Questa ricerca è stata sostenuta dal Dipartimento della Difesa Breast Cancer Research Program con il numero premio (ASH, W81XWH-13-1-0010), NIH CA100324, CA100324 PPG, e il programma di imaging integrato.

Tutte le procedure descritte devono essere eseguite in conformità con le linee guida e regolamenti per l&#39;uso di animali vertebrati, tra cui la preventiva approvazione da parte del Albert Einstein College of Medicine Istituzionale cura degli animali e del Comitato uso. 1. Generazione di fluorescenza Tumori etichettati e macrofagi associati al tumore <ol><li> Generare cellule tumorali fluorescente attraversando il, autoctona, topo geneticamente modificato modello cancro mammario spontaneo in cui la ripetizione lungo terminale virus tumore del mouse mammaria spinge l&#39;antigene polioma mezzo T (MMTV-PyMT) con i topi transgenici con i giornalisti fluorescenti [cioè, una maggiore verde fluorescente proteine ​​(EGFP), una maggiore proteina ciano fluorescente (ECFP) o Dendra2] 8,9. </li><li> Macrofagi etichetta fluorescente negli animali PyMT con fluorescente cellule tumorali attraversando modelli di topo geneticamente con i giornalisti fluorescenti specifiche myeloid- e macrofagi [cioè, MacGreen 15 o topi MacBlue CSF1R -GAL4VP16 / UAS-ECFP 16]. </li><li> In alternativa, generano fluorescente tumori attraverso il trapianto ortotopico di cellule fluorescente PyMT tumorali (singenici), linee di cellule umane di cancro al seno o tumori primari paziente umano derivati ​​(xenotrapianti) 11,17. <ol><li> Implant fluorescente cellule tumorali PyMT in topi singenici con le cellule mieloidi fluorescenti proteina marcato per generare animali con cellule tumorali fluorescenza etichettati e cellule mieloidi 13. </li></ol></li><li> Iniettare 100 ml di 10 mg / kg fluorescente 70 kDa destrano per via endovenosa (iv) la coda vena in grave topi immunodeficienza combinata (SCID) con tumori xenotrapianto di linee cellulari umane o tumori primari del paziente di derivazione a macrofagi etichette fluorescenza 2 ore prima della partenza dell&#39;imaging intravitale. </li></ol> 2. Installazione Microscopio e Imaging Preparazione Nota: Questa procedura descrive il set-upper l&#39;imaging intravitale su un microscopio multiphoton 8. <ol><li> Accendere tutti i componenti del microscopio e laser, tra cui laser a due fotoni e rivelatori. </li><li> Selezionare la casella di riscaldamento a 30 ° C per pre-riscaldare il palco. Questo passaggio è fondamentale per il mantenimento della temperatura fisiologica dell&#39;animale. </li><li> Per l&#39;uso su un posto microscopio invertito la fase di inserimento su misura sul palco microscopio. L&#39;inserto personalizzato è un foglio di 1/8 &quot;in alluminio di spessore lavorato per adattarsi allo spazio inserto palco e con un 1&quot; di diametro foro passante nel centro per l&#39;imaging. <ol><li> Pulire la fase di microscopio e l&#39;inserto palco con il 70% di etanolo e aria secca. </li><li> Mettere un grande goccia d&#39;acqua sulla NA obiettivo 20X, 1,05 microscopio per mantenere il contatto ottico con il vetro di copertura. </li><li> Bicchiere coperchio (# 1.5 spessore) sulla porta di imaging sull&#39;inserto palco microscopio. Fissare in posizione con nastro adesivo di laboratorio. </li><li> Utilizzare un pugno buco per fare un buco due centimetri x 2 cmin un pad in gomma flessibile e posizionare il gommino sul palco microscopio con il foro del pad allineato con il foro dell&#39;inserto fase personalizzato. </li></ol></li></ol> 3. Preparazione della vena della coda catetere e reagenti per iniezione durante Imaging <ol><li> Tagliare una lunghezza di 30 cm in polietilene (PE) tubo. </li><li> Utilizzando pinze, spostare l&#39;ago metallico di un ago G 31 avanti e indietro finché non si rompe fuori del raccordo di plastica. </li><li> Utilizzando pinze, inserire l&#39;estremità smussata dell&#39;ago distaccata nel tubo PE. </li><li> Inserire un ago G 31 nell&#39;altra estremità del tubo PE. </li><li> Riempire una siringa da 1 cc con fosfato sterile salina tamponata (PBS) e inserirlo nel ago G 31 e lavare tutta l&#39;aria dal tubo e ago con PBS. PBS è usato per mantenere l&#39;idratazione e osmolarità del sangue 9,18, soluzione salina isotonica tuttavia possono anche essere utilizzati. </li><li> Riempire una siringa da 1 cc con 200 ml di 3 mg / kg 155 