Questo studio è sostenuto in parte dal premio DOD IDEA Breast Cancer W81XWH-08-1-0583 e NIH / NCI CA141348-01A1 (DZ) e FAMRI premio scienziato clinico (DS). Infrastruttura di imaging è fornito da sud-ovest di piccola Imaging Research Program animali realizzata in collaborazione con U24 CA126608 e Simmons Cancer Center (P30 CA142543) e NIH 1S10RR024757-01.

Tutte le procedure di animali sono stati approvati dalla cura degli animali e del Comitato Istituzionale uso della University of Texas Southwestern Medical Center. 1. In vitro HIF-1α saggio di bioluminescenza <ol><li> Materiali e metodi: <ul><li> Umano tumore metastatico al seno linea di cellule MDA-MB231 transfettate con un romanzo HIF-1-dipendente gene reporter, 5HRE-ODD-luc è stata generata dal Dr. Harada. </li><li> In condizioni di ipossia, l&#39;espressione di ossigeno-dipendente di degradazione del dominio (ODD)-luciferasi proteina di fusione è guidata da 5 copie di ipossia-risposta elemento (5HRE). La presenza di ODD provoca la degradazione di ODD-Luc proteina con conseguente estremamente bassa attività luciferasi sfondo in condizioni di normossia. Pertanto, questo nuovo sistema può essere utilizzato per rilevare le regioni ipossiche in un tumore da imaging in tempo reale. La costruzione di questo 5HRE-ODD-luc vettore di espressione è stata riportata da 1,2 Harada et al. </li></ul></li><li> Cellule in coltura normossia o ipossia: <ul><li> Mantenere la ricombinante cellule MDA-MB231 nel 10% siero fetale bovino (FBS)-DMEM medio contenente 1% glutamina, antibiotici di 400 mg / ml di G418 e 1% di penicillina / streptomicina. </li><li> In vitro per la HIF-1α saggio bioluminescenza, piatto 3 x 10 5 MDA-MB231 cellule che esprimono 5HRE-ODD-luc vettore in ciascun pozzetto di due sei piatto bene. </li><li> Consentono alle cellule per attaccare la parete piatto dopo incubazione durante la notte e poi trasferire un piatto in una camera di ipossia (Billups-Rothenberg, Inc. Del Mar, CA) per gli studi di ipossia, mentre tenere il piatto di altri in condizioni di normossia (21% O 2). </li><li> Rimontare la camera e la camera a gas con 0,1% O 2, collegando il tubo di ingresso porta con una bombola di gas. Nota: sia in ingresso e morsetti porta di uscita deve essere aperto durante questa procedura. </li><li> Scollegare fonte di gas dopo della camera è stato eliminato e la camera di tenuta chiudendo fascette di plastica. </li><li> Posizionare la camera in un incubatore a 37 ° C con 5% di CO 2. Nota: La camera deve essere umidificata per evitare l&#39;eccessiva evaporazione delle culture. Questo può essere ottenuto mettendo 10-20 ml di acqua sterile nella camera. </li></ul></li><li> Bioluminescenza saggio: <ul><li> Dopo 24 ore di incubazione, rimuovere il terreno e lavare le cellule in modo rapido con ghiaccio PBS freddo (2X). </li><li> Aggiungere 1 ml di PBS freddo con 100 ml di Luciferin (Biotecnologie Oro, St Louis, MO). </li><li> BL acquisire immagini (sistema Spectrum IVIS, Caliper Life Sciences, Hopkinton, MA) con tempi di esposizione diversi (1, 30, 60, 180 s). </li><li> Misura l&#39;intensità della luce in ciascun pozzetto utilizzando il software immagine vivente (Caliper Life Sciences). </li></ul></li></ol> 2. Istituzione di un modello di metastasi al seno cancro al cervello <ol><li> Preparazione delle cellule MDA-MB231/5HRE-ODD-luc <ul><li> Recuperare e coltivare le cellule MDA-MB231/5HRE-ODD-luc nel medio DEME contenente 10% FBS, 1% glutamina e 1% di penicillina / streptomicina. </li><li> Sostituire media ogni 2-3 giorni. Tripsinize e lavare le cellule quando raggiungono l&#39;80% confluenza. </li><li> Contare un numero adeguato di cellule e risospendere in siero medio DEME libero con il 25% Matrigel (BD Biosciences, San Jose, CA) con una concentrazione finale di 10 5 cellule in 4 volumi microlitri. </li><li> Cellule posto sul ghiaccio prima dell&#39;iniezione intracranica. </li></ul></li><li> Impianto chirurgico <ul><li> Topi nudi femminili (BALB / c nu / nu; National Cancer Institute, Bethesda, MD) a 4-6 settimane di età sono utilizzati in questo studio. </li><li> Anestetizzare (3% isoflurano / O2 in una camera di induzione; isoflurano da Baxter International Inc., Deerfield, IL, USA) e mantenere gli animali con isoflurano (1%) in ossigeno (1 dm 3 / min) durante la procedura chirurgica 3. </li><li> La pelle parietale destro deve essere preparata con Betadine e poi il 70% di alcol prima di incisione. </li><li> Utilizzando un trapano ad alta velocità, bava di 1 mm foro nell&#39;emisfero destro del cranio, 1 mm anteriore alla sutura coronale e 2 mm laterali di sutura sagittale. </li><li> Draw 4 miscela microlitri di cellule (10 5 cellule) con un 10 microlitri siringa Hamilton (Hamilton Company, Reno, NV). Iniettare le cellule direttamente nel diritto caudale diencefalo, 1,5 mm sotto la dura madre con una misura 32G ago Hamilton. Tenere l&#39;ago in posizione per circa 30 s prima del ritiro. L&#39;utilizzo di un ago 32G sottile minimizza i danni ai