Este estudo é apoiado em parte pelo DOD Prêmio IDEA Câncer de Mama W81XWH-08-1-0583 e NIH / NCI CA141348-01A1 (DZ) e FAMRI prêmio cientista clínico (DS). Infra-estrutura de imagem é fornecido pelo Southwestern Programa de Pequenas Pesquisa Animal de imagem suportados em parte pela U24 CA126608 e Câncer Simmons Center (P30 CA142543) e NIH 1S10RR024757-01.

Todos os procedimentos com animais foram aprovados pelo Institutional Animal Care e do Comitê Uso de University of Texas Southwestern Medical Center. 1. In vitro ensaio HIF-1α bioluminescência <ol><li> Materiais e Métodos: <ul><li> Humana de mama metastático linhagem de células MDA-MB231 câncer transfectadas com um gene repórter romance HIF-1-dependente, 5HRE-ODD-luc foi gerado pelo Dr. Harada. </li><li> Em condições de hipóxia, a expressão aumentada de oxigênio-dependente de domínio degradação (ODD)-luciferase proteína de fusão é impulsionado por 5 cópias de hipóxia-resposta elemento (5HRE). A presença de ODD causas da degradação de ODD-Luc proteína, resultando em muito baixa atividade de fundo luciferase em condições normóxica. Portanto, este novo sistema pode ser usado para detectar regiões hipóxica em um tumor por imagem em tempo real. A construção deste vetor de expressão 5HRE-ODD-luc tem sido relatada por 1,2 Harada et al. </li></ul></li><li> Células de cultura em normóxia ou hipóxia: <ul><li> Manter o MDA-MB231 recombinante células em 10% de soro fetal bovino (FBS), DMEM contendo 1% de glutamina, antibióticos de 400 mcg / ml de G418 e 1% de penicilina / estreptomicina. </li><li> Para o ensaio in vitro HIF-1α bioluminescência, placa de 3 x 10 5 MDA-MB231 células que expressam 5HRE-ODD-luc vetor em cada poço de dois prato seis também. </li><li> Permitem que as células para anexar o prato de parede após incubação overnight e depois transferir um prato em uma câmara de hipóxia (Billups-Rothenberg, Inc. Del Mar, CA) para estudos de hipóxia, enquanto que manter o outro prato sob condição normóxica (21% O 2). </li><li> Remontar a câmara ea câmara de gás com 0,1% do O 2, ligando o tubo porta de entrada com um cilindro de gás. Nota: Ambos entrada e abraçadeiras porta de saída devem ser abertas durante este procedimento. </li><li> Desconecte a fonte de gás após a câmara foi purgado e câmara de vedação, fechando braçadeiras de plástico. </li><li> Coloque a câmara em uma incubadora de 37 ° C com 5% de CO 2. Nota: A câmara deve ser umidificado para evitar a evaporação excessiva de culturas. Isso pode ser feito colocando 10-20 ml de água estéril na câmara. </li></ul></li><li> Ensaio de bioluminescência: <ul><li> Depois de 24 hr de incubação, remova o meio e lavar as células rapidamente com gelado PBS (2X). </li><li> Adicionar 1 ml de PBS frio com 100 l de luciferina (Gold Biotecnologia, St Louis, MO). </li><li> Adquirir BL imagem (IVIS sistema Spectrum, Caliper Life Sciences, Hopkinton, MA) com vários tempos de exposição (1, 30, 60, 180 s). </li><li> Medir a intensidade de luz em cada poço usando o software Image Living (Caliper Life Sciences). </li></ul></li></ol> 2. Estabelecimento de um modelo de metástase de câncer de mama cérebro <ol><li> Preparação das células