare con procedure chirurgiche, la chirurgia può richiedere più tempo. Organizzarele anestesia dosaggio di conseguenza. </li><li> Applicare pomata oftalmica sugli occhi di topi, per evitare gli occhi secchi. </li><li> Pulire la zona rasata utilizzando il batuffolo di cotone imbevuto in etanolo preparato al punto 1.6. Alternando scrub con betadine e alcol tre volte in modo circolare rotante di distanza dal sito di incisione pianificata deve essere eseguita. In alternativa, utilizzare ionoforo come disinfettante. </li><li> Fai una piccola incisione tra il quarto capezzolo e la linea mediana, con una forbice e fare una tasca inserendo il tampone di cotone inumidito con PBS pH 7.4. </li><li> Utilizzare una pinzetta per esporre il cuscinetto adiposo mammario. Osservare il cuscinetto di grasso per il suo colore bianco. </li><li> Spremere il cuscinetto adiposo con l&#39;altra pinzetta dalla sua base; così facendo, esporre completamente il cuscinetto di grasso (Figura 1B) per eseguire iniezioni facilmente. </li><li> Omogeneizzare la miscela di cellule pipettando su e giù. Aspirare delicatamente 50 ml di sospensione cellulare in una siringa da insulina e iniettare inil cuscinetto adiposo mammario tenendo l&#39;ago in senso orizzontale. Confermare iniezione di successo controllando per rigonfiamento del cuscinetto adiposo. </li><li> Rilasciare il cuscinetto adiposo delicatamente. </li><li> Suturare l&#39;incisione utilizzando un driver ago. Effettuare tre combinazioni ruotando la sutura intorno al conducente ago in senso orario, in senso antiorario e in senso orario. Dovrebbero essere utilizzati due nodi per sutura. NOTA: Assicurarsi che i nodi sono stretti altrimenti topi possono aprire i punti di sutura. </li><li> Dopo l&#39;intervento chirurgico, iniettare un analgesico, come Temgesic a 0,05-0,1 mg / kg di peso corporeo, per via sottocutanea al fine di alleviare il dolore. </li><li> Non lasciare gli animali incustoditi fino guadagnano coscienza e mantenere prona sternale. Mettere tutti gli animali in diverse gabbie fino a quando non sono completamente recuperati. </li><li> Per mantenere la sterilità, utilizzare gabbie autoclave e acqua. Modificare le gabbie ogni tre giorni per mantenere l&#39;ambiente pulito. </li></ol> 3. prelievo di organi per l&#39;analisi <ol><li> Allagiorno della raccolta, 8 settimane dopo l&#39;impianto delle cellule, riempire 15 ml provette coniche con 3 ml di soluzione di Bouin per ogni mouse. Inoltre, utilizzare due 15 ml tubi riempiti con 5 ml di soluzione di formalina per topo. </li><li> Anestetizzare gli animali iniettando Xylazin / ketamina, alla dose di 10 mg / kg a 100 mg / kg di peso corporeo per via sottocutanea. Attendere che l&#39;animale perde punta ritiro pizzico reflex. </li><li> Fissare l&#39;animale con aghi su una base. Fare un lungo, verticale mediana un&#39;incisione con le forbici. Fare due incisioni orizzontali proprio sotto la gamba anteriore e al di sopra della gamba posteriore. Esporre il tumore da appuntare la pelle alla base. Misurare il volume del tumore usando un calibro. </li><li> Dissociare il tumore della pelle con le forbici. Snap congelare una parte del tumore in azoto liquido per l&#39;isolamento dell&#39;RNA. Posizionare l&#39;altra parte, nel tubo da centrifuga conica riempita di formalina per eseguire immunoistochimica seguente embedding di paraffina. </li><li> Prendere delicatamente i polmoni. Posizionare ilpolmone sinistro nella soluzione di Bouin. Mantenere il polmone in soluzione per 3 giorni. Osservare superficiale foci metastatici chiaramente all&#39;occhio nudo. Facoltativamente, inserire il polmone destro in formalina di verificare micrometastasi con ematossilina e eosina (H &amp; E) colorazione. NOTA: Anche se, metastasi polmonari osservati frequentemente nel cancro al seno, si potrebbe anche voler raccogliere ossa, fegato, cervello e milza analizzare metastasi. Le cellule presso l&#39;area metastatico sono più densi e morfologicamente diverso e quindi possono distinguere facilmente dal tessuto polmonare. </li><li> Un giorno dopo il raccolto, aspirare la soluzione di formalina da 15 ml tubi e sostituire con il 70% di etanolo. Incorpora i tessuti in paraffina e condurre studi di immunoistochimica. </li><li> Per l&#39;analisi del sangue, aprire la cavità toracica con le forbici. Prelevare 450 ml di sangue tramite puntura cardiaca. Porre il campione di sangue in una provetta contenente 50 ml di 3,2% di sodio citrato. Dopo il sangue viene raccolto, facendo girare a 2.000 g per 10 kmn. Conservare i campioni di plasma a -20 ° C fino all&#39;utilizzo. NOTA: Ci sono diverse altre tecniche di raccolta del sangue comunemente utilizzati nella pratica 20 e la tecnica che deve essere utilizzato dipende apparato sperimentale e la scelta dello scienziato. Se non è richiesta campione di sangue, eutanasia animale in base al protocollo animali o secondo le linee guida dell&#39;istituzione. </li></ol>

