Ringraziamo Cappellani J. e J. Qiu per il supporto tecnico. Questo lavoro è stato sostenuto da fondi del National Cancer Institute (U01 CA141451), il Consorzio Starr Cancro, Susan G. Komen for the Cure, Long Island 2 Un giorno per combattere il cancro al seno, Coalizione Manhasset donne contro il cancro al seno a ME, e un pre-dottorato dal dal Congresso Regia Programma di ricerca sul cancro al seno, Stati Uniti per ESNESN è anche il destinatario del C. Leslie rapida e William Randolph Hearst Borse di studio della Fondazione della Scuola Watson di Scienze Biologiche. HAA è stato sostenuto da fondi del Consiglio norvegese per la ricerca (160698/V40 e 151882), e del sud-est autorità sanitarie regionali (2.007.060).

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Gli autori non hanno conflitti di interesse da dichiarare. Tutti gli esperimenti sugli animali sono stati condotti in conformità con IACUC protocolli approvati.

Le risposte dei tumori a terapie sistemiche in vivo può essere notevolmente diverse da quelle delle cellule tumorali in vitro, come il microambiente influenza sia la risposta acuta e ricaduta 5. Uno dei maggiori ostacoli alla esplicitazione nei percorsi di chemioresistenza in vivo è la complessità delle interazioni tra cellule tumorali e il loro micro. In particolare, poiché tumori solidi hanno sviluppato un equilibrio tra cancro e cellule stromali, perturbazioni di un componente, come la morte cellulare che si verifica durante il trattamento antitumorale, può provocare effetti drammatici sulla organizzazione dei tessuti. La tecnica di imaging intravitale qui fornite permette la visualizzazione diretta del cancro cellula-stroma interazioni all&#39;interno dei tumori di topi anestetizzati dal vivo, durante il trattamento con farmaci anti-cancro. Usiamo microscopia confocale disco rotante, che ha una profondità relativa penetrazione limitata (v. 17 </sup&gt;), ma le tecniche chirurgiche ed etichettatura può essere utilizzato con altri tipi di microscopia. Filmati acquisiti da questi esperimenti possono essere utilizzati per monitorare i processi che includono cambiamenti di intensità di fluorescenza (ad esempio a causa di infiltrazioni di una popolazione etichettati o induzione della morte cellulare), la motilità cellulare, distribuzione di iniettabili (ad esempio per monitorare perdita vascolare), e co- localizzazione di segnali fluorescenti 5,12,17,20. Noi di routine topi di immagine per più di 6 ore e di imaging eseguita anche per oltre 18 ore. Ciò richiede mantenendo i topi continuamente sotto anestesia per questi periodi di tempo lunghi. Con l&#39;anestesia adeguata, oltre il 90% dei nostri animali sopravvivono 6 ore, e circa l&#39;80% sopravvive più di 18 ore. I dettagli su come eseguire a lungo termine anestesia sono stati pubblicati in precedenza 19. Nella nostra esperienza, cinque fattori sono fondamentali: evitare l&#39;ipotermia, evitare la disidratazione, mantenendo fisiologiche nel sangue i livelli di saturazione di ossigeno, ucantare gas umidificato vettore per isoflurano, e mantenere gli animali al più basso livello di anestesia in cui essi non mostrano segni di dolore. Per mantenere la temperatura corporea, teniamo topi sotto una coperta riscaldata (a 38 ° C). Per evitare la disidratazione, si iniettano piccole quantità di soluzione salina ip durante tutta la procedura di imaging intero. Per mantenere fisiologici livelli di saturazione dell&#39;ossigeno nel sangue, si parte con il 21% di ossigeno nel gas di trasporto (piuttosto che il comunemente usato 100%). Questo ci permette di aumentare i livelli di ossigeno per via inalatoria se un topo - ore in un esperimento - mostra una diminuzione della saturazione di ossigeno nel sangue. Nella nostra esperienza, aumentare i livelli di ossigeno per via inalatoria in quel momento (ad esempio, per il 30-40%), quasi sempre si stabilizzerà l&#39;animale consentendo ore di immagini aggiuntive. Il livello ottimale di anestesia è per la maggior parte dei mouse conseguiti con 1-1,5% isoflurano e si traduce in tassi di 60-65/min respiro e pulsazioni di 400-450/min. Nei nostri esperimenti di imaging, si utilizzano principalmente modello di topo transgenicos (MMTV-PyMT e C3 [1]-Tag) in cui i tumori sono multifocali, consentendo l&#39;imaging di lesioni multiple in diverse fasi della progressione tumorale in parallelo alle posizioni x, y multipli durante una sessione di imaging. Questo diminuisce preoccupazioni riguardanti mouse-a-mouse variazione rispetto a esperimenti volti a identificare risposte terapeutiche stadio-dipendenti, e riduce il numero di animali necessari per l&#39;imaging. Il MMTV-PyMT modello (e di