kDa destrano-tetramethylrhodamine (TMR) O punti quantici per l&#39;etichettatura vascolare. </li><li> Preparare eventuali altre proteine ​​iniettabili come ad esempio un acido amino 165 isoforma del fattore di crescita vascolare endoteliale A (VEGFA 165) in una siringa da 1 cc e posto sul ghiaccio. Iniettare 0,2 mg / kg VEGFA 165 19 alla concentrazione di 0,05 mg / ml. </li></ol> 4. Inserimento della coda Indwelling Vena catetere <ol><li> Posto l&#39;animale in anestesia utilizzando una camera di induzione con 5% isoflurano con O 2 come gas di trasporto. </li><li> Trasferire il mouse per la piattaforma chirurgica una volta che è completamente anestetizzato e non risponde ad un pizzico dito del piede. </li><li> Linea aperta isoflurano alla piattaforma chirurgica, chiudere la linea anestesia alla camera di induzione e posizionare il cono sopra muso dell&#39;animale. Ridurre il isoflurano dal 5% al ​​2,5%. </li><li> Applicare una pomata oftalmica agli occhi del mouse per prevenire la secchezza durante l&#39;anestesia. </li><li> Scaldare l&#39;animale sotto la lampada di riscaldamentoper altri 2 minuti per aumentare la circolazione nella coda come circolazione rallenta con anestesia profonda. </li><li> Sterilizzare la vena della coda del mouse con il 70% di etanolo. </li><li> Inserire la punta di un ago G 31 dal catetere costruito nella Sezione 3 nella vena caudale laterale dell&#39;animale e spingere l&#39;ago 2-3 mm. </li><li> Tirare indietro sulla siringa per vedere il sangue, garantendo il catetere viene inserito nella vena della coda. </li><li> Utilizzare una lunghezza 1 cm di nastro lab posto sopra l&#39;ago per mantenere l&#39;ago in posizione parallelamente alla vena lungo la lunghezza della coda. </li></ol> 5. Pelle Flap procedura chirurgica per esporre il tumore mammario <ol><li> Con l&#39;animale sulla piattaforma chirurgica tamponare il tumore e la superficie ventrale con il 70% di etanolo. Nota: Come descritto, questa procedura non viene eseguita completamente asettico. Per le sessioni lunghe di imaging in cui l&#39;infezione può diventare un fattore di confusione, si consiglia di utilizzare una corretta tecnica asettica. </li><li> utilizzando steriLe pinze, sollevare la pelle ventrale e con le forbici sterili fanno un&#39;incisione sottocutaneo lungo la linea mediana ventrale di circa 1 cm di lunghezza. Evitare la puntura del peritoneo durante l&#39;incisione. </li><li> Delicatamente tagliare il tessuto connettivo fissare la ghiandola mammaria e tumori lontano dal peritoneo per esporre il tumore mammario. </li><li> Usando forbici e pinze, rimuovere delicatamente e tagliare via la fascia e grasso dalla superficie esposta del tumore mantenendo l&#39;integrità del sistema vascolare del tumore e minimizzando sanguinamento. Questo passaggio è fondamentale nel mantenere l&#39;architettura tumore e vascolarizzazione fornitura del tumore. </li></ol> 6. Preparazione degli animali per la microscopia <ol><li> Trasferire l&#39;animale alla fase di imaging pre-riscaldato con il tumore esposta collocato sul vetrino sull&#39;inserto palcoscenico sul obiettivo microscopio per l&#39;imaging. Fare attenzione a trasferire il catetere vena della coda delicatamente con l&#39;animale in modo da non staccare l&#39;ago. </li><li> ° postoe anestesia naso cono sopra il muso dell&#39;animale per mantenere insieme l&#39;anestesia al 3% isoflurano. </li><li> Posizionare l&#39;animale in modo che il tumore poggia nel foro del gommino ed entra in contatto con il vetro coprioggetto. </li><li> Utilizzare due tamponi in gomma per tenere delicatamente il tumore al suo posto e fissarli al palco microscopio con del nastro laboratorio per ridurre il movimento durante l&#39;acquisizione dell&#39;immagine. </li><li> Riempire la camera dal pad di gomma con PBS per mantenere il tessuto idratato e mantenere il contatto ottico con il vetrino. </li><li> Inizia il monitoraggio dei segni vitali dell&#39;animale con una sonda pulsossimetro. Collegare un sensore clip alla coscia dell&#39;animale. In alternativa un collare che è configurato per adattarsi intorno al collo può essere utilizzato. </li><li> Posizionare la scatola del riscaldamento sopra l&#39;animale per mantenere 30 ° C 20. </li><li> Lentamente ridurre il livello di isoflurano al 0,5-1% per mantenere l&#39;anestesia e mantenere il flusso