MDA-MB231/5HRE-ODD-luc <ul><li> Recuperar e cultura as células MDA-MB231/5HRE-ODD-luc em meio DEME contendo 10% FBS, 1 glutamina% e 1% de penicilina / estreptomicina. </li><li> Substituir média a cada 2-3 dias. Tripsinize e lavar as células quando atingirem confluência de 80%. </li><li> Contar o número adequado de células e ressuspender-los em meio isento de soro DEME com Matrigel 25% (BD Biosciences, San Jose, CA) com uma concentração final de 10 5 células em 4 mL de volume. </li><li> Células colocar no gelo antes da injeção intracraniana. </li></ul></li><li> Implantação cirúrgica <ul><li> Feminino camundongos nude (BALB / c nu / nu; National Cancer Institute, Bethesda, MD), 4-6 semanas de idade são usadas neste estudo. </li><li> Anestesiar (3% isoflurano / O2 em uma câmara de indução; isoflurano da Baxter International Inc., Deerfield, IL, EUA) e manter os animais com isoflurano (1%) em oxigênio (1 dm 3 / min) durante o procedimento cirúrgico 3. </li><li> A pele parietal direito deve ser preparado com betadine e depois álcool 70% antes da incisão. </li><li> Utilizando uma broca de alta velocidade, burr um buraco de 1 mm no hemisfério direito do crânio, 1mm anterior à sutura coronal e 2 mm lateral à sutura sagital. </li><li> Draw 4 mistura de células mL (10 5 células) usando um seringa 10 ml Hamilton (Hamilton Company, Reno, NV). Injetar as células diretamente no direito caudal diencéfalo, 1,5 mm abaixo da dura-máter usando um custom-made agulha 32G Hamilton. Mantenha a agulha no local por cerca de 30 s antes da retirada. Uso de uma agulha fina 32G minimiza dano tecidual. </li><li> Preencher o buraco com cera de osso rebarbas e fechar o couro cabeludo com suturas absorvíveis. </li><li> Preparar a região da incisão com álcool a 70%. </li><li> Aplicar buprenorfina analgesia a cada 12 horas durante dois dias. </li></ul></li></ol> 3. Na imagem de bioluminescência in vivo de tumor na mama dinâmica hipóxia metástase do câncer de cérebro <ul><li> Iniciado imagem de bioluminescência longitudinal (IVIS sistema Spectrum) duas semanas após o implante intracraniana e repetir uma vez por semana durante 8-10 Semanas. </li><li> Anestesiar três ratos em um momento (3% isoflurano / O 2 em uma câmara de indução) </li><li> Administrar uma solução de D-luciferina (120 mg / kg em PBS em um volume total de 80 mL; Biotecnologia Gold) por via subcutânea na região do pescoço de cada rato, como descrito em detalhes anteriormente 4. </li></ul> D-luciferina não é tóxico e tem se mostrado capaz de penetrar barreira hemato-encefálica intacta (BBB) ​​e membranas celulares 3,5. <ul><li> Coloque os 3 ratos na câmara de imagem e mantida com isoflurano (1%) em oxigênio (1 dm 3 / min) durante o exame. </li><li> Cinco minutos após a injeção de luciferina, adquirir BL imagem com uma matriz de tempo de exposição diversas (1, 30, 60, 180 s). </li></ul> Nossas observações mostram que o tempo de emissão de pico de luz é de cerca de 5 minutos após a administração subcutânea de luciferina na região do pescoço 3,4. <ul><li> Analisar os dados com o software de imagem Living (Caliper Life Sciences) usando contagem absoluta de fótons (fótons / s) em uma região