Diese Studie ist Teil von DOD Breast Cancer IDEA Award unterstützt W81XWH-08-1 bis 0583 und NIH / NCI CA141348-01A1 (DZ) und FAMRI klinischen Scientist Award (DS). Imaging-Infrastruktur wird durch Southwestern Small Animal Imaging Research Program zum Teil durch U24 CA126608 und Simmons Cancer Center (P30 CA142543) und NIH 1S10RR024757-01 unterstützt werden.

Alle tierischen Verfahren wurden vom Institutional Animal Care und Verwenden Ausschuss der University of Texas Southwestern Medical Center genehmigt. 1. In-vitro-HIF-1α Biolumineszenzassay <ol><li> Materialien und Methoden: <ul><li> Menschliche metastasierendem Brustkrebs-Zelllinie MDA-MB231 mit einem neuartigen HIF-1-abhängigen Reportergen transfiziert wurde 5HRE-ODD-luc durch Dr. Harada generiert. </li><li> In hypoxischen Zustand ist die verstärkte Expression von Sauerstoff-abhängigen Abbau Domain (ODD)-Luciferase-Fusionsprotein von 5 Exemplare von Hypoxie-Response-Element (5HRE) angetrieben. Die Anwesenheit von ODD bewirkt, dass der Abbau von ODD-Luc resultierende Protein in extrem niedrigen Hintergrund Luciferaseaktivität in normoxischen Bedingungen. Daher kann dieses neue System verwendet, um hypoxische Regionen eines Tumors durch Echtzeit-Bildgebung zu erkennen. Der Bau dieser 5HRE-ODD-luc Expressionsvektor wurde von Harada et al 1,2 berichtet worden. </li></ul></li><li> Kultur-Zellen unter Normoxie oder Hypoxie: <ul><li> Pflegen Sie die rekombinante MDA-MB231-Zellen in 10% fötalem Rinderserum (FBS)-DMEM-Medium mit 1% Glutamin, Antibiotika von 400 pg / ml G418 und 1% Penicillin / Streptomycin. </li><li> Für die in vitro HIF-1α Biolumineszenzassay, Platte 3 x 10 5 MDA-MB231-Zellen, die 5HRE-ODD-luc-Vektor in jede Vertiefung von zwei sechs gut Schüssel. </li><li> Lassen Sie Zellen in die Schale Wand nach Inkubation über Nacht befestigen und dann übertragen ein Gericht in eine Hypoxie Kammer (Billups-Rothenberg, Inc. Del Mar, CA) für Hypoxie-Studien, während die übrigen Gericht unter normoxischen Bedingungen (21% O 2). </li><li> Montieren Sie die Kammer und Gas der Kammer mit 0,1% O 2, indem Sie den Einlass-Schlauch mit einer Gasflasche. Hinweis: Sowohl Ein-und Auslass-Anschluss Klemmen muss während dieser Verhandlung zu eröffnen. </li><li> Trennen Gasquelle nach Kammer wurde gespült und Dichtungsraum durch Schließen Kunststoffklammern. </li><li> Setzen Sie die Kammer in einem 37 ° C Inkubator mit 5% CO 2. Hinweis: Die Kammer muss befeuchtet werden, um übermäßige Verdunstung der Kulturen zu verhindern. 20 ml steriles Wasser in die Kammer - Dies kann, indem 10 erreicht werden. </li></ul></li><li> Biolumineszenz-Assay: <ul><li> Nach 24 Stunden Inkubationszeit, entfernen Sie das Medium und die Zellen schnell mit eiskaltem PBS (2X). </li><li> 1 ml kaltem PBS mit 100 ul Luciferin (Gold Biotechnologie, St Louis, MO). </li><li> Erwerben BL-Tomographie (IVIS Spectrum System Caliper Life Sciences, Hopkinton, MA) mit verschiedenen Belichtungszeiten (1, 30, 60, 180 s). </li><li> Messen Sie die Lichtintensität in jedem gut mit dem Leben Image Software (Caliper Life Sciences). </li></ul></li></ol> 2. Einrichtung einer Brustkrebs Hirnmetastasen Modell <ol><li> Vorbereitung