We thank Nguyen H. Nguyen for her technical help and Audrey O&#8217;Neill for support with the Zeiss Cell Observer spinning-disk confocal microscope. This work was supported by a Department of Defense postdoctoral fellowship (W81XWH-11-01-0139) and the Weizmann Institute of Science-National Postdoctoral Award Program for Advancing Women in Science (to V.P.).

Tutte le procedure descritte devono essere eseguite in conformità con le linee guida e regolamenti per l&#39;uso di animali vertebrati, tra cui l&#39;approvazione preventiva della Istituzionale Cura locali animali e del Comitato Usa (IACUC). 1. Generazione di polmone metastasi per ex vivo l&#39;imaging dal vivo (transgenico o la coda Vein Injection) NOTA: metastasi polmonari possono essere generati utilizzando modelli murini geneticamente o per via endovenosa (ev) delle cellule tumorali. <ol><li> Generare metastasi polmonari per l&#39;imaging attraversando un modello murino di tumore geneticamente modificato in un topo giornalista transgenico, ad esempio, si attraversa il cancro al seno modello di topo, mouse virus tumore mammario lunga terminal repeat-polyoma mezzo T antigene (MMTV-PyMT) 11 in ACTB-ECFP modello di topo 12. NOTA: Il modello ACTB-ECFP esprime migliorato proteina fluorescente ciano (ECFP) sotto il β-actnel promotore in modo tale che tutte le cellule fluorescenti nel blu, il canale PCP. Tuttavia, le cellule tumorali sono di gran lunga i più importanti e appaiono come una massa di cellule ECFP-positive al microscopio. Il modello di topo MMTV-PyMT sviluppa una malattia progressiva, in cui la crescita del tumore mammario è associata con la diffusione delle cellule tumorali alla periferia, soprattutto ai polmoni. Nei topi MMTV-PyMT sul FVB / n sfondo, micrometastasi possono osservare intorno 10-11 settimane di età. In generale, questi progressi macrometastasi a circa 14 settimane di 13 anni. O </li><li> Generare metastasi sperimentali utilizzando cellule primarie o linee cellulari singenici. Utilizzare nelle cellule tumorali in vitro primaria manipolato o linee cellulari (ad es., Trasduzione) seguito da iniezione endovenosa 14. <ol><li> In breve, in questo protocollo, iniettare una proteina fluorescente verde (GFP) esprimente (+) linea cellulare MMTV-PyMT in topi fluorescenti Reporter (ACTB-ECFP) o di tipo selvatico mice. Poi,visualizzare queste cellule denominate cellule VO-PyMT 15 utilizzando il verde, il canale GFP. NOTA: La linea cellulare originale VO-PyMT è stato derivato al Vanderbilt Ortopedia a Nashville, TN. VO acronimo di Vanderbilt Ortopedia. </li><li> Dopo l&#39;iniezione di 10 6 cellule (in 200 microlitri), osservare stravaso cellula tumorale immediatamente e fino a poche ore dopo l&#39;iniezione; osservare micrometastasi tra 1-3 settimane dopo l&#39;iniezione e rilevare macrometastasi 3 settimane dopo l&#39;iniezione 15. NOTA: minor numero di cellule possono essere iniettati per prolungare il tempo di iniezione per la crescita metastatica. </li></ol></li></ol> 2. L&#39;etichettatura dei componenti di interesse per il metastatico Microenvironment (transgenici e / o iniettabili) NOTA: etichettatura può essere ottenuto topi transgenici e / o vari iniettabili. Assicurarsi di utilizzare i colori fluorescenti diversi per l&#39;etichettatura dei vari tipi di cellule. <ol><li> componenti Label del microambiente metastatico utilizzando topi transgenici. Attraversate il modello di tumore del mouse precedentemente menzionato (ad es., MMTV-PyMT x ACTB-ECFP) in un modello di topo transgenico in cui le cellule stromali di interesse sono etichettati da una proteina fluorescente che non è ECFP, ad es., C-fms-EGFP 4,16. NOTA: Oltre alla visualizzazione delle cellule tumorali nel canale PCP, questo permette la visualizzazione di cellule mieloidi nel canale GFP 4. E / O </li><li> Etichettare i vari componenti del microambiente metastatico utilizzando iniettabili in topi transgenici reporter fluorescente o topi di tipo selvatico (non fluorescenti). NOTA: Diversi composti può essere iniettato per etichettare vari componenti del microambiente metastatico, ad esempio, un AF647-coniugato Gr-1 anticorpo viene qui utilizzato per etichettare neutrofili e sono utilizzati alcuni monociti 13 e diversi destrani peso molecolareper etichettare capillari polmonari. Per la preparazione di questi iniettabili vedere il punto 4. </li></ol> 3. Preparazione