Somos gratos aos drs. Diane Palmieri e Patricia Steeg de NCI por nos fornecer as células MDA-MB231Br. Agradecemos ao Dr. Ralph Mason, Sr. Jason Reneau ea Sra. Ramona Lopes para o suporte técnico e colegial. Este trabalho foi financiado em parte pelo IDEA DOD Prêmios W81XWH-08-1-0583 e W81XWH-12-1-0317. Experimentos de ressonância magnética foram realizados no Centro de Pesquisa de Imagem avançada, uma facilidade NIH BTRP # P41-RR02584 e injeção intracardíaca guiada por ultra-som foi realizada com VisualSonics Vevo 770 sob 1S10RR02564801.

Todos os procedimentos com animais foram aprovados pelo Comitê de University of Texas Southwestern Medical Center Animal Care Institucional e uso. 1. Preparação das Células MDA-MB231/Br-GFP <ol><li> Recuperar e cultura das células MDA-MB231/Br-GFP (gentilmente cedidas pelos Drs. Palmieri e Steeg, NCI) em meio DMEM contendo 10% de FBS, 1% de glutamina e 1% de penicilina / estreptomicina. </li><li> Observar o estado das células e cor de meio, e substituir meio cada 2-3 dias. </li><li> Tripsinizar e recolher as células, quando 80% de confluência é alcançado como descrito nas etapas 1.3.1-1.3.5: <ol><li> Remover o meio antigo por completo e adicione 5 ml de PBS (1x) para lavar as células suavemente. Remover o PBS a partir do prato. </li><li> Adicionar 1,5 ml de tripsina no prato; inclinar o prato suavemente para assegurar que todas as células são cobertas por tripsina. Coloque o prato de volta na incubadora celular e mantê-la a 37 ° C durante 1 min. </li><li> Leve o prato de incubadora e observaras células em microscópio óptico para assegurar que as células separar do prato. </li><li> Adicionar 3 ml de meio para parar o efeito de tripsina. Recolhe-se a mistura de células num tubo de centrífuga. Centrifugar a mistura a 2000 rpm durante 5 min. </li><li> Retire cuidadosamente o meio e ressuspender as células em 5 ml de soro meio livre até ficar homogéneo. </li></ol></li><li> Contar o número apropriado de células e ressuspender-los em meio DMEM isento de soro com uma concentração final de 1,75 x 10 5 células em 100 uL de volume. <ol><li> Tome 50 mistura de células mL e adicionar 50 ul Trypan Blue. Após a mistura, levar de 10 mL mistura e adicione cuidadosamente para o slide contagem das células. Use várias amostras para garantir a estimativa do número de células precisa. </li><li> Fique no slide contagem de células, uma extremidade de cada vez eo contador de células começa a contar automaticamente. Tome média de todos os resultados de contagem e calcular o número total de células. </li><li> Centrifugar as células novamente e ressuspender-los emo volume apropriado de meio isento de soro para resultar na concentração final de 1,75 x 10 5 células/100 uL de volume. </li></ol></li><li> Colocar as células em gelo antes da injecção intracardíaca. </li></ol> 2. Ecografia-guiada injeção intracardíaca <ol><li> Use camundongos nu feminino (BALB / c nu / nu) entre 6-8 semanas de idade. </li><li> Entrar no sistema de imagem. Inicializar o transdutor 704 (40 MHz). </li><li> Iniciar um novo estudo e preencher as informações. </li><li> Anestesiar (3% isoflurane/100% O 2 em uma câmara de indução) e manter os animais com isofluorano (2%) em 100% de O2 (1 dm 3 / min) durante todo o processo por meio de um cone de nariz. A anestesia é confirmado quando há reflexo de retirada é observado com toe pitada. </li><li> Regule a temperatura da tabela de imagem de 37 ° C. Tape o rato anestesiado com a tabela de imagem aquecido em posição supina. </li><li> Manter o gel de ultra-sons a 37 ° C antes de imenvelhecimento. Aplique o gel para o peito do mouse. </li><li> Monte o transdutor no suporte. Diminuir o transdutor até a profundidade da imagem desejada seja atingida. Mover a etapa até que o ventrículo esquerdo é identificado com a aorta ascendente, como o ponto de referência (Figura 1A). Bloquear o estágio em que uma visão clara do ventrículo esquerdo é visualizado. </li><li> Desenhar 100 ul de mistura de células para uma seringa de 1 ml com uma agulha G 22. Coloque e fixar a seringa na seringa de montagem. </li><li> Mova a seringa para a frente em direção ao peito do mouse e mova-o com cuidado lado para o outro até que a ponta da agulha está no campo de imagem de visão (antes de entrar no mouse). </li><li> Ajuste a altura da agulha e ângulo para apontar para baixo do ventrículo esquerdo. </li><li> Penetrar a agulha da seringa no espaço intercostal prontamente através de camadas da pele e do músculo para o ventrículo esquerdo, sob a orientação de ultra-sonografia. Uma indicação de uma inserção bem sucedida da agulha no interior do ventrículo esquerdocle é o refluxo do sangue arterial fresco (cor-de-rosa, em contraste com o sangue venoso vermelho escuro) com a seringa </li><li> Injectar a mistura de células lentamente (Figura 1B). </li><li> Após a conclusão, retirar a agulha, levante o transdutor, limpe o gel de ultra-som com gaze umedecida e remova a fita. </li><li> Prepare uma gaiola limpa com um bloco pré-aquecido. </li><li> Colocar o animal na almofada e observar o animal até a recuperação total. </li><li> Monitorar o comportamento do animal a cada 24 horas, por dois dias. </li><li> Condições gerais e sinais neurológicos de complicações em ratinhos experimentais são rotineiramente monitorados. </li></ol> 3. Monitoramento MRI de desenvolvimento do tumor intracraniano <ol><li> Use um ímã 9,4 Tesla para monitorar o desenvolvimento intracraniana de metástases cerebrais. </li><li> Iniciado MRI duas semanas após a implantação do tumor e repita uma vez por semana por até três semanas. </li><li> Sedar os animais com 3% de isoflurano e mantê-los sob geneanestesia geral (1,5% de isoflurano). </li><li> Monitorar e manter a temperatura do corpo do animal e constante da respiração durante todo o experimento. </li><li> Multislice de alta resolução (14 fatias com 1 mm de espessura, sem intervalo) T 1 - T 2 e ponderada imagens coronais, que abrange a região do lobo frontal para a fossa posterior, são adquiridos com os seguintes parâmetros: 1-T imagens ponderadas: girar eco fatia múltipla (SEMS), TR / TE = 400 ms msec/20, matriz: 256 x 256, FOV 20 x 20 mm, na resolução do plano: 78 x 78 mM 2. T imagens 2-ponderada: rotação rápida eco fatia múltipla (FSEMS) seqüências, TR / TE = 2500 msec/48 ms, 8 trens eco, matriz: 256 x 256, FOV 20 x 20 mm, na resolução do plano: 78 x 78 ^ M 28,9. Lesões tumorais parecem mais brilhantes do que os tecidos normais do cérebro em t imagens 2-ponderados. </li><li> O tamanho do tumor é determinado em t imagens 2-ponderados por delinear manualmente o acesciamento porção da massa em cada imagem usando programas MATLAB escritos por nós 8. Dado a maior parte do tumor diâmetros são menores do que a espessura da fatia (1 mm), o tamanho do tumor apresenta-se como na área do plano, em vez de o volume. </li></ol> 4. H &amp; E coloração Confirmando as metástases <ol><li> Sacrificar os ratos imediatamente após a última MR varredura, dissecar os cérebros portadores de tumor e incorporá-lo em meio outubro e congelado em -80 ° C. </li><li> Seção de uma série de 10 m de espessura cérebro coronal amostras com criostato. </li><li> Realize H &amp; E coloração nas seções do cérebro. </li></ol>