der MDA-MB231/5HRE-ODD-luc Zellen <ul><li> Abrufen und Kultur der MDA-MB231/5HRE-ODD-luc Zellen in DEME-Medium mit 10% FBS, 1% Glutamin und 1% Penicillin / Streptomycin. </li><li> Ersetzen Medium alle 2-3 Tage. Tripsinize und waschen Sie die Zellen, wenn sie 80% Konfluenz erreichen. </li><li> Graf entsprechende Anzahl von Zellen und resuspendieren sie in serumfreien Medium DEME mit 25% Matrigel (BD Biosciences, San Jose, CA) mit einer Endkonzentration von 10 5 Zellen in 4 ul Volumen. </li><li> Legen Zellen auf Eis vor intrakranielle Injektion. </li></ul></li><li> Chirurgische Implantation <ul><li> Weiblicher Akt Mäusen (BALB / c nu / nu; National Cancer Institute, Bethesda, MD) bei 4-6 Wochen alt sind in dieser Studie verwendet. </li><li> Anesthetize (3% Isofluran / O2 in einer Induktion Kammer; Isofluran von Baxter International Inc., Deerfield, IL, USA) und pflegen die Tiere mit Isofluran (1%) in Sauerstoff (1 dm 3 / min) während des chirurgischen Verfahren 3. </li><li> Die rechte parietal Haut sollte mit betadine und dann 70% igem Alkohol vor Einschnitt vorbereitet werden. </li><li> Mit einem High-Speed-Bohrer, Fräse ein 1 mm Loch in der rechten Hemisphäre des Schädels, 1mm anterior der Kranznaht und 2 mm lateral saggital Naht. </li><li> Draw 4 ul Zellgemisch (10 5 Zellen) mit einem 10 ul Hamilton-Spritze (Hamilton Company, Reno, NV). Spritzen Sie die Zellen direkt in die rechte kaudale Zwischenhirn, 1,5 mm unter der Dura mater mit einem maßgeschneiderten 32G Hamilton Nadel. Halten Sie die Nadel in Platz für ca. 30 s vor dem Rückzug. Die Nutzung eines 32G feinen Nadel minimiert Gewebeschäden. </li><li> Füllen Sie das Bohrloch mit Knochenwachs und in der Nähe der Kopfhaut mit resorbierbaren Fäden. </li><li> Bereiten Sie den Schnitt Region mit 70% Alkohol. </li><li> Bewerben Buprenorphin Analgesie alle 12 Stunden für zwei Tage. </li></ul></li></ol> 3. In-vivo-Biolumineszenz-Bildgebung von Tumorhypoxie Dynamik bei Brustkrebs Hirnmetastasen <ul><li> Initiieren Längs-Biolumineszenz-Bildgebung (IVIS Spectrum System) zwei Wochen nach intrakraniellen Implantation und wiederholen Sie einmal in der Woche für 8-10 Wochen. </li><li> Anesthetize drei Mäusen zu einer Zeit (3% Isofluran / O 2 in einer Induktion Kammer) </li><li> Verwalten einer Lösung von D-Luciferin (120 mg / kg in PBS in einem Gesamtvolumen von 80 ul; Gold-Biotechnologie) subkutan im Halsbereich jeder Maus, wie im Detail beschrieben bisher 4. </li></ul> D-Luciferin ist ungiftig und hat sich gezeigt, dass zu durchdringen vermag intakte Blut-Hirn-Schranke (BHS) und Zellmembranen 3,5. <ul><li> Legen Sie die 3 Mäuse in der Imaging-Kammer und gepflegt mit Isofluran (1%) in Sauerstoff (1 dm 3 / min) während der Bildgebung. </li><li> Fünf Minuten nach Injektion Luciferin, erwerben BL Bildgebung mit einer Reihe von verschiedenen Belichtungszeiten (1, 30, 60, 180 s). </li></ul> Unsere Beobachtungen zeigen, dass die Peak-Lichtemission ca. 5 Minuten nach subkutaner Verabreichung von Luciferin im Halsbereich 3,4 ist. <ul><li> Analysieren von Daten mit dem lebendigen Imaging-Software (Caliper Life Sciences), indem Sie absolute Photonen (Photonen / s) in einer Region of