Name: in vivo bioluminescent imaging of mammary tumors using ivis spectrum
Uploaded: 2009-04-29T00:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum at JoVE.com

Eine Vielzahl von Luciferase exprimierenden Zelllinien können für die präklinische Forschung in Maus-Modellen verwendet werden. Diese Zellen werden als pathogen-freien gefrorenen Kultur, die sich leicht in Standard-Medien wachsen, ohne dass für Selektionsmarker zur Verfügung gestellt. Für unser Experiment verwenden wir die 4T1-luc2 murine Brusttumor-Zelllinie, die das Luciferase-Gen, das als eine optische Anzeige der Genexpression oder Tumorgenese in vivo dient zum Ausdruck bringt. Wir verwenden Luciferaseexpression zum Wachstum des Primärtumors nicht-invasiv Spur, aber sie können auch verwendet werden, zu lokalisieren und zu überwachen metastatischen Läsionen werden. Luciferaseaktivität überprüft werden müssen vor der Injektion, und um dies tun zu können, ist eine 90% konfluent Kolben durch Trypsinierung geerntet und dann gezählt. 50.000 Zellen werden dann in einer einzigen Vertiefung einer Mikrotiterplatte und Verdünnungsreihen verzichtet werden durchgeführt. Luciferin wird in die Vertiefungen an 150ug pro ml zugegeben und für 2 Minuten. Die Mikrotiterplatte kann in IVIS oder einer lumineszierenden Platte Leser Expression bestimmen abgebildet werden. Diese Zellinie exprimiert bis 6500 Photonen pro Sekunde pro Zelle, aber jeder Ausdruck Ebene oberhalb von 500 Photonen pro Sekunde pro Zelle können erfolgreich in vivo dargestellt werden. Jetzt, da wir Zellen optimale Aktivität haben, können wir auf die subkutane Injektion Schritt fort. Um eine optimale Erkennung des Tumors zu erleichtern, werden wir die Verwendung eines athymischen immungeschwächten Nacktmaus Belastung. Vor der Injektion werden die Tiere betäubt, um für tiefe Betäubung Wirkung. Als nächstes werden wir spritzen bis zu 250.000 Zellen in 100 ul PBS subkutan in die Flanke injiziert. Laden Sie die Zellen in einer 1 ml Spritze eine 26 Gauge Nadel. Heben Sie die Haut vorsichtig mit einer Pinzette ein &quot;Zelt&quot; zu machen und injizieren die Zellen an der Basis. Die neu injizierten Zellen können sofort abgebildet werden. In jedem Imaging-Sitzung insgesamt 150 mg Luciferin pro kg Körpergewicht wird dann über zwei Injektionen in die Bauchhöhle verabreicht. In dieser Studie werden die Tiere 10 Minuten nach Injektion Luciferin abgebildet, um konsistente Photonenfluss zu gewährleisten. Wir zeigen Ihnen, diese im nächsten Schritt. Luciferin Kinetik von Tag zu Tag variieren. In diesem Modell, Messung 10 Minuten nach Injektion Luciferin gab ein Ergebnis innerhalb eines Bereiches von 15% Variabilität. Für unser Experiment werden wir die IVIS Spectrum in vivo Imaging-System verwenden wird ein Back-ausgedünnt charge coupled device gekühlt auf -90 ° C bis maximale Empfindlichkeit zu erreichen. Zur Unterstützung der absolute Quantifizierung, misst das System dunklen Ladung während down-Zeit und führt eine Selbstkalibrierung während der Initialisierung. So starten Bildgebung, initialisieren Sie die IVIS System (ein Klick) und stellen Sie die Imaging-Parameter für das Experiment. Wählen Sichtfeld für die Anzahl der Tiere abgebildet. Bis zu 5 Tiere können in das Gerät mit dem integrierten Anästhesie vielfältig gepflegt werden. Die Bühne ist mit einer konstanten 37 ° C auf Körpertemperatur der Tiere aufrecht zu erhalten. Ein EKG-Anschluss ist vorgesehen, um die Tiergesundheit in stressigen Verfahren zu überwachen. In Living Image Software, Belichtungszeit, Blende und Pixel-Binning können basierend auf der Expression der Zelllinie optimiert werden. Diese Einstellungen können jederzeit während eines Experiments werden ohne Auswirkungen auf das quantitative Ergebnis verändert. IVIS erwirbt ein fotografisches Bild des Tieres unter weißem Licht und einer quantitativen Biolumineszenz oder Fluoreszenz-Signal, das auf dem Bild überlagert ist. Die Biolumineszenz-Signal wird in Photonen pro Sekunde und als einer Intensität Karte. Die Bildanzeige wird angepasst, um optimale Kontrast und Auflösung des Bildes, ohne die Quantifizierung bieten. Lumineszenz aus den Zellen können an der Injektionsstelle mit einem region of interest-Tool gemessen werden. Die Messdaten werden in der Tabelle zusammen mit allen experimentellen Parameter im Zusammenhang mit der Aufnahme von Bildern, die gespeichert oder exportiert werden kann für die Analyse angezeigt Mehrere Bilder können erworben werden und im Vergleich in Längsschnittstudien für Sekunden oder Monaten, je nach der Art des Experiments. Wir werden Photonenfluss aus dem Tumor zu messen zum Zeitpunkt Null und Monitor für 4 Wochen mit Bildgebung bei bi-wöchentlichen Abständen. Photonenfluss aus dem Tumor ist proportional zur Anzahl der lebenden Zellen, die Luciferase so Biolumineszenz korreliert direkt mit der Tumorgröße. Bei 5 Tage nach der Implantation des Tumors ist noch nicht greifbar, aber die Zellen können durch Biolumineszenz und der Tumor gesehen werden aktiv wachsenden quantifiziert werden. In diesem Stadium der Biolumineszenz-Signal ist viel stronger. Die Belichtungszeit, Blende und Pixel-Binning kann so eingestellt, dass das Bild klar ist und die Kamera nicht zu sättigen. IVIS kompensiert automatisch die Änderungen im Lichte Sammlung, so dass diese Messungen mit denen früher und später im Experiment gesammelten verglichen werden kann. An 7 Tagen nach der Implantation Tumoren sind spürbar zum ersten Mal und Biolumineszenz-Messung bereits 7 Tage der Daten erzeugt hat. Nach 28 Tagen nach der Implantation, sind Tumoren es nekrotisch und Zellen beginnen zu sterben. Tumorgröße durch Dickenmessung geschätzt nicht merklich verändert, aber Lumineszenz aus dem Tumor verringert Angabe Zelltod. Caliper Messungen und Biolumineszenz-Messung kann fortgesetzt werden, bis eine humane Endpunkt erreicht ist. Tumor-Nekrose durch Hypoxie oder Behandlungen wird durch reduzierte Biolumineszenz angegeben werden, auch wenn sie reduzieren nicht die Tumormasse.