altri modelli di cancro spontanee) passare attraverso stadi progressivi istopatologici che possono essere rilevate durante l&#39;esposizione intravitale, anche se la messa in scena patologica delle lesioni tumorali che si possono fare durante l&#39;esposizione intravitale è diverso e meno dettagliata, di quella di sezioni istologiche 5,17. Al contrario, i modelli di trapianto tendono a riflettere i tumori in fase avanzata meglio, come le cellule tumorali in genere sono isolati da tumori avanzati. Tali modelli sono quindi più suscettibili allo studio del tumore-stromainterazioni di tumori in fase avanzata che differenze in queste interazioni tra diverse fasi. Mostriamo alcuni esempi di come immagine cambia nucleari strutturali che si verificano dopo la morte cellulare indotta dalla terapia. In assenza di un trattamento anti-cancro, questa strategia etichettatura può invece essere utilizzata per la divisione cellulare immagine in situ, chiarire, ad esempio, come la proliferazione cellulare e dormienza è influenzata dal microambiente. In futuro, marcatura fluorescente di componenti mitocondriali possono consentire di immagine cambia mitocondriali che si verificano durante la morte cellulare per apoptosi. In questo protocollo, abbiamo anche mostrato esempi di come immagine del cancro delle cellule immuni interazioni delle cellule dopo la terapia, ma altri componenti microambientali possono anche essere visualizzati e creata l&#39;immagine. Esistenti linee giornalista transgeniche per i componenti stromali includono quelli per i fibroblasti (ad es., FSP1-EGFP 17, αSMA-RFP 21, COL1A1-EGFP 21) o cellule del sistema vascolare (es. Tie2-GFP 22; VEGF-GFP 23). Intravitale imaging consente la visualizzazione in tempo reale delle interazioni e processi che avvengono in vivo somministrazione di chemioterapia seguente. Con la tecnologia attualmente disponibile, abbiamo fatto importanti progressi nella comprensione di come cellule mieloidi influenzare la risposta terapeutica, tuttavia, perché il microambiente tumorale è così complesso, grandi progressi sono ancora necessari per iniziare a scavare meccanicamente nei processi sottostanti ambiente-mediata resistenza ai farmaci. Uno dei progressi più importanti ancora richiesti è l&#39;identificazione di migliori marcatori per specifiche popolazioni cellulari. L&#39;importanza di migliori marcatori è ben illustrato con due dei componenti più importanti cellule stromali del microambiente tumorale mammario, fibroblasti e cellule immunitarie. α-muscolo liscio actina (αSMA) viene spesso utilizzato per identificarefibroblasti, ma la proteina è espressa da cellule muscolari lisce vascolari e cellule mioepiteliali 24. Così, anche se αSMA-GFP topi reporter esiste, determinare il tipo specifico di essere cellula seguente durante un esperimento di imaging intravitale è impegnativo. Analogamente, un singolo marcatore fluorescente come EGFP espresso sotto il c-fms promotore più spesso identificare sottopopolazioni di cellule immunitarie 12,17. Così, anche se uno vorrebbe idealmente utilizzare un singolo marcatore per identificare un tipo specifico cellulare la complessità della biologia sottostante rappresenta una sfida importante nell&#39;utilizzo questo approccio. Una parziale soluzione a questo problema è di utilizzare etichette iniettabili per differenziare ulteriormente sottopopolazioni di cellule che esprimono il reporter fluorescente stessa. Per esempio, destrani fluorescenti sono ripreso da macrofagi e può essere usato per etichettare le sottopopolazioni di macrofagi che sono etichettati da c-fms-EGFP e Fsp1-EGFP topi reporter transgenici <sup&gt; 17. Anticorpi coniugati a sonde fluorescenti sono stati utilizzati per identificare le sottopopolazioni specifiche (ad esempio il Gr1-positivo popolazione di cellule mieloidi etichettati da c-fms-EGFP giornalista 17). A differenza di curve di risposta del tumore generati dal calibro di misura, che forniscono poche informazioni sui meccanismi su come e perché i tumori rispondono alla terapia somministrata, o una serie istologico derivati ​​da tessuti raccolte in diversi momenti che richiedono grandi coorti di animali, la nostra tecnica fornisce diretta, informazioni quantificabili sulla dinamica della morte cellulare e le interazioni di cellule stromali con cellule tumorali in coorti piccole. Così, il vantaggio di utilizzare la procedura riportata qui è che fornisce informazioni dinamiche sulle risposte farmacologiche nel contesto di un microambiente tumorale intatta in tempo reale. Tali informazioni possono portare a intuizioni importanti nei processi di fondo che guidano le risposte terapeutiche 5 &lt;/ Sup&gt;.