Interest (ROI), manuell gezogen, um die BLI-Signal des Gehirns zu skizzieren. </li><li> Plot Zeitverlauf Kurve der Photonen, um anzuzeigen, Tumorhypoxie Dynamik. </li><li> Unmittelbar nach dem letzten BLI, verwalten pimonidazole, die Hypoxie-Marker und 1 Stunde später, opfern die Mäuse und sezieren die Gehirne. Embed das ganze Gehirn in Optimal Cutting Temperature (OCT) mittel-und frieren in -80 ° C Gefrierschrank. Nachfolgende histologische Kresylviolett-Färbung und die immunhistochemische Färbung gegen Luciferase, Hypoxie-Marker pimonidazole und HIF-1α durchgeführt, um Imaging-Beobachtungen bestätigen. </li></ul> 4. Repräsentative Ergebnisse: Wie in Abb. 1 dargestellt, war signifikant höher BLI-Signal nach der transfizierten Zellen in der hypoxischen Kammer (1% O 2) für 24 Stunden inkubiert beobachtet. Schwache Lichtemission aus dem Kontroll-Zellen unter Normoxie beobachtet. Dies kann aus der überfüllten Zelle Bevölkerung nach 24 Stunden Kultur der 3 x 10 5 Zellen, die einen Stress-Signal zu den Zellen induziert HIF-1α überexprimieren führen. Dennoch wurden in vivo BLI Ergebnisse durch immunhistochemische Färbung zeigt Kolokalisation zwischen Tumor Hypoxie und Luciferaseexpression validiert. Eine automatisierte Auswahl an Belichtungszeiten ermöglicht eine kontinuierliche Akquisitionen von einer Reihe von Bildern, die Erfassung von einem schwachen Signal erleichtert durch längere Belichtungszeit, und starke Signale mit einer kürzeren Zeit ohne Sättigung des CCD. <img alt="Abbildung 1" src="/files/ftp_upload/3175/3175fig1.jpg" /> Abbildung 1 In-vitro-HIF-1α Biolumineszenzassay Obere Reihe:. 3 x 10 5 MDA-MB231/5HRE-ODD-luc Zellen in jeder Vertiefung einer 6-well-Schale in einer Hypoxie Kammer (0,1% O 2) für 24 inkubiert hr, bevor das Medium entfernt, gewaschen und ersetzt mit 1 ml PBS. Unmittelbar nach 100 ul Luciferin in jedes Well zugegeben wurde BLI mit einer Reihe von Belichtungszeiten (1, 30, 60, 180 s) erworben. Starke Lumineszenz wurde von repräsentativen Brunnen beobachtet Untere Reihe:. Als eine Kontrolle, 3 x 10 5 Zellen unter Normoxie (21% O 2) emittiert schwachem Licht inkubiert. <img alt="Abbildung 2" src="/files/ftp_upload/3175/3175fig2.jpg" /> Abbildung 2 In-vivo-Biolumineszenz-Bildgebung von Tumorhypoxie Dynamik. A) Eine schwache Lichtsignal von der rechten Maus-Gehirn wurde erstmals visualisiert 5 Wochen nach der intrakraniellen Implantation MDA-MB231/5HRE-ODD-luc Brustkrebszellen. Erhöhte optisches Signal wurde über weitere 6 Wochen beobachtet, was auf erhöhte Tumor-Hypoxie. B) Die Handlung des zeitlichen Verlaufs Kurve quantitative Photonen des Lichts Signal zeigten. <img alt="Abbildung 3" src="/files/ftp_upload/3175/3175fig3.jpg" /> Abbildung 3 Colocaliztion von Luciferase und Hypoxie durch immunhistochemische Färbung nachgewiesen. Eine gefrorene Gehirn der Maus trägt eine Metastase des Brustkrebses MDA-MB231/5HRE-ODD-luc in OCT eingebettet wurde geschnitten. A 10 pm Abschnitt immungefärbt mit hypoxischen Marker, pimonidazole zeigte intratumoralen Heterogenität der Hypoxie, die gefunden räumlich korrelieren mit Luciferase-Expression durch anti-Luciferase Färbung erkannt wurde. Scale-bar, 100 um.