sanguigno. </li></ol> 7. Acquisizione di immagini e l&#39;iniezione di flent Coloranti e iniettabili Proteine <ol><li> Utilizzando il fuoco del microscopio oculare sulle cellule tumorali fluorescenti sulla superficie del tumore. </li><li> Selezionare un&#39;area di interesse trovando zone con che scorre i vasi sanguigni. La selezione di una zona di interesse con scorre vasi sanguigni è critico per valutare vasi tumorali. </li><li> Una volta che è stata selezionata una regione di interesse passare al microscopio in modalità di imaging multiphoton. </li><li> Impostare i limiti superiore e inferiore di una serie z. Impostare il limite superiore della serie Z utilizzando il regolatore di messa a fuoco per spostare l&#39;obiettivo nella posizione di partenza desiderato e facendo clic sul pulsante di posizione Z &quot;Top&quot;. Determinare il limite superiore della serie Z visualizzando la rete in fibra di collagene sulla superficie del tumore nel canale generazione di seconda armonica (SHG) e osservando quando nessuna cellula ma solo fibre di collagene sono visibili. <ol><li> Impostare il limite inferiore della serie Z spostando l&#39;obiettivo alla profondità di imaging desiderata (typically 50 - 150 micron) e il tasto &quot;basso&quot; cliccando la posizione Z. </li><li> Impostare la dimensione del passo digitando il valore desiderato nel campo dimensione del passo. Determinare dimensione del passo sulla base di considerazioni di risoluzione e tempo di acquisizione (in genere 2 micron viene utilizzato per la ricostruzione 3D ad alta risoluzione e 5 micron altro). </li></ol></li><li> Impostare l&#39;intervallo di time-lapse passando al pannello Time-Lapse e inserendo il lasso di tempo desiderato nel campo Time-Lapse. Per risoluzione temporale ottimale impostare l&#39;intervallo di tempo a 0 sec per l&#39;imaging continuo. Nota: Per lungo time-lapse imaging si consiglia di impostare l&#39;intervallo di tempo di 10 secondi tra le acquisizioni per ricostituire il liquido di immersione obiettivo. </li><li> Una volta che i parametri per l&#39;imaging sono stati determinati, iniettare lentamente 155 kDa destrano-TMR. <ol><li> Rimuovere la siringa con PBS nel catetere coda vena e sostituirlo con la siringa contenente 155 kDa destrano-TMR facendo attenzione a non introdurre bolle nel Line. </li><li> Iniettare lentamente 155 kDa destrano-TMR nel mouse, con un volume massimo di 200 ml, e sostituire la siringa con siringa contenente PBS. PUNTO CRITICO: eseguire l&#39;iniezione lentamente e prendere precauzioni per evitare di ottenere una soluzione al di fuori della vena. </li></ol></li><li> Avviare l&#39;acquisizione del time-lapse imaging Z-stack facendo clic sui pulsanti Time-lapse Z-Stack e per deprimere loro e poi cliccando sul pulsante di registrazione. </li><li> Se altri destrani fluorescenti, vale a dire, isotiocianato 10 kDa destrano-fluoresceina (FITC), o le proteine, cioè, VEGFA 165, sono stati preparati in anticipo, li iniettare dopo l&#39;inizio di acquisizione di immagini per a = 0 min inizio. <ol><li> Rimuovere la siringa con PBS nella vena della coda del catetere e sostituirlo con la siringa contenente 10 kDa destrano-FITC o VEGFA 165 facendo attenzione a non introdurre bolle nella linea. </li><li> Iniettare lentamente iniettabili nel mouse attraverso il catetere vena della codae sostituire la siringa con una siringa contenente PBS. </li></ol></li><li> Ogni 30-45 min, iniettare lentamente 50 ml di PBS o soluzione salina per mantenere l&#39;idratazione dell&#39;animale. </li></ol> 8. L&#39;eutanasia <ol><li> Al termine della acquisizione delle immagini, eutanasia dell&#39;animale. <ol><li> Aumentare la isoflurano al 5%. </li><li> Mantenere l&#39;animale sotto il 5% isoflurano fino al 30 sec dopo che cessa di respirare e rimuovere l&#39;animale dal palco. </li><li> Eseguire dislocazione cervicale per garantire la completa eutanasia. </li></ol></li></ol> Processing 9. Immagine <ol><li> Acquisire le immagini in file TIFF a 16 bit per ogni singolo canale ad ogni tempo e salvare in modo sequenziale. </li><li> Eseguire la separazione di sovrapposizione spettrale (cioè, GFP e PCP), l&#39;eliminazione di deriva xy e la generazione di film da sequenze time-lapse con metodi stabiliti in ImageJ 9. Eseguire ricostruzioni di superficie 3D di immagini ad alta risoluzione 13. </li></ol>