Tumori solidi sono costituiti da due compartimenti principali: le cellule tumorali e le componenti stromali (sia cellulare e non cellulare) che aiutano a mantenere un ambiente adatto per la crescita tumorale 1. Questi componenti stromali giocano un ruolo critico nello sviluppo, progressione e metastasi di molti tipi di tumori, compresi quelli del seno 2-5. L&#39;ambiente stromale influenza anche le risposte terapeutiche 2,4,5. Così, determinando come le cellule tumorali rispondono alle terapie convenzionali e romanzo nel contesto del microambiente intatto è importante per la nostra comprensione della biologia del cancro e migliorare attuali strategie terapeutiche. Inoltre, definendo le modalità di cellule di cancro-stroma interazioni cambiano in seguito alla somministrazione della terapia è fondamentale per la comprensione della biologia di recidiva tumorale. Organotipiche co-coltura di sistemi sono stati utili per lo studio delle interazioni tumore-stroma <sup&gt; 6, ma è evidente che i metodi attualmente disponibili non correttamente ricapitolare il microambiente intatta tumorale in vitro, in particolare per quanto riguarda la funzione vascolare e reclutamento di cellule immunitarie. Infatti, le risposte delle cellule tumorali a terapie che vengono osservate in vitro sono spesso significativamente differenti da quelle osservate in vivo, in cui il microambiente è intatto 5. Così, in modelli in vivo per lo studio del tumore-stroma interazioni e il loro impatto sulla risposta terapeutica può rispecchiare meglio i meccanismi clinicamente rilevanti 7. Il mezzo principale di studiare le risposte a terapia antitumorale in vivo sono passati attraverso le misurazioni di dimensione del tumore e la valutazione istologica dei tessuti provenienti da animali trattati o pazienti. Tuttavia, i progressi nella microscopia a fluorescenza e giornalisti nel corso degli ultimi due decenni hanno permesso la visualizzazione di processi inter-e intracellularead alta risoluzione in un animale vivo, anestetizzati (intravitale imaging) 8. Queste tecnologie microscopia intravitale hanno dimostrato di essere particolarmente utile per spazialmente e temporalmente la dissezione nella dinamica in vivo del tumore-stroma interazioni con risoluzione cellulare e sub-cellulare 8,9. Processi che sono stati svelati da imaging intravitale includono antitumorale citotossicità delle cellule T 10,11, interazioni tra cellule T e cellule mieloidi 12, la dinamica dei cambiamenti nella composizione collagene e organizzazione dopo il trattamento terapeutico 13, macrofagi-dipendente intravasation cellula tumorale e metastasi 14, e la permeabilità vascolare 15. Ci sono tre requisiti principali per l&#39;imaging intravitale. Questi includono la preparazione adeguata e l&#39;esposizione di tessuti per l&#39;imaging, l&#39;etichettatura fluorescente delle componenti del tessuto di interesse, e di una coppia di microscopia-camera sistema in grado di acquisire immagini 16. Le strategie più comuni utilizzati per rispondere a queste esigenze sono: 1) preparati eterotopiche (ad esempio, l&#39;inoculazione del orbitale orecchio o con gli occhi), permanente Imaging per Windows o preparati esteriorizzato (ad esempio, lembo di pelle dorsale), 2) transgenici fluorescenti e giornalisti.. iniettabili fluorescenti di etichettare componenti del tessuto e 3) sistemi di microscopia confocale multiphoton o in coppia con tubi fotomoltiplicatori o charge-coupled device (CCD), rispettivamente 9. Usiamo un modello preparato chirurgicamente lembo cutaneo per esporre la ghiandola mammaria inguinale per l&#39;imaging in animali anestetizzati che esprimono transgeni codificanti reporter fluorescenti. Immagini sono ottenute in un periodo di 12 a 40 ore utilizzando un CCD intensificata (ICCD) fotocamera accoppiato con quattro laser filatura sistema microscopio confocale disco 17. Imaging in questo modo ci ha permesso di studiare i processi, come nella distribuzione di farmaci in vivo, la fase-dipendente chrisposte emotherapeutic, il tipo di chemioterapia morte cellulare, e il comportamento delle cellule mieloidi 5. Forniamo un protocollo per l&#39;imaging intravitale delle risposte delle cellule tumorali di terapia antitumorale e tumore-stroma interazioni in modelli murini di cancro al seno. Questo protocollo può essere utilizzato per monitorare i comportamenti e la morte di entrambe le cellule tumorali e componenti stromali con una vasta gamma di etichette fluorescenti transgenici e iniettabili sia in modelli transgenici e il trapianto per periodi fino a 40 ore in una singola sessione di imaging.