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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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Brustkrebs Hirnmetastasen tritt in 30% der Patientinnen mit Brustkrebs im Stadium IV. Es ist mit hoher Morbidität und Mortalität verbunden und hat eine mediane Überlebenszeit von 13 Monaten 6. Es besteht ein Bedarf an geeigneten Tiermodellen für diese klinisch verheerenden Krankheit vortäuschen, um unser Verständnis der intrakraniellen Initiation und Progression sowie pathophysiologischen Profilen zu erleichtern. Hier haben wir einen orthotopen Brustkrebs Hirnmetastasen Modell durch die Injektion von me...

<table border="1"><tbody><tr><th> Name des Reagenzes </th><th> Firma </th><th> Katalog-Nummer </th><th> Kommentare (optional) </th></tr><tr><td> D-Luciferin </td><td> Gold-Biotechnologie </td><td> L-123 </td><td> 120 mg / kg in PBS in einem Gesamtvolumen von 80 ul in-vivo-Studie </td></tr><tr><td> Isofluran </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-Spritze </td><td> Hamilton Company </td><td> 1701 </td><td></td></tr><tr><td> 32G Hamilton Nadel </td><td> Hamilton Company </td><td> 7803-04 </td><td></td></tr><tr><td> Hypoxie Kammer </td><td> Billups-Rothenberg, Inc. </td><td> MIC-101 </td><td></td></tr><tr><td> Biolumineszenz Imaging-System </td><td> Caliper Life Sciences </td><td> IVIS Spectrum-System </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

Es ist allgemein anerkannt, dass Tumor-Hypoxie eine wichtige Rolle bei der Förderung der malignen Progression und beeinflussen therapeutische Reaktion negativ spielt. Es gibt wenig Wissen über in situ, in vivo, Tumor-Hypoxie während intrakranieller Entwicklung von bösartigen Hirntumoren wegen des Mangels an effizienten Mitteln, um es in diesen tief sitzenden Tumoren orthotopen überwachen. Biolumineszenz-Bildgebung (BLI), auf der Detektion von Licht durch lebende Zellen, die ein Luciferase-Gen emittiert basiert, wurde schnell für die Krebsforschung hat, insbesondere, um das Tumorwachstum oder Tumorgröße Veränderungen in Reaktion auf die Behandlung in präklinischen Tierstudien zu bewerten. Darüber hinaus durch die Expression eines Reportergens unter der Kontrolle einer Promotorsequenz kann die Genexpression nicht-invasiv durch BLI überwacht werden. Unter hypoxischen Stress, signalisieren Reaktionen hauptsächlich über die Hypoxie-induzierbaren Faktors-1α (HIF-1α) vermittelt, um die Transkription verschiedener Gene zu fahren. Deshalb haben wir eine HIF-1α Reporter-Konstrukt, 5HRE-ODD-luc, stabil in menschlichen Brustkrebszellen MDA-MB231-Zellen (MDA-MB231/5HRE-ODD-luc). In-vitro-HIF-1α Biolumineszenzassay ist durchgeführt transfizierten verwendet Inkubation der transfizierten Zellen in einer hypoxischen Kammer (0,1% O 2) für 24 Stunden vor dem BLI, während die Zellen in Normoxie (21% O 2) als Kontrolle dienen. Deutlich höhere Photonenfluss für die Zellen unter Hypoxie beobachtet schlägt eine erhöhte HIF-1α Bindung an seinen Promotor (HRE Elemente), wie diese in Normoxie verglichen. Die Zellen werden direkt in das Gehirn der Maus injiziert, um eine Brustkrebs Hirnmetastasen Modell zu etablieren. In-vivo-Biolumineszenz-Bildgebung von Tumorhypoxie Dynamik ausgelöst wird 2 Wochen nach der Implantation und wiederholte einmal pro Woche. BLI zeigt zunehmende Licht Signale aus dem Gehirn, da der Tumor fortschreitet, was darauf hinweist erhöhten intrakraniellen Tumor Hypoxie. Histologische und immunhistochemische Untersuchungen werden verwendet, um die in-vivo-Bildgebung Ergebnisse zu bestätigen. Hier werden wir Ansätze von In-vitro-HIF-1α Biolumineszenzassay, chirurgische Einrichtung einer Brustkrebs Hirnmetastasen in einer Nacktmaus und Anwendung von In-vivo-Biolumineszenz-Bildgebung zur intrakraniellen Tumorhypoxie Monitor vorstellen.