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<li>Yano, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial-temporal+FUCCI+imaging+of+each+cell+in+a+tumor+demonstrates+locational+dependence+of+cell+cycle+dynamics+and+chemoresponsiveness%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Spatial-temporal FUCCI imaging of each cell in a tumor demonstrates locational dependence of cell cycle dynamics and chemoresponsiveness.</a> Cell Cycle. 13 (13), 2110-2119 (2014).</li>
<li>Yang, M., Jiang, P., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-body+subcellular+multicolor+imaging+of+tumor-host+interaction+and+drug+response+in+real+time%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">Whole-body subcellular multicolor imaging of tumor-host interaction and drug response in real time.</a> Cancer Res. 67 (11), 5195-5200 (2007).</li>
<li>Manning, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intravital+imaging+reveals+conversion+between+distinct+tumor+vascular+morphologies+and+localized+vascular+response+to+Sunitinib%5BTitle%5D)+AND+%22Intravital%22%5BJournal%5D)">Intravital imaging reveals conversion between distinct tumor vascular morphologies and localized vascular response to Sunitinib.</a> Intravital. 2 (1), 24790(2013).</li>
<li>Alexander, S., Koehl, G. E., Hirschberg, M., Geissler, E. K., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+imaging+of+cancer+growth+and+invasion%3A+a+modified+skin-fold+chamber+model%5BTitle%5D)+AND+%22Histochem+Cell+Biol%22%5BJournal%5D)">Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model.</a> Histochem Cell Biol. 130 (6), 1147-1154 (2008).</li>
<li>Brown, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo.+measurement+of+gene+expression%2C+angiogenesis+and+physiological+function+in+tumors+using+multiphoton+laser+scanning+microscopy%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">In vivo. measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy.</a> Nat Med. 7 (7), 864-868 (2001).</li>
</ol>