tessuti. </li><li> Riempire il foro con della cera bava osso e chiudere il cuoio capelluto con punti di sutura riassorbibili. </li><li> Preparare la regione incisione con il 70% di alcol. </li><li> Applicare l&#39;analgesia buprenorfina ogni 12 ore per due giorni. </li></ul></li></ol> 3. Nell&#39;imaging bioluminescenza in vivo del tumore dinamiche ipossia in metastasi cerebrali tumore al seno <ul><li> Avviare l&#39;imaging longitudinale bioluminescenza (sistema Spectrum IVIS) due settimane dopo l&#39;impianto intracranica e ripetere una volta a settimana per 8-10 Settimane. </li><li> Anestetizzare tre topi in un periodo (3% isofluorano / O 2 in una camera di induzione) </li><li> Somministrare una soluzione di D-luciferina (120 mg / kg in PBS in un volume totale di 80 microlitri; Biotecnologie Oro) per via sottocutanea nella regione del collo di ogni mouse come descritto in dettaglio in precedenza 4. </li></ul> D-luciferina non è tossico ed ha dimostrato di essere in grado di penetrare intatta barriera emato-encefalica (BBB) ​​e le membrane delle cellule 3,5. <ul><li> Mettere i 3 topi in camera di imaging e mantenuti con isoflurano (1%) in ossigeno (1 dm 3 / min) durante l&#39;imaging. </li><li> Cinque minuti dopo l&#39;iniezione luciferina, BL acquisire immagini con una gamma di tempo di esposizione diversi (1, 30, 60, 180 s). </li></ul> Le nostre osservazioni mostrano che il tempo di picco di emissione di luce è di circa 5 minuti dopo la somministrazione sottocutanea di luciferina nella regione del collo 3,4. <ul><li> Analizzare i dati con il software Imaging Living (Caliper Life Sciences) utilizzando conta assoluta dei fotoni (fotoni / s) in una regione di interesse (ROI), disegnata a mano a delineare il segnale BLI del cervello. </li><li> Tempo curva trama corso della conta fotone per indicare la dinamica ipossia tumorale. </li><li> Immediatamente dopo l&#39;ultima BLI, amministrare pimonidazole, il marcatore ipossia e 1 ora dopo, il sacrificio dei topi e sezionare il cervello. Incorpora il cervello tutto in temperatura ottimale di taglio (OCT), medie e congelare in freezer -80 ° C. Successiva colorazione istologica violetto cresolo e colorazione immunoistochimica contro la luciferasi, marcatore pimonidazole ipossia, e HIF-1α eseguita per convalidare le osservazioni di imaging. </li></ul> 4. Rappresentante dei risultati: Come mostrato in Fig.1, significativamente più alto segnale BLI è stata osservata dopo che le cellule transfettate incubate nella camera di ipossia (1% O 2) per 24 ore. Emissione di luce debole è stata osservata da parte delle cellule di controllo in normossia. Ciò può derivare da popolazione di cellule sovraffollata cultura dopo 24 ore di 3 x 10 5 cellule, che ha indotto un segnale di stress per le cellule di iperespressione di HIF-1α. Tuttavia, in vivo BLI risultati sono stati convalidati dalla colorazione immunoistochimica mostrando colocalizzazione tra ipossia tumorale ed espressione luciferasi. Un array automatico dei tempi di esposizione permette di acquisizioni continue di una serie di immagini, che facilita la cattura di un segnale debole con il tempo di esposizione più lunghi e forti segnali con un tempo più breve senza saturare il CCD. <img alt="Figura 1" src="/files/ftp_upload/3175/3175fig1.jpg" /> Figura 1 in vitro HIF-1α saggio bioluminescenza Top riga:. 3 x 10 5 cellule MDA-MB231/5HRE-ODD-luc incubate in ciascun pozzetto di una ben-6-piatto in una camera di ipossia (0,1% O 2) per 24 ora prima del mezzo è stato rimosso, lavato e riutilizzato con 1 ml di PBS. Immediatamente dopo 100 luciferina microlitri è stato aggiunto in ciascun pozzetto, BLI è stata acquisita con una serie di tempi di esposizione (1, 30, 60, 180 s). Luminescenza forte è stata osservata da pozzi rappresentante fila in basso:. Come controllo, 3 x 10 5 cellule incubate sotto normossia (21% O 2) emessa luce debole. <img alt="Figura 2" src="/files/ftp_upload/3175/3175fig2.jpg" /> Figura 2 Nell&#39;imaging bioluminescenza in vivo delle dinamiche di ipossia tumorale. A) Un segnale debole luce dalla parte destra del cervello di topo è stato visualizzato 5 settimane dopo l&#39;impianto di cellule intracranica MDA-MB231/5HRE-ODD-luc cancro al seno. Aumento segnale ottico è stato osservato per altre 6 settimane, che indica l&#39;ipossia tumore aumentato. B) La trama ha mostrato la curva di durata di quantitative conta fotone di segnale luminoso. <img alt="Figura 3" src="/files/ftp_upload/3175/3175fig3.jpg" /> Figura 3 Colocaliztion della luciferasi e ipossia rilevati da immunohisto-chimici della colorazione. Un cervello di topo congelato recanti una metastasi del cancro al seno MDA-MB231/5HRE-ODD-luc incorporato in ottobre è stato sezionato. A 10 micron sezione immunostained con pennarello ipossia, pimonidazole, ha rivelato l&#39;eterogeneità intratumorale di ipossia, che è stato trovato di correlare spazialmente con l&#39;espressione luciferasi rilevato da anti-luciferasi colorazione. Scala grafica, 100 micron.