dei Materiali prima della dissezione <ol><li> 2% agarosio <ol><li> Pesare 0,2 g di agarosio ed aggiungere a 10 ml 1 x PBS. Riscaldare la soluzione per sciogliere l&#39;agarosio. Agarose solidificherà a RT, quindi mantenerlo a bagnomaria C 37 ° fino utilizzato per il riempimento. </li></ol></li><li> CO 2 e regolatore di temperatura <ol><li> Controllare DDH 2 O nella camera di umidificazione. Riempire quando necessario. Inserire piastra di configurazione nel portatarga fase temperatura (camera climatica). Accendere il controller di CO 2 e impostare CO 2 al 5%. Assicurarsi che la portata d&#39;aria è fissato a 0,4 Nl / min. </li><li> Aprire l&#39;aria e CO 2 valvole. Accendere il regolatore di temperatura. Assicurarsi che la temperatura della camera climatica e il coperchio sono fissati a 37 ° C. </li><li> Scaricare la pressione dell&#39;aria sul CO 2 metri. Controllare CO 2 in aumento, equilibration può richiedere fino a 30 min. </li></ol></li><li> Spinning disco microscopio confocale NOTA: I dettagli del microscopio set-up sono stati precedentemente descritti 4,17. <ol><li> Attivare i laser (laser argon 488 nm di eccitazione e lo stato solido 405 nm, 561 nm e 640 nm laser). Accendere il microscopio, la telecamera, l&#39;unità di controllo disco rotante, la AOTF, l&#39;unità di controllo laser e il controllore fotocamera. </li><li> Aprire l&#39;otturatore microscopio, accendere il computer che esegue il microscopio e aprire il software. </li></ol></li><li> Preparazione degli strumenti e la piattaforma dissezione. <ol><li> Accendere lo sterilizzatore a caldo tallone e lasciarlo raggiungere i 250 ° C. Pulire le 2 paia di forbici chirurgiche e pinze con acqua e sapone. Sterilizzare gli strumenti per almeno 30 sec. Lasciate che gli strumenti raffreddare. Utilizzare un coperchio di polistirolo come piattaforma di dissezione. Coprire con un pezzo di laboratorio soaker. </li></ol></li></ol> 4. Preparazione di iniezioni NOTA: A seconda del tempo di dimezzamento e la risposta preferita, iniettare fluorescente anticorpi e / o molecole fluorescenti o immediatamente prima del sacrificio animale o un paio di ore o giorni prima. <ol><li> Per neutrofili immagine Gr1-positive e monociti, prepara una siringa con 7 ml di brodo Gr-1 anticorpi AF647-coniugato (1 mg / ml) in 100 ml di PBS sterile sotto il cofano. Posizionare un 27 G ½ ago sulla siringa. </li><li> Per capillari polmonari immagine, preparare una seconda e terza siringa con 100 ml di o 70 kD rodamina-destrano coniugato (4 mg / ml) o destrano 10 kD AF647-coniugato (4 mg / ml). Posizionare un 27 G ½ ago sulle siringhe. </li><li> Iniettare l&#39;AF647 coniugato soluzione di anticorpi iv 5 ore prima di escissione polmoni. </li><li> Iniettare una o entrambe le soluzioni di destrano iv immediatamente precedenti l&#39;escissione polmoni &#39;. </li></ol> 5. Preparazione dei polmoni per ex vivo l&#39;imaging dal vivo NOTA: Prova a lavorare come sterile e accurato possibile per evitare inutili sfide delle cellule immunitarie nei polmoni. <ol><li> Iniettare il intraperitoneale mouse (ip) con un sovradosaggio letale di anestetico consentita dal protocollo animale approvato da IACUC, ad es., 1 ml di 2,5% Avertin. Attendere il mouse per smettere di respirare e di essere completamente non-responsivi agli stimoli nocivi (zampa posteriore pinch). NOTA: dislocazione cervicale e biossido di carbonio l&#39;eutanasia dovrebbe essere evitato in quanto può influenzare negativamente la vitalità delle cellule del polmone. </li><li> Immobilizzare il mouse su una tavola di dissezione e sterilizzare il mouse con il 70% di etanolo. </li><li> Utilizzare forbici chirurgiche prima fare una incisione epigastrica trasversale attraverso la pelle, seguita da un&#39;incisione simile attraverso il peritoneo. Tenere la tavola di dissezione in posizione verticale e tagliare l&#39;aorta discendente, in modo che le piscine sangue giù nell&#39;addome e non nella cavità toracica. </li><li> Snip una piccola apertura nel diaframma per rilasciare il vuoto. Tagliare lungo la nervatura 10 e 12 di asportare il diaframma e ottenere accesso visivo ai polmoni. </li><li> Usare le forbici chirurgiche per tagliare la pelle fino alla trachea sopra la gabbia toracica, ma lascia intatta la gabbia toracica. Separare la pelle dalla cassa toracica. Esporre la trachea rimuovendo il tessuto connettivo circostante, facendo