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The authors declare that they have no competing financial interests.

Iniezione ortotopico di cellule di cancro al seno è un modello potente per studiare tutti gli aspetti della crescita del cancro. Impianto di queste cellule in tampone grasso mammario dei topi deve essere attentamente eseguito per evitare variazioni nella crescita tumorale. Soprattutto, iniettando la stessa quantità di cellule di ogni topo è cruciale. Per farlo, si dovrebbe trypsinize celle rigorosamente senza compromettere la vitalità delle cellule. Le cellule non vitali devono essere prese in considerazione durante...

Il cancro è una malattia molto complessa che è stata oggetto di studi per più secoli. Il cancro al seno è il tipo più comune di cancro; si verifica principalmente nelle donne, ma può verificarsi anche sporadicamente nei maschi 27. La malattia è causata principalmente dalla perdita di meccanismo di controllo divisione cellulare governo che a sua volta porta ad una crescita infinita di cellule nel corpo. Questi guasti possono essere causate da diversi meccanismi: in primo luogo, le cellule sane devono segnali di crescita delle cellule circostanti per proliferare mentre le cellule tumorali fanno loro fattori di crescita e aumentano l&#39;espressione dei fattori di crescita recettori ottenendo un tasso proliferativa superiore 1; secondo, le cellule tumorali sono meno sensibili ai segnali anti-proliferativi 8; terzo, per bilanciare il numero di cellule nella morte cellulare corpo è anche richiesto; Tuttavia, le cellule tumorali sfuggono dalla morte cellulare programmata, apoptosi chiamati 14; quarto, le cellule aderiscono matrici extracellulariper sopravvivere ma le cellule tumorali possono crescere senza la necessità di attaccamento e resistenti alle anoikis 19; Quinto, l&#39;attivazione della telomerasi aggira il accorciamento dei telomeri e impedisce la senescenza replicativa 21; ultimo ma non meno importante, saltando di DNA di controllo di qualità dopo i risultati mitosi nel contenuto genetico alterato 15,16. Per identificare oncogeni o soppressori tumorali che svolgono un ruolo in questa proliferazione non regolamentati, esperimenti di crescita tumorale nei topi sono cruciali. La crescita del tumore primario in genere non è la ragione principale della morte. La migrazione delle cellule tumorali dal sito primario a un sito secondario, chiamato metastasi, è la principale causa di morte nella maggior parte dei pazienti affetti da cancro 22. Metastasi comporta l&#39;invasione delle cellule tumorali, intravasation, viaggiando attraverso la circolazione, evitando immune attacco, stravaso e la crescita nel sito secondario. La transizione epitelio mesenchimale (EMT) è un processo fondamentale inmetastasi e coinvolge un interruttore in profili di espressione genica producendo cellule con maggiore motilità e l&#39;invasività, che sono pre-requisiti per la cella metastasi 12. Poiché il processo canceroso è la risultante di una combinazione di diverse azioni, tra cui le interazioni reciproche tra cellule tumorali, le cellule stromali e cellule pro- ed anti-infiammatorie, un approccio in vitro al cancro spesso non fornisce piena comprensione del processo canceroso. Analogamente, trattamenti antitumorali impatto ai vasi tumorali spesso non possono essere studiate in vitro, così l&#39;uso di approcci in vivo è inevitabile. Per studiare la progressione del cancro della mammella, sono stati sviluppati diversi metodi sperimentali. Il modello più diffuso è l&#39;iniezione sottocutanea di cellule di carcinoma mammario in topi 5. In questa configurazione sperimentale, il ricercatore può introdurre una vasta gamma di alterazioni di una linea cellulare di scelta in vitro (cioèupregulation, downregulation di proteine) e iniettare le cellule sotto la pelle. Anche se questo metodo è semplice e il processo di iniezione è semplice, senza necessità di eseguire un intervento chirurgico su topi, il sito in cui il tumore viene iniettato non rappresenta l&#39;ambiente tumore mammario locale e l&#39;assenza di questo ambiente può causare lo sviluppo del cancro al seno che differisce da quella osservata nella patologia umana. In secondo luogo, i topi geneticamente ingegnerizzati sono usati spesso come uno strumento in vivo per studiare la progressione del cancro al seno. In questo modello, oncogene (cioè PyMT, Neu) espressione è azionato da un promotore specifico tessuto mammario che porta alla formazione di spontanea del tumore mammario s. Questa configurazione sperimentale è utile per studiare l&#39;aspetto trattamento della malattia iniettando farmaci o anticorpi, mentre il controllo del formato del tumore in tempo 3. Tuttavia, l&#39;allevamento di questi topi con altri ceppi di topi carenti o mutati in un gene di interesse potrebbe anche dare insiritti nel ruolo di diverse proteine ​​in crescita del tumore al seno 24. Il rovescio della medaglia di questo modello è che è soggetta a variazioni