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	<li>Harada, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combination+of+hypoxia-response+enhancers+and+an+oxygen-dependent+proteolytic+motif+enables+real-time+imaging+of+absolute+HIF-1+activity+in+tumor+xenografts.">The combination of hypoxia-response enhancers and an oxygen-dependent proteolytic motif enables real-time imaging of absolute HIF-1 activity in tumor xenografts.</a> Biochem Biophys Res Commun. 360, 791-796 (2007).</li><li>Ou, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+HIF-1+imaging+for+determining+optimal+timing+of+combining+bevacizumab+and+radiotherapy.">Usefulness of HIF-1 imaging for determining optimal timing of combining bevacizumab and radiotherapy.</a> Int J Radiat Oncol Biol Phys. 75, 463-467 (2009).</li><li>Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, e8051-e8051 (2009).</li><li>Contero, A., Richer, E., Gondim, A., Mason, R. 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Metastasi del cancro al seno del cervello si verifica nel 30% dei pazienti con carcinoma mammario in stadio IV. E &#39;associata ad alta morbilità e mortalità ed ha una sopravvivenza mediana di 13 mesi 6. Vi è la necessità di avere modelli animali appropriati per simulare questa malattia clinicamente devastante al fine di facilitare la comprensione della sua iniziazione e la progressione intracranica e profili fisiopatologiche. Qui, abbiamo sviluppato un modello ortotopico tumore al seno metastasi al cerv...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </th></tr><tr><td> D-luciferina </td><td> Oro Biotecnologie </td><td> L-123 </td><td> 120 mg / kg in PBS in un volume totale di 80 microlitri per studio in vivo </td></tr><tr><td> Isoflurano </td><td> Baxter International Inc. </td><td> 1001936060 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td></td></tr><tr><td> Hamilton siringa </td><td> Hamilton Azienda </td><td> 1701 </td><td></td></tr><tr><td> 32G Hamilton ago </td><td> Hamilton Azienda </td><td> 7803-04 </td><td></td></tr><tr><td> Camera di ipossia </td><td> Billups-Rothenberg, Inc. </td><td> MIC-101 </td><td></td></tr><tr><td> Bioluminescenza sistema di imaging </td><td> Caliper Life Sciences </td><td> IVIS Spectrum sistema </td><td></td></tr><tr><td> G418 </td><td> Fisher Scientific </td><td> SV3006901 </td><td></td></tr></tbody></table>

in vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model

È ben noto che l&#39;ipossia tumorale svolge un ruolo importante nel promuovere la progressione maligna e colpisce negativamente la risposta terapeutica. C&#39;è poca conoscenza in situ, in vivo, ipossia tumorale durante lo sviluppo cerebrale dei tumori maligni al cervello a causa della mancanza di mezzi efficaci per il monitoraggio in questi tumori ortotopico profonde. Di imaging bioluminescenza (BLI), basato sul rilevamento della luce emessa dalle cellule viventi che esprimono un gene luciferasi, è stato rapidamente adottato per la ricerca sul cancro, in particolare, per valutare la crescita del tumore o cambiamenti dimensioni del tumore in risposta al trattamento negli studi su animali preclinici. Inoltre, esprimendo un gene reporter sotto il controllo di una sequenza promotore, l&#39;espressione del gene specifico può essere monitorata in modo non invasivo da BLI. Sotto stress ipossico, le risposte di segnalazione sono mediati principalmente attraverso il fattore ipossia-inducibile 1α (HIF-1α) a guidare la trascrizione di vari geni. Pertanto, abbiamo utilizzato un HIF-1α costruire giornalista, 5HRE-ODD-luc, stabilmente transfettate in cancro mammario umano MDA-MB231 cellule (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1α saggio bioluminescenza è eseguita da incubando le cellule transfettate in una camera di ipossia (0,1% O 2) per 24 ore prima di BLI, mentre le cellule in normossia (21% O 2) Come controllo. Significativamente più alto flusso di fotoni osservati per le cellule in ipossia suggerisce un aumento di HIF-1α vincolante per i suoi promotori (elementi HRE), rispetto a quelli normossia. Le cellule vengono iniettate direttamente nel cervello di topo per stabilire un modello di cancro al seno metastasi al cervello. Nell&#39;imaging bioluminescenza in vivo delle dinamiche ipossia tumore è iniziato 2 settimane dopo l&#39;impianto e ripetute una volta alla settimana. BLI rivela sempre più segnali di luce dal cervello come il tumore progredisce, indicando un aumento ipossia intracranica del tumore. Studi istologici ed immunoistochimici sono utilizzati per confermare i risultati di imaging in vivo. Qui, introdurremo approcci in vitro di HIF-1α saggio bioluminescenza, creazione chirurgica di una metastasi cerebrali tumore al seno in un topo nudo e in applicazione della bioluminescenza di imaging in vivo di monitorare ipossia intracranica del tumore.

Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

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

L&#39;imaging bioluminescenza di ipossia-inducibile fattore 1α attività è applicata a monitorare lo sviluppo del tumore ipossia cerebrale in un modello di cancro al seno del mouse metastasi al cervello.

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

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

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Research

JoVE Journal

Medicine

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

19.5K Views.  University of Texas Southwestern Medical Center. L'imaging bioluminescenza di ipossia-inducibile fattore 1α attività è applicata a monitorare lo sviluppo del tumore ipossia cerebrale in un modello di cancro al seno del mouse metastasi al cervello.

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Ricerca

Medicina

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

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

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

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

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

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Stereotassica impianto intracranico e<em&gt; In vivo</em&gt; Imaging Bioluminescent di xenotrapianti di tumori in un sistema modello del mouse di Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

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

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

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

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

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

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 ...

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

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

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

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

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

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Un romanzo ad alta risoluzione<em&gt; In vivo</em&gt; Tecnica di imaging per studiare la risposta dinamica di strutture intracraniche alla crescita del tumore e Terapeutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Ultrasound Imaging guidata intracardiaca iniezione di sviluppare un modello murino di Breast Cancer metastasi cerebrali Seguito da longitudinale MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

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

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

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

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

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

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imaging ottico dei tumori cerebrali e artrite Uso fluorescente SAPC-DOPS nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...