attenzione a non danneggiare la trachea stessa (Figura 1A). </li><li> Snip una piccola apertura di circa 1 mm di diametro nella trachea esposto parallelamente agli anelli cartilaginei, più vicino alla laringe possibile (Figura 1B). Fare attenzione a non tagliare completamente attraverso la trachea. </li><li> Prendere un ago G 20 e inserire delicatamente l&#39;ago mm 4-5 nella trachea, senza alcuna forza contatore (Figura 1D). L&#39;estremità dell&#39;ago deve essere visibile attraverso la trachea (Figura 1C). Utilizzare pinze per stabilizzare l&#39;ago nella trachea. In alternativa, una sutura può essere legato aroUnd la trachea per tenere l&#39;ago in posizione. NOTA: Inserendo troppo profonda, la carena può essere traumatizzato o solo un lato dei polmoni potrebbe essere gonfiato. </li><li> Riempire una siringa con 400 ml di 37 ° C 2% agarosio a bassa temperatura di fusione (presa direttamente da un bagno a temperatura costante). Assicurarsi che la scheda di dissezione è in piedi e infondere lentamente l&#39;agarosio caldo attraverso l&#39;ago nei polmoni, usare ~ 400 microlitri per gonfiare i polmoni. NOTA: Guarda i polmoni gonfiano all&#39;interno della cassa toracica. Non gonfiare eccessivamente il polmone come sarà rottura. </li><li> Una volta che i polmoni sono gonfiati, riempiendo ~ ⅔ della gabbia toracica, staccare la siringa e l&#39;ago mantenere all&#39;interno della trachea per evitare la fuoriuscita di agarosio. </li><li> Versare circa 50 ml di 20 ° C PBS oltre i polmoni gonfiati per consentire l&#39;agarosio all&#39;interno dei polmoni per impostare e solidificare. Lentamente rimuovere l&#39;ago e chiudere la trachea con una pinza per evitare non solidificato agarosio da perdite. &lt;/ Li&gt; <li> Esporre i polmoni eseguendo una sternotomia e successivamente asportare i polmoni. Per escissione dei polmoni, trattenere la trachea durante il taglio attraverso la trachea completamente. Tirare delicatamente la trachea up, tagliare il tessuto connettivo e dell&#39;esofago tirando i polmoni dalla cavità toracica fino polmoni è separato dal mouse (Figura 1E). </li><li> Immergere i polmoni a caldo RPMI-1640 per lavare via il sangue eccessivo e separare delicatamente i lobi utilizzando forbici e pinze per tagliare fusto principale bronco lobi &#39;in ilo (Figura 1F). </li><li> Posizionare i lobi, con la superficie piatta giù per massimizzare la superficie di imaging, in un pozzetto di una piastra di imaging 24 pozzetti (Figura 1G). Aggiungere 100 pl di 37 ° C RPMI-1640 in cima dei lobi. Posizionare diversi 15 mm vetrini di copertura microscopio circolare sulla parte superiore dei lobi per evitare che galleggianti. </li><li> Versare caldo PBS nei pozzetti circostanti per evitare che i media RPMI-1640 da evaporante. Inserire la piastra 24 pozzetti nella camera climatica equilibrato e mantenere i lobi polmonari a 37 ° C con aria e 5% CO 2. Inserire la camera climatica sul palco del microscopio confocale. NOTA: Altre miscele di gas (ad esempio, 5% O 2, 5% CO 2 in N 2 per esaminare il comportamento delle cellule in condizioni di ipossia / ossigeno inferiore) potrebbe anche essere considerato. </li></ol><img alt="Figura 1" src="/files/ftp_upload/53741/53741fig1.jpg" /> Figura 1. Protocollo per la preparazione dei polmoni per l&#39;imaging in tempo reale. (A) esposizione della trachea dopo la preparazione del mouse. (B) Piccola snip fatto nella trachea esposto parallelamente agli anelli cartilaginei. (C) 20 ago G inserita 4-5 mm nella trachea. (D) instillazione di 400 microlitri 2% a bassa temperatura di fusione agarosio nei polmoni. (E) Inflpolmoni ATED separati dal mouse. (F) lobi separati dopo l&#39;inflazione. (G) Lobi posto in un pozzo di una piastra di imaging 24-bene. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53741/53741fig1large.jpg" target="_blank">Fai clic qui per vedere una versione più grande di questa figura.