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

Biolumineszenz-Bildgebung des Hypoxie-induzierbaren Faktors-1α Aktivität wird angewandt, um intrakranielle Tumorhypoxie Entwicklung in einer Brustkrebs Hirnmetastasen Mausmodell zu überwachen.

In-vivo-Biolumineszenz-Bildgebung von Tumorhypoxie Dynamics of Breast Cancer Hirnmetastasen in einem Mausmodell

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

in-vivo-bioluminescence-imaging-tumor-hypoxia-dynamics-breast-cancer

Research

JoVE Journal

Medicine

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

19.5K Aufrufe.  University of Texas Southwestern Medical Center. Biolumineszenz-Bildgebung des Hypoxie-induzierbaren Faktors-1α Aktivität wird angewandt, um intrakranielle Tumorhypoxie Entwicklung in einer Brustkrebs Hirnmetastasen Mausmodell zu überwachen.

Serie: 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-Photonen-Imaging der Tumorzellinvasion in einem orthotopen Mausmodell des oralen Plattenepithelkarzinoms

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

Forschung

Medizin

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.

Kombination Strahlentherapie in einem orthotopen Maus Brain Tumor-Modell

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.

Stereotaktische Intrakranielle Implantation und<em&gt; In vivo</em&gt; Bioluminescent Imaging der Tumorxenotransplantaten in einem Maus-Modell-System des 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.

Multimodale Bildgebung von Angiogenese in einem Nude Rattenmodell der Brustkrebs Knochenmetastasen mittels Magnetresonanztomographie, volumetrische Computertomographie und Ultraschall

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.

Live-Imaging of Drug Responses in der Tumor-Mikroumgebung in Mausmodellen von Brustkrebs

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.

Zeitraffer-Imaging of Primary Präkanzeröse Brustepithelzellen aus gentechnisch veränderten Mausmodellen der Breast Cancer Abgeleitet

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.

A Novel Hochauflösende<em&gt; In vivo</em&gt; Bildtechnik, um die Dynamik der intrakranielle Strukturen zu Tumorwachstum und Therapeutics Studieren

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.

Ultraschall-Imaging-guided Intra Injection zur Entwicklung eines Mausmodells von Brustkrebs Hirnmetastasen durch Längs MRI Gefolgt

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.

Initiation Metastasierende Mammakarzinom durch Targeting des duktalen Epithel mit Adenovirus-Cre: Eine neue transgene Maus-Modell der Brustkrebs

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 Optical Imaging von Hirntumoren und Arthritis mit fluoreszierenden SapC-DOPS Nanovesicles

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