de interesse (ROI), manualmente desenhados para traçar o sinal BLI do cérebro. </li><li> Plot curva curso de tempo de contagem de fótons para indicar a dinâmica hipóxia tumor. </li><li> Imediatamente após a última BLI, administrar pimonidazole, o marcador de hipóxia e 1 hr depois, o sacrifício dos camundongos e dissecar o cérebro. Incorporar o cérebro inteiro na temperatura ideal de corte (OCT) de médio e congelar em freezer -80 ° C. Coloração violeta cresil histológicas subseqüentes e coloração imuno-histoquímica contra a luciferase, hipóxia marcador pimonidazole e HIF-1α realizada para validar observações de imagem. </li></ul> 4. Resultados representativos: Como mostrado na Fig.1, significativamente maior sinal de BLI foi observada após as células transfectadas incubados na câmara hipóxica (1% O 2) durante 24 horas. Emissão de luz fraca foi observada a partir de células controle sob normóxia. Isto pode resultar da população cela superlotada após 24 h de cultura 3 x 10 5 células, o que induziu um sinal de estresse para as células a HIF-1α superexpressam. No entanto, os resultados in vivo BLI foram validados por coloração imuno-histoquímica mostrando co-localização entre hipóxia tumor e expressão luciferase. Um array automatizada de tempos de exposição permite aquisições contínua de uma série de imagens, o que facilita a captura de um sinal fraco, com maior tempo de exposição, e sinais de forte usando um tempo mais curto sem saturar o CCD. <img alt="Figura 1" src="/files/ftp_upload/3175/3175fig1.jpg" /> Figura 1 ensaio in vitro HIF-1α bioluminescência linha Top:. 3 x 10 5 células MDA-MB231/5HRE-ODD-luc incubadas em cada poço de uma 6-bem-prato em uma câmara de hipóxia (0,1% O 2) para 24 hr antes do meio foi retirado, lavado, substituído com 1 ml de PBS. Imediatamente após 100 mL luciferina foi adicionado em cada poço, BLI foi adquirido com uma série de tempos de exposição (1, 30, 60, 180 s). Luminescência forte foi observada a partir de poços representante Linha inferior:. Como controle, 3 x 10 5 células incubados em normóxia (21% O 2) a luz emitida fraco. <img alt="Figura 2" src="/files/ftp_upload/3175/3175fig2.jpg" /> Figura 2 Na imagem de bioluminescência vivo da dinâmica hipóxia tumor. A) Um sinal de luz fraca do lado direito do cérebro do rato foi visualizada 5 semanas após o implante de células de câncer intracraniano MDA-MB231/5HRE-ODD-luc mama. Aumento do sinal óptico foi observada ao longo de seis semanas adicionais, indicando hipóxia tumor aumentou. B) O enredo mostrou a curva de evolução no tempo da contagem quantitativa fóton de sinal luminoso. <img alt="Figura 3" src="/files/ftp_upload/3175/3175fig3.jpg" /> Colocaliztion figura 3 do luciferase e hipóxia detectados pela imuno-químico de coloração. Um cérebro de camundongo congelado tendo uma metástase de câncer de mama MDA-MB231/5HRE-ODD-luc embutidos em outubro foi seccionado. Uma seção de 10 mM imunocoradas com encefalopatia hipóxico marcador, pimonidazole, revelou heterogeneidade intratumoral de hipóxia, que foi encontrado para correlacionar espacialmente com expressão luciferase detectado pelo anti-luciferase coloração. Barra de escala, 100 mm.