</a> 6. Acquisizione e analisi di immagini NOTA: Le immagini possono essere acquisite con una varietà di filatura del disco microscopio confocale supportati da vari programmi software. In questo protocollo, sia μManager con una filatura disco microscopio confocale misura o Zen con un disco di filatura microscopio confocale disponibile in commercio viene utilizzato per l&#39;acquisizione dell&#39;immagine, mentre Imaris viene utilizzato per l&#39;editing di film e di analisi. <ol><li> Acquisire le immagini utilizzando μManager. Un passo dettagliata da protocollo passo per l&#39;acquisizione di immagini utilizzando il software μManager è descritto in precedenza 18. O </li><li> acquisire le immaginiutilizzando il software di analisi delle immagini, come Zen (vedi Figura S1). <ol><li> Fare clic sulla scheda &#39;Locate&#39; e scegliere obiettivo (10x o 20x) nello strumento di &#39;Light Path&#39; (Figura S1A, scatola rossa). Successivamente, clicca su &#39;Occhi - DAPI&#39; a guardare il canale PCP attraverso l&#39;oculare (Figura S1A, scatola blu). Localizzare il campione manualmente utilizzando il microscopio. Fare clic su &#39;All Off&#39;after il tessuto è centro del campo visivo. </li><li> Fare clic sulla scheda &#39;Acquisizione&#39; per impostare tutti i parametri per l&#39;acquisizione delle immagini. </li><li> Nello strumento di &#39;canali&#39;, fare clic sul pulsante &#39;+&#39; (Figura S1B, scatola rossa). Appare un menu pop-up e cercare il colorante (s) presente nel campione nel &#39;Dye Database&#39; (Figura S1B). Selezionare il colorante e fare clic su &#39;Aggiungi&#39;. NOTA: Il programma impostato tutti i filtri di ottimizzare. Un colorante può essere cancellatoselezionandolo seguito facendo clic sul pulsante del cestino (Figura S1B, scatola gialla). </li><li> Nel menu &#39;Acquisition Mode&#39;, impostare &#39;Binning&#39; a 5x5. Fare doppio clic sul ECFP nel menu canali per selezionarla. Abbassare la potenza del laser al 20% in modo che il campione non verrà sbiancato durante l&#39;impostazione dei parametri di acquisizione delle immagini. </li><li> Selezionare la casella &#39;piastrelle&#39; nella sezione &#39;Experiment Manager&#39; e lo strumento piastrelle compare nel gruppo di strumenti &#39;multidimensionale Acquisition&#39; (Figura S1C). Fare clic sul pulsante &#39;Impostazioni avanzate&#39; per visualizzare l&#39;immagine dal vivo dalla telecamera. Fare clic sul pulsante &#39;Aggiungi&#39; nella sezione &#39;posizioni&#39; aggiungere da 4 a 6 posizioni per l&#39;esperimento. Per cancellare una posizione, selezionare quella posizione e fare clic sul pulsante del cestino. </li><li> Nel gruppo strumento &#39;acquisizione dei parametri&#39;, aprire lo strumento &#39;Strategia Focus&#39;, e selezionare &#39;Absolute fissa Z-position &#39;dal menu a discesa. </li><li> Selezionare la casella Z-Stack nella sezione &#39;Experiment Manager&#39; e lo strumento Z-Stack appare nel gruppo di strumenti del &#39;multidimensionale Acquisition&#39; (Figura S1D). Fare doppio clic su una delle posizioni nella sezione &#39;posizioni&#39; e premere &#39;Live&#39;. Impostare manualmente prima e impostare ultima posizione del campo dell&#39;imaging. Impostare l&#39;intervallo di 4 micron. NOTA: Il programma determina il numero di fette per l&#39;intervallo e l&#39;intervallo selezionato. Idealmente, 5-7 fette sono convenienti per permettere la visualizzazione sufficiente e rapida acquisizione delle immagini. </li><li> Controllare la casella &#39;Time Series&#39; nella sezione &#39;Experiment Manager&#39;. Set desiderato &#39;Durata&#39; e tempi &#39;intervallo&#39; in funzione &#39;Time Series&#39; che è apparso nel gruppo di strumenti &#39;multidimensionale Acquisition&#39; (Figura S1E). </li><li> Nel &#39;acquisiModalità ne &#39;di menu, impostare&#39; Binning &#39;a 2x2. Doppio click su un fluoroforo nel menu canali per selezionare e aumentare la potenza del laser al 100%. Premere &#39;Live&#39; e regolare il &#39;tempo di esposizione&#39;. Ripetere questa operazione per ogni fluoroforo. </li><li> Selezionare la casella &#39;Enable Auto Save&#39;. Selezionare una cartella e digitare il nome del file. Tutte le immagini acquisite verranno salvate automaticamente in questa cartella. </li><li> Fare clic su &#39;Start Experiment&#39; nella sezione &#39;Experiment Manager&#39; per avviare l&#39;acquisizione delle immagini. </li></ol></li><li> Dopo l&#39;acquisizione delle immagini, compilare i dati grezzi nel software Imaris. Convertire immagini in .ims i file e le regolazioni possono essere effettuate. Un passo dettagliata da protocollo passo per la conversione di file, le regolazioni e film risparmio utilizzando Imaris è descritto in precedenza 18. </li><li> Quando si salva il film, impostare la &#39;Frame Rate&#39; a 5 fotogrammi al secondo (fps). </li></ol>