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interazioni cellulari che si verificano spontaneamente nel microambiente tumorale possono portare a cambiamenti nella motilità delle cellule tumorali e intravasation. High-Resolution Imaging intravitale del tessuto tumorale dal vivo permette la visualizzazione delle dinamiche pluricellulari che possono essere 10,13,24 altamente transitorio. End-point in vivo o immagini time-lapse acquisiti con i punti di tempo discreto grado di fornire informazioni essenziali sui meccanismi molecolari dei processi n...

Diffusione di cellule tumorali dal tumore mammario primario ha dimostrato di coinvolgere non solo le cellule tumorali, ma ospitare cellule stromali compresi macrofagi e cellule endoteliali. Inoltre, il tumore vascolare è anormale con aumento della permeabilità 1. Così, determinare come le cellule tumorali, macrofagi e cellule endoteliali interagiscono per mediare la permeabilità vascolare e intravasation delle cellule tumorali nel microambiente tumorale primaria è importante per la comprensione delle metastasi. Comprendere la cinetica della permeabilità vascolare, intravasation delle cellule tumorali e il meccanismo di segnalazione alla base delle interazioni multicellulari nel microambiente tumorale in grado di fornire informazioni cruciali per lo sviluppo e la gestione delle terapie anti-cancro. I mezzi primari di studiare tumore permeabilità vascolare in vivo è stata la misura di coloranti extravascolare come Evans blue 2, destrani ad alto peso molecolare (155 kDa)3 e fluoroforo o proteine ​​radiotracciante coniugato (tra cui l&#39;albumina) 4 in punti fissi di tempo dopo l&#39;iniezione del colorante. I progressi nella microscopia, modelli animali e coloranti fluorescenti hanno permesso la visualizzazione dei processi cellulari e permeabilità vascolare di animali vivi attraverso la microscopia intravitale 5. Immagini di animali vivi con l&#39;acquisizione di immagini statiche, o sequenze di breve time-lapse in diversi punti di tempo non permette la completa comprensione di eventi dinamici nel microambiente tumorale 6,7. Infatti, l&#39;acquisizione di immagini statiche nel corso di 24 ore ha dimostrato che tumore vascolarizzazione è a tenuta, tuttavia la dinamica della permeabilità vascolare non è stata osservata 6. Così, lungo l&#39;imaging continuo time-lapse fino a 12 ore cattura la cinetica di eventi dinamici nel microambiente tumorale. Questo protocollo descrive l&#39;uso di prolungato multiphot time-lapsesulla microscopia intravitale per studiare eventi multicellulari dinamici nel microambiente tumorale. Più tipi di cellule del microambiente tumorale sono etichettati con coloranti iniettabili o utilizzando animali transgenici che esprimono proteine ​​fluorescenti. Utilizzando un catetere venoso coda coloranti vascolari o proteine ​​possono essere iniettati dopo l&#39;inizio di imaging per acquisire dati cinetici di eventi multicellulari nel microambiente tumorale. Per l&#39;imaging cellulare dal vivo il tumore mammario è esposta attraverso la preparazione chirurgica di un lembo cutaneo. Le immagini vengono acquisite fino a 12 ore utilizzando un microscopio multiphoton dotato di molteplici tubi fotomoltiplicatori (PMT) rilevatori 8. Utilizzando opportuni filtri, un algoritmo di sottrazione consente 4 rivelatori PMT per acquisire 5 segnali fluorescenti nel microambiente tumorale contemporaneamente 9. Ad alta risoluzione multiphoton intravitale microscopia cattura di imaging singola risoluzione delle cellule di interazioni tumore-stroma nel microambiente tumorale, portando ad una migliore understanding della permeabilità vascolare e del tumore delle cellule intravasation 10-13. In particolare, rivelato altamente localizzati, transitori eventi permeabilità prolungati di imaging intravitale vascolari che si verificano selettivamente nei siti di interazione tra una cellula tumorale, un macrofago e una cellula endoteliale (definito come il microambiente tumorale delle metastasi, TMEM 14) 13. Inoltre, intravasation cellule tumorali si verifica solo in TMEM ed è spazialmente e temporalmente correlata con la permeabilità vascolare 13. risoluzione singola cella della dinamica di questi eventi è stato reso possibile attraverso l&#39;utilizzo di prolungato time-lapse multifotonica microscopio di cellule fluorescente nel microambiente tumorale.