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De mama câncer de metástase cerebral ocorre em 30% dos pacientes com câncer de mama no estágio IV. Ela está associada com alta morbidade e mortalidade, com sobrevida mediana de 13 meses 6. Há uma necessidade de ter modelos animais apropriados para imitar esta doença clinicamente devastadora, a fim de facilitar a nossa compreensão da sua iniciação intracraniana e progressão, bem como perfis fisiopatológicos. Aqui, temos desenvolvido um modelo ortotópico de câncer de mama metástase cerebral atrav...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> D-luciferina </td><td> Biotecnologia ouro </td><td> L-123 </td><td> 120 mg / kg em PBS em um volume total de 80 mL para estudo 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 seringa </td><td> Empresa Hamilton </td><td> 1701 </td><td></td></tr><tr><td> Hamilton 32G agulha </td><td> Empresa Hamilton </td><td> 7803-04 </td><td></td></tr><tr><td> Câmara de hipóxia </td><td> Billups-Rothenberg, Inc. </td><td> MIC-101 </td><td></td></tr><tr><td> Sistema de imagem de bioluminescência </td><td> Caliper Life Sciences </td><td> IVIS sistema Spectrum </td><td></td></tr><tr><td> G418 </td><td> Fisher científica </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

É bem reconhecido que a hipóxia tumor desempenha um papel importante na promoção da progressão maligna e afetando negativamente a resposta terapêutica. Há pouco conhecimento sobre in situ, em hipóxia, vivo tumor intracraniano durante o desenvolvimento de tumores cerebrais malignos por causa da falta de meios eficientes para monitorá-lo nestes tumores profundos ortotópico. Imagem de bioluminescência (BLI), baseado na detecção de luz emitida por células vivas expressando um gene da luciferase, foi rapidamente adotado para a investigação do cancro, em particular, para avaliar o crescimento do tumor ou mudanças tamanho do tumor em resposta ao tratamento em estudos com animais pré-clínicos. Além disso, ao expressar um gene repórter sob o controle de uma seqüência promotora, a expressão de genes específicos podem ser monitorados de forma não invasiva por BLI. Sob estresse hipóxico, as respostas de sinalização são mediadas principalmente através da indução de hipóxia factor-1α (HIF-1α) para dirigir a transcrição de vários genes. Portanto, temos utilizado uma construção repórter HIF-1α, 5HRE-ODD-luc, estavelmente transfectadas em câncer de mama humano MDA-MB231 células (MDA-MB231/5HRE-ODD-luc). In vitro ensaio HIF-1α bioluminescência é realizada por incubando as células transfectadas em uma câmara de hipóxia (0,1% O 2) por 24 horas antes de BLI, enquanto as células em normóxia (21% O 2) servir como controle. Significativamente maior fluxo de fótons observados para as células em hipóxia sugere uma ligação de HIF-1α aumentou para seu promotor (elementos HRE), em comparação com aqueles em normóxia. As células são injetadas diretamente no cérebro de camundongo para estabelecer um modelo de câncer de mama metástase cerebral. Na imagem de bioluminescência vivo da dinâmica hipóxia tumor é iniciado 2 semanas após o implante e repetido uma vez por semana. BLI revela aumento sinais de luz a partir do cérebro como o tumor progride, indicando aumento da hipóxia tumor intracraniano. Estudos histológicos e imunohistoquímicos são usados ​​para confirmar a no resultado de imagem in vivo. Aqui, vamos introduzir abordagens de ensaio in vitro HIF-1α bioluminescência, criação cirúrgica de uma metástase cerebral de câncer de mama em camundongos nude e aplicação de imagem de bioluminescência em vivo para monitorar hipóxia tumor intracraniano.

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

Imagem de bioluminescência de hipóxia induzida a atividade do fator-1α é aplicada para monitorar o desenvolvimento hipóxia intracraniana tumor em um câncer de mama modelo de cérebro de rato metástase.

Na imagem de bioluminescência vivo Tumor de mama Dynamics Hipóxia metástase do cancro cerebral em um modelo de rato

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 visualizações.  University of Texas Southwestern Medical Center. Imagem de bioluminescência de hipóxia induzida a atividade do fator-1α é aplicada para monitorar o desenvolvimento hipóxia intracraniana tumor em um câncer de mama modelo de cérebro de rato metástase.

Vídeo: 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-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

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

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.

Radioterapia em uma combinação ortotópico modelo de tumor de cérebro de camundongos

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.

Implantação estereotáxica intracraniana e<em&gt; Em vivo</em&gt; Imaging Bioluminescent de xenoenxertos de tumores em um Sistema Modelo de Rato 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 de imagem de angiogênese em um modelo de rato nu de Câncer de mama utilizando a ressonância magnética, tomografia computadorizada volumétrica e ultra-som

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.

Viver de imagem de Respostas de drogas no microambiente tumoral em modelos do rato do cancro da mama

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.

Lapso de tempo de imagem de pré-neoplásicas primárias células epiteliais mamárias derivados de modelos de rato geneticamente modificado do cancro da mama

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 de alta resolução<em&gt; In vivo</em&gt; Técnica de Imagem para estudar a resposta dinâmica de estruturas intracranianas ao crescimento do tumor e Terapêutica

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.

Ecografia-guiada injeção intracardíaca para desenvolver um modelo do rato de mama metástases do cancro cerebral Seguido por 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 ...

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.

Início do Carcinoma de mama metastático por Segmentação do ductal Epithelium com Adenovírus-Cre: Um Modelo Novel de camundongo transgênico do Câncer de Mama

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 Imagem Óptico de tumores cerebrais e artrite Usando fluorescente SAPC-DOPS nanovesículas

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