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J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic,+long-term+in+vivo+imaging+of+tumor-stroma+interactions+in+mouse+models+of+breast+cancer+using+spinning-disk+confocal+microscopy.">Dynamic, long-term in vivo imaging of tumor-stroma interactions in mouse models of breast cancer using spinning-disk confocal microscopy.</a> Cold Spring Harb Protoc. (2), (2011).</li><li>Bonnans, C., Lohela, M., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+of+myeloid+cells+dynamics+in+ApcMin/++intestinal+tumors+by+spinning+disk+confocal+microscopy.">Real-time imaging of myeloid cells dynamics in ApcMin/+ intestinal tumors by spinning disk confocal microscopy.</a> J Vis Exp. (92), (2014).</li><li>Nakasone, E. S., Askautrud, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tumor-stroma+interactions+during+chemotherapy+reveals+contributions+of+the+microenvironment+to+resistance.">Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance.</a> Cancer cell. 21 (4), (2012).</li><li>Cheng, N., Lambert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+transplantation+of+stromal+cells+and+carcinoma+cells+in+C57BL/6J+mice.">Mammary transplantation of stromal cells and carcinoma cells in C57BL/6J mice.</a> J Vis Exp. (54), (2011).</li><li>Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., Muschel, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+origin+of+metastasis+from+the+proliferation+of+endothelium-attached+tumor+cells:+a+new+model+for+metastasis.">Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.</a> Nat Med. 6 (1), 100-102 (2000).</li><li>Wong, C. W., Song, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+location+of+breast+cancer+cells+after+spontaneous+metastasis+to+the+lung.">Intravascular location of breast cancer cells after spontaneous metastasis to the lung.</a> Am J Pathol. 161 (3), 749-753 (2002).</li><li>Liang, C. -. C., Park, A. Y., Guan, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat protoc. 2 (2), 329-333 (2007).</li><li>Nelson, K., Bobba, C., Ghadiali, S., Hayes, D. J., Black, S. M., Whitson, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ex+vivo+lung+perfusion+as+a+platform+for+transplantation+research.">Animal models of ex vivo lung perfusion as a platform for transplantation research.</a> World J Exp Med. 4 (2), (2014).</li><li>Magness, S. T., Bataller, R., Yang, L., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+reporter+gene+transgenic+mouse+demonstrates+heterogeneity+in+hepatic+fibrogenic+cell+populations.">A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.</a> Hepatology. 40 (5), 1151-1159 (2004).</li><li>Motoike, T., Loughna, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+GFP+reporter+for+the+study+of+vascular+development.">Universal GFP reporter for the study of vascular development.</a> Genesis. 28 (2), (2000).</li><li>Srivastava, M. K., Andersson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+suppressor+cells+and+immune+modulation+in+lung+cancer.">Myeloid suppressor cells and immune modulation in lung cancer.</a> Immunotherapy. 4 (3), (2012).</li><li>Craig, A., Mai, J., Cai, S., Jeyaseelan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+recruitment+to+the+lungs+during+bacterial+pneumonia.">Neutrophil recruitment to the lungs during bacterial pneumonia.</a> Infect Immun. 77 (2), 568-575 (2009).</li><li>Kreisel, D., Nava, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+imaging+reveals+monocyte-dependent+neutrophil+extravasation+during+pulmonary+inflammation.">In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation.</a> Proc Natl Acad Sci USA. 107 (42), 18073-18078 (2010).</li></ol>