Ich möchte versichern, dass alle Experimente durchgeführt, um die Zeitschrift Visualisierte Experimente Artikel produzieren im Einklang waren mit dem Tier-Protokoll 060 vom Institutional Animal Care genehmigt durchgeführt und Verwenden Committee (IACUC) bei Caliper Life Sciences, Alameda CA Jae Kim, Ph . D., IACUC Chair, Caliper Life Sciences.

in vivo bioluminescent imaging of mammary tumors using ivis spectrum

4T1 Maus Mamma Tumorzellen subkutan in nu / nu-Mäusen implantiert werden, um tastbare Tumoren in 15 bis 20 Tage bilden. Diese Xenograft-Tumor-Modell ist für die präklinische in vivo Evaluierung von mutmaßlichen Antitumorverbindungen wertvoll. Die 4T1-Zelllinie wurde entwickelt, um konstitutiv die Glühwürmchen-Luciferase-Gen (luc2). Wenn Mäuse mit 4T1-luc2 Tumoren sind mit Luciferin die Tumoren strahlen eine visuelle Lichtsignal, überwacht mit Hilfe eines empfindlichen optischen Imaging-System wie die IVIS Spectrum kann injiziert. Der Photonenfluss aus dem Tumor ist proportional zur Anzahl der lichtemittierenden Zellen und das Signal kann gemessen werden, um das Tumorwachstum und die Entwicklung zu überwachen. IVIS kalibriert ist, damit absolute Quantifizierung der Biolumineszenz-Signal und Längsschnittstudien über viele Monate und über mehrere Aufträge von Signalgröße kann, ohne dabei das quantitative Ergebnis durchgeführt werden. Das Tumorwachstum kann für mehrere Tage durch Biolumineszenz beobachtet werden, bevor die Tumorgröße wird spürbar oder messbar traditionellen physikalischen Mitteln. Diese schnelle Überwachung kann Einblick in frühe Ereignisse in der Tumor-Entwicklung bereitzustellen oder führen zu kürzeren experimentellen Verfahren. Tumor Zelltod und Nekrose durch Hypoxie oder medikamentöse Behandlung ist eine frühzeitige durch eine Reduktion der Biolumineszenz-Signal angezeigt. Das Zelltod möglicherweise nicht durch eine Reduktion der Tumorgröße von materiellen Mitteln gemessen begleitet werden. Die Fähigkeit zur frühen Ereignisse in Tumor-Nekrose-sehen hat erhebliche Auswirkungen auf die Auswahl und Entwicklung von Therapeutika. Quantitative Bildgebung des Tumorwachstums mit IVIS bietet eine präzise Quantifizierung und beschleunigt den experimentellen Prozess, um Ergebnisse zu erzielen.

Watch this Scientific Journal Video about In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum at JoVE.com

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Brusttumor-Zellen, die Luciferase werden subkutan in Mäuse implantiert und visualisiert mit Hilfe der optischen Bildgebung, das Tumorwachstum und die Entwicklung nicht-invasiv in einer Längsschnittstudie zu überwachen.

In vivo Biolumineszenz Imaging von Brusttumoren mit IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

in-vivo-bioluminescent-imaging-of-mammary-tumors-using-ivis-spectrum

Research

JoVE Journal

Biology

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

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Serie: In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Gründung Intrakranielle Brain Tumor Xenografts mit anschließender Analyse des Tumorwachstums und Ansprechen auf die Therapie mit Biolumineszenz-Bildgebung

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Forschung

Neurowissenschaften

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Überwachung Tumormetastasen und Osteolysen mit Biolumineszenz und Micro-CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Medizin

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

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

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

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

In-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 Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo Dual-Substrat Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

Intrakranielle Implantation mit anschließender 3D In Vivo Bioluminescent Imaging von Murine Gliome

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Bakterielle Imaging<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Immunologie und Infektion

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Überwachung des Brustkrebswachstums und der metastasierenden Koloniebildung bei Mäusen mittels Biolumineszenz

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Krebsforschung

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

<meta charset="utf8"></head><body>In-vivo-Bildgebung zur Messung spontaner Lungenmetastasen von orthotopisch injizierten Brusttumorzellen

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Im lebenden Organismus Bildgebung zur Messung der spontanen Lungenmetastasierung von orthotopisch injizierten Brusttumorzellen

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Kombiniert<em&gt; In-vivo-</em&gt; Optische und μCT Imaging-Infektion, Entzündung und Knochenanatomie in einem Orthopädie Implantat-Infektion in Mäuse Monitor

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

In vivo Biolumineszenz Imaging von Brusttumoren mit IVIS Spectrum

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