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagente </td></tr><tr><td> MMTV-PyMT topi </td><td> Jackson Laboratory </td><td> 2374 </td><td></td></tr><tr><td> C3 (1) Tag-topi </td><td> Jackson Laboratory </td><td> 13591 </td><td></td></tr><tr><td> ACTB-ECFP topi </td><td> Jackson Laboratory </td><td> 3773 </td><td></td></tr><tr><td> ACTB-H2B-EGFP topi </td><td> Jackson Laboratory </td><td> 5418 </td><td></td></tr><tr><td> 1 x PBS </td><td> Made in-house </td><td></td><td></td></tr><tr><td> 2 MD Destrano, fluoresceina coniugato </td><td> Invitrogen </td><td> D-7137 </td><td> Ricostituire in dH 2 O (4 mg / ml, conservare a -20 ° C), diluire a 1 mg / ml in 1 x PBS </td></tr><tr><td> 10 kD destrano, Alexa Fluor 647-coniugato </td><td&gt; Invitrogen </td><td> D-22.914 </td><td> Ricostituire in dH 2 O (4 mg / ml, conservare a -20 ° C), diluire a 1 mg / ml in 1 x PBS </td></tr><tr><td> Doxorubicina cloridrato </td><td> Sigma-Aldrich </td><td> 44583 </td><td> Ricostituire in dH 2 O e conservare a 4 ° C; diluire in 1 x PBS </td></tr><tr><td> Isothesia (isoflurano) </td><td> Butler Animal Health di alimentazione </td><td> 029450 </td><td> 250 ml </td></tr><tr><td> Propidio ioduro </td><td> Invitrogen </td><td> P3566 </td><td> 1 mg / ml, diluito 1:15 in 1 x PBS </td></tr><tr><td> Azoto </td><td></td><td></td><td></td></tr><tr><td> Ossigeno </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Attrezzatura </td></tr><tr><td> 18G x 1 ½ &quot;ago smussatura regolare </td><td> BD </td><td> 305196 </td><td></td></tr><tr><td> μManager </td><td> Vale Lab, UCSF </td><td> <a href="www.micro-manager.org" target="_blank">www.micro-manager.org</a> </td><td> Software open-source </td></tr><tr><td> Tampone imbevuto di alcool </td><td> BD </td><td> 326895 </td><td> 70% di alcol isopropilico tamponi </td></tr><tr><td> Anestesia sistema </td><td> Imaging Products molecolari, Co. </td><td></td><td></td></tr><tr><td> Copertura in vetro </td><td> Corning </td><td> 2940-245 </td><td> No. 1 ½, 24x50 mm, disinfettare con il 70% salviette isopropanolo </td></tr><tr><td> Rezza spugne garza (sterile) </td><td> Kendall </td><td> 6939 </td><td></td></tr><tr><td> Vetro microscopio diapositive </td><td> Corning </td><td> 2948-75x25 </td><td> Pre-pulite, se non pre-lavaggio, disinfettare con il 70% salviette isopropanolo </td></tr><tr><td> Temprati forbici sottili </td><td> Strumenti Scienza Belle </td><td> 14090-11 </td><td> 2 coppie, in acciaio inossidabile, forte affilati come punte, punta diritta, 26 tagliente mm, 11 cm di lunghezza </td></tr><tr><td> Coperta riscaldata </td><td> Gaymar Industries </td><td></td><td></td></tr><tr><td> Hot tallone sterilizzatore </td><td> Strumenti Scienza Belle </td><td> 18000-45 </td><td> Attivare circa 30 minuti prima dell&#39;uso; sterilizzare strumenti a&gt; 200 ° C per 30 sec </td></tr><tr><td> Imaris </td><td> Bitplane </td><td> <a href="www.bitplane.com" target="_blank">www.bitplane.com</a> </td><td></td></tr><tr><td> Krazy Glue </td><td> Prodotti di Elmer </td><td> KG484 </td><td></td></tr><tr><td> Laboratorio nastro (½ &quot;) </td><td></td><td></td><td></td></tr><tr><td> Laboratorio nastro (1 &quot;) </td><td></td><td></td><td></td></tr><tr><td> Coperchio diuna spedizione polistirolo raffreddamento </td><td></td><td></td><td> Questo sarà utilizzato come piattaforma chirurgica </td></tr><tr><td> Micro dissettore </td><td> Roboz </td><td> RS-5153 </td><td> 1x2 denti, una leggera curva, 0,8 mm di larghezza, punta 4 &quot;lunghezza </td></tr><tr><td> Micro dissettore </td><td> Roboz </td><td> RS-5135 </td><td> Dentata, leggera curva, 0.8 larghezza punta mm 4 &quot;lunghezza </td></tr><tr><td> Microscopio </td><td></td><td></td><td> Spinning-disco confocale, XYZ fase Piezo, capablility epifluourescence 17,25 </td></tr><tr><td> Microscopio fase di inserimento </td><td> Applicata strumentazione scientifica </td><td></td><td> Personalizzato fabbricato, con due porte di imaging circolari </td></tr><tr><td> MouseOx ossimetro, software e sensori </td><td> STARR Life Sciences </td><td> <a href="www.starrlifesciences.com" target="_blank">www.starrlifescience</a>s.com </td><td></td></tr><tr><td> Nalgene Versi Super-Dry lab inzuppatori </td><td> Thermo Scientific </td><td> 74218-00 </td><td> Tagliare in quarti per il rivestimento piattaforma chirurgica </td></tr><tr><td> Nebulizzatore </td><td> Salter Labs </td><td> 8901 </td><td> Utilizzato per umidificare i gas, previene le irritazioni dei tessuti polmonari </td></tr><tr><td> Slip-siringa monouso tubercolina (1 ml) </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> Surflo alato set d&#39;infusione </td><td> Terumo </td><td> SV-23BLK </td><td> X 23G ¾ &quot;ultra sottile ago, 12&quot; tubo </td></tr><tr><td> Betadine Spray </td><td> Purdue Pharma </td><td> BASP3H </td><td></td></tr></tbody></table>