The authors have no conflicts of interest to disclose. All animal experiments were conducted in accordance with IACUC approved protocols, UCSF.

Questo manoscritto descrive un metodo dettagliato per ex vivo l&#39;imaging dal vivo di metastasi polmonare in modelli murini di metastasi. Questo protocollo di imaging fornisce una visualizzazione diretta delle interazioni tumore a cellule-stroma dinamici e spaziali all&#39;interno del microambiente polmonare. Si tratta di un metodo relativamente semplice e veloce che permette l&#39;imaging affidabile di metastasi polmonari per almeno 4 ore. Film acquisiti da questi esperimenti possono essere utilizzati per mo...

The deadliest aspect of cancer is metastasis, which accounts for more than 90% of cancer-related morbidity and mortality1. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular mechanisms that govern metastatic dissemination and growth are still elusive. To metastasize, tumor cells in the primary tumor must detach from their neighboring cells and basement membrane, cross through the extracellular matrix, intravasate, travel via blood or lymphatic vessels, extravasate at the secondary site, and finally, survive and establish secondary tumors. In addition to the properties of the tumor cells, the contribution from the microenvironment, which includes the adjacent stroma along with the normal counterparts of the cancer cells, is crucial for the seeding and establishment of metastatic lesions2.
Traditional methods to study metastatic seeding and growth examine static states, as tissues are excised and sectioned for histology. These data only generate a snapshot of this highly dynamic process. Although some useful information can be gained from these studies, the complicated process by which tumor and stromal cells interact during metastatic formation cannot be adequately assessed by these methods. Furthermore, it is not possible to gain insights into tumor or stromal cell migration patterns, which are important in establishing a colony at the distant site. In order to effectively study the metastatic process, it is essential to visualize various interactions between cancer cells and their microenvironment in a continuous manner and at real time. 
The lung is a common site for metastases from solid tumors as breast, colorectal, pancreatic cancer, melanoma and sarcoma3. Intravital imaging was previously used to study cell-cell interaction in various primary tumor and metastatic models4,5. Methods of lung imaging in mice, including intravital imaging, lung section imaging, and an ex vivo pulmonary metastasis assay have been published6&#8211;9. Intravital imaging of mouse lungs utilizes a thoracic suction window to stabilize the lungs6. This method is used for time-lapse imaging of the lung microcirculation and alveolar spaces. The anatomical location of the lungs poses a challenge to intravital imaging. In order to access the lungs, the chest cavity must be opened which leads to loss of negative pressure and collapsed lungs. This method only allows the visualization of a small part of the lungs and is technically demanding; an unnecessary complication in studies that examine processes that are independent of blood flow. Moreover, this method also requires gating out movement caused by breathing. This is done either by collecting images between breaths or during post image acquisition analyses10. The alternative ex vivo lung section imaging provides stability and depth, and also prepares lung parenchyma for immunostaining7. However, the lengthy sectioning process leads to an extensive delay between the time of animal sacrifice and the start of the imaging session. Moreover, the process of sectioning a mouse lung causes considerable amount of cell death8, thus interfering with the quality and quantity of imaging samples and perhaps needlessly altering tumor-stroma interactions. In order to technically bridge between the methods of intravital imaging and lung section imaging, while exploiting the advantages of the two techniques, a relatively fast and easy method for ex vivo lung imaging was developed. This method was achieved by imaging of non-sectioned whole lung lobes. 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.