<table><tbody><tr><td>155 kDa dextran-tetramethylrhodamine isothiocyanate</td><td>Sigma Aldrich</td><td>T1287</td><td>reconstitute at 20 mg/ml in 1x PBS</td></tr><tr><td>70 kDa dextran-Texas Red</td><td>Life Technologies</td><td>D-1830</td><td>reconstitute at 10 mg/ml in 1x PBS</td></tr><tr><td>10 kDa dextran-fluorescein isothyocyanate</td><td>Sigma Aldrich</td><td>FD10S</td><td>reconstitute at 20 mg/ml in 1x PBS</td></tr><tr><td>Qdot 705 ITK Amino (PEG) Quantum Dots</td><td>Life Technologies</td><td>Q21561MP</td><td>Dilute 25 &amp;#956;l in 175 &amp;#956;l of 1x PBS for injection</td></tr><tr><td>MMTV-PyMT mice</td><td>Jackson Laboratory</td><td>2374</td><td></td></tr><tr><td>Csf1r-ECFP mice (Csf1r-Gal4/VP16,UAS-ECFP)</td><td>Jackson Laboratory</td><td>26051</td><td></td></tr><tr><td>Csf1r-EGFP mice</td><td>Jackson Laboratory</td><td>18549</td><td></td></tr><tr><td>1x PBS</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Isoethesia (isoflurane)</td><td>Henry Schein Animal Health</td><td>50033</td><td>250 ml</td></tr><tr><td>Oxygen</td><td>AirTech</td><td></td><td></td></tr><tr><td>1 ml syringe, tuberculin slip tip</td><td>BD</td><td>309659</td><td></td></tr><tr><td>30 G x 1 (0.3 mm x 25&amp;nbsp;mm) needle</td><td>BD</td><td>305128</td><td></td></tr><tr><td>Polyethylene micro medical tubing&amp;nbsp;</td><td>Scientific Commodities Inc</td><td>BB31695-PE/1</td><td>0.28 mm I.D. x 0.64 mm O.D.</td></tr><tr><td>Microscope coverglass</td><td>Corning</td><td>2980-225</td><td>thickness 1.5, 22 x 50&amp;nbsp;mm</td></tr><tr><td>PhysioSuite MouseSTAT pulse oximeter, software and sensors</td><td>Kent Scientific</td><td></td><td></td></tr><tr><td>Laboratory tape</td><td>Fisher Scientific</td><td>159015R</td><td></td></tr><tr><td>soft rubber pad</td><td>McMaster-Carr</td><td>8514K62</td><td>Ultra-Soft Polyurethane Film, 3/16&amp;rdquo; Thick, 12&quot; x 12&quot;, 40 Oo Durometer, Plain Back</td></tr><tr><td>hard rubber pad</td><td>McMaster-Carr</td><td>8568K615</td><td>High-Strength Neoprene Rubber Sheet 1/4&quot; Thick, 12&quot; x 12&quot;, 50&amp;nbsp;A Durometer</td></tr><tr><td>Microscope</td><td>Olympus</td><td></td><td>The microscope is a custom built two laser multiphoton microscope based on an Olympus IX-71 stand utilizing a 20X 1.05NA objective lens.</td></tr><tr><td>7-Punch set</td><td>McMaster-Carr</td><td>3429A12</td><td>1/4&quot; to 1&quot; Hole Diameter, for Hammer-Driven Hole Punch</td></tr></tbody></table>

extended time-lapse intravital imaging of real-time multicellular dynamics in the tumor microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

Esteso microscopia time-lapse intravitale permette l&#39;imaging singola cella risoluzione dei processi multicellulari nel microambiente tumorale. Da parte delle cellule tumorali fluorescenza etichettatura, i macrofagi, lo spazio vascolare, e visualizzare la rete di fibre di collagene con il secondo segnale di generazione di armoniche, comparti multipli nel microambiente tumorale sono contemporaneamente monitorati durante l&#39;imaging. Le cellule tumorali marcate con proteine ​​fluo...

Watch this Scientific Journal Video about Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment at JoVE.com

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Questo protocollo descrive l&#39;uso della microscopia multiphoton effettuare prolungato time-lapse imaging di interazioni multicellulari in tempo reale, in vivo a risoluzione singola cellula.

Esteso Time-lapse Imaging intravitale del Real-time multicellulare Dynamics nel microambiente tumorale

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

extended-time-lapse-intravital-imaging-real-time-multicellular

Research

JoVE Journal

Medicine

8.1K Views.  Albert Einstein College of Medicine.  Questo protocollo descrive l'uso della microscopia multiphoton effettuare prolungato time-lapse imaging di interazioni multicellulari in tempo reale, in vivo a risoluzione singola cellula. 

Video: Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

A lungo termine ad alta risoluzione intravitale Microscopia nel polmone con un vuoto stabilizzato finestra Imaging

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Ricerca

Ricerca sul cancro

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

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

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

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

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

Medicina

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Imaging intravitale longitudinale del comportamento delle cellule tumorali cerebrali in risposta a una biopsia chirurgica invasiva

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

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

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

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

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

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

A lungo termine intravitale immunofluorescenza Imaging di tessuto componenti della matrice con Epifluorescenza e due fotoni Microscopia

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Bioingegneria

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Una finestra permanente per indagare sulle metastasi del cancro al polmone

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

Un approccio di segmentazione senza etichette per l'imaging intravitale del microambiente del tumore mammario

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

In tempo reale Imaging di cellule mieloidi Dynamics in<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Intestinale tumori da Spinning Disk Microscopia confocale

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

Immunologia e infezioni

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

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

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

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

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

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

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Videomicroscopia microscopia del sistema vascolare del tumore-associati utilizzando avanzate dorsale dello Skinfold finestra Chambers su topi transgenici fluorescenti