in termini di dimensioni del tumore e il numero. Inoltre, il livello di espressione del transgene dipende dal sito di integrazione nel genoma e può cambiare da un ceppo di topi all&#39;altro 4. In questo modello, l&#39;espressione del transgene può essere ottenuto da tutte le cellule con origine epiteliale che nella malattia umana, solo una sottopopolazione di cellule esprimono l&#39;oncogene o downregulate livelli oncosoppressori 26. Per studiare le metastasi, le cellule del cancro al seno possono essere iniettati per via endovenosa (un modello chiamato metastasi sperimentale) 25. Tuttavia, questo approccio ricapitola solo il processo metastatico parzialmente; elude il requisito per le cellule tumorali di invadere e intravasate, e inizia dal punto in cui le cellule tumorali sono facilmente presenti nella circolazione. Nel nostro lavoro, utilizziamo una iniezione ortotopicomodello di ioni per studiare il coinvolgimento dei geni di interesse nella progressione del cancro al seno 13. Abbiamo iperespressione della proteina nelle cellule di cancro al seno umano e iniettarli nel cuscinetto adiposo mammario di NOD / SCID gamma (NSG) topi. Questo metodo è vantaggioso in molti modi: permette cambiamenti genetici molto rapidi e diverse nella linea cellulare iniettato, che copre l&#39;intero processo di progressione del cancro al seno dalla crescita del tumore primario metastasi in siti patologicamente pertinenti, e fornisce anche un buon modello sperimentale per studiare l&#39;impatto di trattamenti terapeutici nelle fasi precoci o tardive della malattia. Inoltre, utilizzando questo modello si può studiare il ruolo di stromale contro proteine ​​cellulari derivate da cancro in progressione della malattia utilizzando topi o cellule geneticamente modificati. Sebbene iniezioni sottocutanee sono più facili da eseguire, modelli ortotopico danno luogo ad una popolazione di cellule di cancro più tumorigenica più metastatico. Pertanto, i risultati ottenuti mediante iniezione sottocutaneas potrebbe essere falsi negativi o falsi positivi 6,17 incoraggiare l&#39;uso di modelli ortotopici per studiare la crescita del tumore.

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			<td height="17" style="height:17px;width:185px;">Name of material/equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:95px;">Catalog number</td>
			<td style="width:641px;">Comments/Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bouin&#39;s solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT10132</td>
			<td>Used for investigating the metastasis on lungs</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>Used to fix the tissues</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Matrigel, growth factor reduced</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Cells can be resuspended in matrigel for injection</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mosquito forceps</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td>Used for stiching</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Angled forceps</td>
			<td>Electron microscopy sciences</td>
			<td>72991-4c</td>
			<td>These make the exposure of mammary fat pad easier</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissors</td>
			<td>B Braun Medicals</td>
			<td>BC056R</td>
			<td>Used to cut open the mice</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Straight forceps</td>
			<td>B Braun Medicals</td>
			<td>BD025R</td>
			<td>This is used to open up the skin to expose mammary fat pad</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NOD scid gamma mice</td>
			<td>Charles River</td>
			<td>005557</td>
			<td>Experimental animal used for experiment</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MDA-MB-231</td>
			<td>Sigma-Aldrich</td>
			<td>92020424</td>
			<td>Experimental cells used for injections</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oculentum simplex</td>
			<td>Teva Pharmachemie</td>
			<td></td>
			<td>Opthalmic ointment used to prevent drying out of eyes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Betadine</td>
			<td>Fischer Scientific</td>
			<td>19-898-859</td>
			<td>Ionophore, used to disinfect the surgical area</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazin/Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>X1251, K2753</td>
			<td>Use injected anesthesia as 10mg/kg and 100mg/kg body weight respectively</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Temgesic</td>
			<td>Schering-Plough</td>
			<td></td>
			<td>Use the painkiller as 0,05-0,1mg/kg body weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Life sciences</td>
			<td>11995</td>
			<td>For trypsin neutralization,use media with serum(FBS:media 1:10 volume); for injection, use media with no serum</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Buffered sodium citrate</td>
			<td>Aniara</td>
			<td>A12-8480-10</td>
			<td>Use the volume ratio as citrate:blood; 1:9</td>
		</tr>
	</tbody>
</table>

orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth.

<html> La crescita del cancro al seno può essere studiato nei topi con una pletora di modelli. Manipolazione genetica di cellule di cancro al seno può fornire intuizioni le funzioni delle proteine ​​coinvolte nella progressione oncogeno o aiuto per scoprire nuovi soppressori tumorali. Inoltre, l&#39;iniezione di cellule tumorali in topi con differenti genotipi potrebbe fornire una migliore comprensione dell&#39;importanza del compartimento stromale. Molti modelli possono essere utili per approfondire alcuni aspetti della progressione della malattia, ma non ricapitolare l&#39;intero processo canceroso. Al contrario, le cellule del cancro al seno attecchimento al cuscinetto adiposo mammario dei topi migliori racchiude in sintesi il percorso della malattia e la presenza del vano stromale corretto e imita quindi migliori malattia tumorale umano. In questo articolo, si descrive come impiantare le cellule del cancro al seno in topi ortotopicamente e spiegare come raccogliere i tessuti per analizzare la: 14px; line-height:. 28px; &quot;&gt; tumore milieu e metastasi in organi distanti Utilizzando questo modello, per molti aspetti (crescita, angiogenesi e metastasi) di cancro possono essere indagati semplicemente fornendo un ambiente adeguato per le cellule tumorali di crescere.

Applicazione di successo del &quot;modello di cancro al seno ortotopico&quot; si basa su una corretta iniezione di cellule nel cuscinetto adiposo mammario. Errori sperimentali come inoculo imprecisa di cellule o perdite potrebbero portare a variazioni nelle dimensioni del tumore o addirittura l&#39;assenza di un tumore che porta alla formazione di una struttura simile alla ricerca di un pad grasso mammario iniettato con un buffer di controllo (Figura 2A). Il tasso di crescita del tumore dipende dalla na...

Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth. at JoVE.com

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Il cancro è una malattia complessa che è influenzata dal tessuto circostante il tumore nonché pro- locale e anti-infiammatori mediatori. Pertanto, modelli iniezione ortotropici, anziché modelli sottocutanee possono essere utili per studiare la progressione del cancro in un modo che imita migliori patologia umana.

Iniezione ortotopico di cellule di carcinoma mammario nel mammaria Fat Pad di Mice per studiare la crescita del tumore.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

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Medicine

78.3K Views.  Leiden University Medical Center.  Il cancro è una malattia complessa che è influenzata dal tessuto circostante il tumore nonché pro- locale e anti-infiammatori mediatori. Pertanto, modelli iniezione ortotropici, anziché modelli sottocutanee possono essere utili per studiare la progressione del cancro in un modo che imita migliori patologia umana. 

Video: Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Esecuzione ed elaborazione di FNA anteriore Fat Pad per Amiloide

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

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Medicina

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Un In Vitro Sistema per studio dormienza tumorale e l&#39;interruttore di crescita metastatica

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Un modello ortotopico tumore della vescica e la valutazione del trattamento intravescicale sarna

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

Aortica trapianto ortotopico in Mouse per lo studio della malattia vascolare

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

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 ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Tre Culture dimensionali: strumento per studiare normale acinose Architettura vs trasformazione maligna delle cellule mammarie

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

Monitoraggio delle mammarie caratteristiche architettoniche e Rilevamento cancro al seno con risonanza magnetica del tensore di diffusione Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Valutazione della solidità del grasso umano iniezione in topi nudi con Micro-Tomografia Computerizzata

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Un<em&gt; Ex vivo</em&gt; Modello per studiare Hormone azione in seno umano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...