<table>
	<tbody>
		<tr>
			<td>MMTV-PyMT/FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>2374</td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>ACTB-ECFP/FVB mice</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>c-fms-EGFP/FVB mice</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>FVB mice</td>
			<td>Jackson Laboratory</td>
			<td>1800</td>
			<td>Female mice</td>
		</tr>
		<tr>
			<td>GFP+ VO-PyMT cells</td>
			<td>UCSF Werb lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70,000 kDa Dextran, rhodamine-conjugated</td>
			<td>Invitrogen</td>
			<td>D1818</td>
			<td>Dilute to&#160; 4mg/ml in 1 x PBS and store at -20&#160; &#176;C. Use 0.4 mg per animal.&#160;</td>
		</tr>
		<tr>
			<td>10,000 kDa Dextran, Alexa Fluor 647 conjugated</td>
			<td>Invitrogen</td>
			<td>D22914</td>
			<td>Dilute to&#160; 4mg/ml in 1 x PBS and store at -20&#160; &#176;C. Use 0.4 mg per animal.&#160;</td>
		</tr>
		<tr>
			<td>Anti-mouse Gr-1 antibody Alexa Fluor 647</td>
			<td>UCSF Monoclonal antibody core</td>
			<td></td>
			<td>Stock 1mg/ml. Use 7 ug per animal.</td>
		</tr>
		<tr>
			<td>Anesthetic</td>
			<td></td>
			<td></td>
			<td>Anesthesia approved by IACUC, used for anesthesia and/or euthanesia</td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>UCSF cell culture facility</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, USP sterile&#160;</td>
			<td>Amresco INC</td>
			<td>K813-500ML</td>
			<td>Ultra pure grade for i.v. injection</td>
		</tr>
		<tr>
			<td>Styrofoam platform</td>
			<td></td>
			<td></td>
			<td>Will be used as dissection board</td>
		</tr>
		<tr>
			<td>Fine scissors sharp&#160;</td>
			<td>Fine Science Tools</td>
			<td>14060-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz Surgical Store</td>
			<td>RS-5135</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>18000-45</td>
			<td>Turn ON 30min before use</td>
		</tr>
		<tr>
			<td>Air</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide</td>
			<td>UCSF</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL syringe without needle&#160;</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>27 G x 1/2 needle&#160;&#160;</td>
			<td>BD</td>
			<td>305109</td>
			<td>for i.v. injection</td>
		</tr>
		<tr>
			<td>20 G x 1 needle, short bevel&#160;&#160;</td>
			<td>BD</td>
			<td>305178</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting-temperature agarose&#160;</td>
			<td>Lonza</td>
			<td>50111</td>
			<td>To make 10 ml of solution, weigh 0.2 g of agarose, add to 10 ml 1 x PBS, and heat to dissolve. Agarose will solidify at room temperature, so maintain in a 37 &#176;C water bath until used for inflation.</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium without phenol red</td>
			<td>Life Technologies</td>
			<td>11835-030</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well Imaging plate&#160;</td>
			<td>E&#38;K scientific</td>
			<td>EK-42892</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slides, 15 mm&#160;</td>
			<td>Fisher Scientific</td>
			<td>22-031-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital CO2 and temperature controller</td>
			<td>Okolab</td>
			<td>DGTCO2BX</td>
			<td>http://www.oko-lab.com</td>
		</tr>
		<tr>
			<td>Climate chamber</td>
			<td>Okolab</td>
			<td></td>
			<td>http://www.oko-lab.com</td>
		</tr>
		<tr>
			<td>Cell Observer spinning disk confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zen software</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Carl Zeiss Inc</td>
			<td>Zeiss Axiovert 200M</td>
			<td></td>
		</tr>
		<tr>
			<td>ICCD camera</td>
			<td>Stanford Photonics</td>
			<td>XR-Mega-10EX S-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk confocal scan-head</td>
			<td>Yokogawa Corporation</td>
			<td>CSU-10b</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mManager</td>
			<td>Vale lab, UCSF</td>
			<td></td>
			<td>Open-source software</td>
		</tr>
	</tbody>
</table>