live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer

Il microambiente tumorale gioca un ruolo fondamentale nella iniziazione del tumore, la progressione, metastasi, e la risposta alle terapie anti-tumore. Tridimensionali di co-coltura sistemi sono spesso utilizzati per spiegare le interazioni tumore-stroma, compreso il loro ruolo nella risposta ai farmaci. Tuttavia, molte delle interazioni che avvengono in vivo nel microambiente intatto non può essere completamente replicata in queste impostazioni in vitro. Pertanto, la visualizzazione diretta di questi processi in tempo reale, è diventato uno strumento importante per capire le risposte alle terapie tumorali e individuare le interazioni tra cellule tumorali e lo stroma che possono influenzare queste risposte. Qui forniamo un metodo per utilizzare la microscopia confocale filatura disco di vivo, topi anestetizzati osservare direttamente la distribuzione di droghe, le risposte delle cellule tumorali e le variazioni tumore-stroma interazioni in seguito alla somministrazione di terapia sistemica in modelli di cancro al seno. Si descrivono le procedure per labeling componenti tumorali diverse, il trattamento degli animali per l&#39;osservazione di risposte terapeutiche, e la procedura chirurgica per l&#39;esposizione di tessuti tumorali per immagini fino a 40 ore. I risultati ottenuti da questo protocollo sono filmati time-lapse, in cui i processi come l&#39;infiltrazione di droga, morte delle cellule tumorali e la migrazione delle cellule stromali possono essere valutati utilizzando software di analisi delle immagini.

Watch this Scientific Journal Video about Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer at JoVE.com

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Si descrive un metodo per l&#39;imaging di risposta anti-cancro<em&gt; In vivo</em&gt; E alla risoluzione di singola cellula.

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

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

live-imaging-drug-responses-tumor-microenvironment-mouse-models

Research

JoVE Journal

Medicine

24.6K Views.  Watson School of Biological Sciences. Si descrive un metodo per l'imaging di risposta anti-cancro In vivo E alla risoluzione di singola cellula.

Video: Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Metodo per Novel Anti-Cancer Drug Development utilizzando Espianti Tumore di campioni chirurgici


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

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 ...

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 ...

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

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

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

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

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal Imaging di angiogenesi in un modello di ratto Nudo di metastasi ossee cancro al seno utilizzando la risonanza magnetica, tomografia computerizzata volumetrica e Ultrasuoni

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse imaging di primaria preneoplastiche cellule epiteliali mammarie Derivato da modelli murini geneticamente modificati di cancro al seno

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

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 ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

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

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

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

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

Ad alta frequenza di Ecografia del mouse cervicali Linfonodi

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

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In vivo ed ex vivo approcci per studiare il cancro ovarico metastatico colonizzazione delle strutture Lattea Spot in peritoneale adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...