ex vivo live imaging of lung metastasis and their microenvironment

Metastasi è una delle principali cause di morbilità e mortalità per cancro. Metastasi è un processo a più fasi e causa della sua complessità, le esatte processi cellulari e molecolari che regolano la diffusione e la crescita metastatica sono ancora sfuggente. Immagini dal vivo permette la visualizzazione delle interazioni dinamiche e spaziali delle cellule e il loro microambiente. I tumori solidi comunemente metastasi ai polmoni. Tuttavia, la posizione anatomica dei polmoni rappresenta una sfida per l&#39;imaging intravitale. Questo protocollo fornisce un metodo relativamente semplice e veloce per ex vivo l&#39;imaging dal vivo delle interazioni dinamiche tra le cellule tumorali e la loro stroma circostante all&#39;interno di metastasi polmonari. Utilizzando questo metodo, la motilità delle cellule tumorali, nonché le interazioni tra cellule tumorali e cellule stromali nel loro microambiente possono essere visualizzati in tempo reale per diverse ore. Utilizzando topi transgenici reporter fluorescente, una linea cellulare a fluorescenza, iniettabili fluorescentemolecole e / o anticorpi, componenti multipli del microambiente polmonare possono essere visualizzati, come i vasi sanguigni e cellule immunitarie. Per immagine diversi tipi cellulari, è stato usato un disco rotante microscopio confocale che permette a lungo termine di imaging continuo con acquisizione delle immagini rapida, a quattro colori. filmati time-lapse compilati da immagini raccolte su più posizioni e piani focali mostrano le interazioni tra metastatico dal vivo e le cellule immunitarie per almeno 4 ore. Questa tecnica può essere ulteriormente utilizzato per testare chemioterapia o terapia mirata. Inoltre, questo metodo potrebbe essere adattato per lo studio di altre patologie polmonari correlati che possono influenzare il microambiente polmonare.

Utilizzando la microscopia confocale spinning-disk, i vari sistemi modello mouse e iniettabili, il microambiente metastatico possono essere visualizzati e monitorati nel tempo. L&#39;utilizzo di un MMTV-PyMT; ACTB-ECFP; c-fms-EGFP tripla modello di topo transgenico, diversi componenti cellulari sono fluorescente (Figura 2A, Film 1). La struttura tipica del parenchima polmonare può essere visualizzato nel canale PCP poiché tutte le cellule esprimono ECFP sotto...

Watch this Scientific Journal Video about Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment at JoVE.com

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

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

Descriviamo un metodo relativamente semplice per ex vivo l&#39;imaging dal vivo del tumore interazioni cellula-stroma all&#39;interno di metastasi polmonari, utilizzando i reporter fluorescenti nei topi. Utilizzando la microscopia confocale spinning-disk, questa tecnica consente la visualizzazione di cellule vive per almeno 4 ore e potrebbe essere adattato per studiare altre malattie polmonari infiammatorie.

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

ex-vivo-live-imaging-of-lung-metastasis-and-their-microenvironment

Research

JoVE Journal

Medicine

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

12.3K Views.  University of California.  Descriviamo un metodo relativamente semplice per ex vivo l'imaging dal vivo del tumore interazioni cellula-stroma all'interno di metastasi polmonari, utilizzando i reporter fluorescenti nei topi. Utilizzando la microscopia confocale spinning-disk, questa tecnica consente la visualizzazione di cellule vive per almeno 4 ore e potrebbe essere adattato per studiare altre malattie polmonari infiammatorie. 

Video: Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex infezione Vivo del tessuto Live con virus oncolitici

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Ricerca

Medicina

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 ...

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 ...

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 ...

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

Metodo di isolato<em&gt; Ex Vivo</em&gt; Lung Perfusion in un modello di ratto: Lezioni apprese da Sviluppo di un programma EVLP Rat

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

Validazione di Nanobody e anticorpi Based<em&gt; In Vivo</em&gt; Esperimenti tumore xenotrapianto NIRF imaging nei topi mediante<em&gt; Ex Vivo</em&gt; Citometria di flusso e microscopia

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Tumore primario e isolamento delle cellule MEF per studiare Lung Metastasi

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

Fette polmonari tagliate con precisione come strumento efficiente per studi ex vivo sulla struttura dei vasi polmonari e sulla contrattilità

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.

Colture organotipiche di fette come modelli preclinici del microambiente tumorale nel carcinoma pancreatico primario e nelle metastasi

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Perfusione polmonare ex situ di ventilazione a pressione negativa normothermica: valutazione della funzione polmonare e del metabolismo

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...