<ol>
	<li>Schouten, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94, 2698-2705 (2002).</li><li>Lin, N. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J. Clin. Oncol. 22, 3608-3617 (2004).</li><li>Eichler, A. 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Cancer Res. 16, 5664-5678 (2010).</li><li>Yoneda, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bone-seeking+clone+exhibits+different+biological+properties+from+the+MDA-MB-231+parental+human+breast+cancer+cells+and+a+brain-seeking+clone+in+vivo+and+in.">A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in.</a> 16, 1486-1495 (2001).</li><li>Palmieri, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Her-2+overexpression+increases+the+metastatic+outgrowth+of+breast+cancer+cells+in+the+brain.">Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.</a> Cancer Res. 67, 4190-4198 (2007).</li><li>Percy, D. 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Radiol. 46, 718-725 (2011).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+MRI+evaluation+of+intracranial+development+and+vascular+characteristics+of+breast+cancer+brain+metastases+in+a+mouse+model.">Longitudinal MRI evaluation of intracranial development and vascular characteristics of breast cancer brain metastases in a mouse model.</a> PLoS One. 8, (2013).</li><li>Zhou, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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, (2009).</li><li>Zhang, R. 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No presente estudo, demonstramos que a injeção ventricular esquerda guiada por ultra-sonografia garante a precisão de modo que todos os animais do estudo desenvolveram metástases cerebrais e morte do animal não foi observada. A beleza da injeção guiada por imagem é que o caminho da penetração da agulha da pele e, finalmente, para o ventrículo esquerdo pode ser monitorado e ajustado sob a imagem, o que é distinto do procedimento manual que exige rigorosos marcos anatômicos para seguir. <p class="jove_con...

Metástase cerebral é a doença maligna intracraniana mais comum em adultos. O prognóstico é extremamente pobre, com uma sobrevida média de 4-6 meses, mesmo com o tratamento agressivo. O câncer de mama é um dos três principais tipos de câncer primário com alta morbidade da metástase cerebral 1-3. Várias linhas de câncer de mama cérebro-trópicos são capazes de desenvolver metástases cerebrais após intracardíaca ou injeção intracarotídeo 4. Injeção direta de células tumorais para o ventrículo esquerdo pode ignorar o leito capilar pulmonar e, assim, aumentar a incidência de formação de metástases cerebrais, minimizando metástases viscerais. A linha MDA-MB231Br é uma das linhas de cancro da mama humanas mais utilizados para desenvolver metástase cerebral em modelos de roedores 5,6. Como muitos outros estudos 4,7, temos realizado um procedimento manual de injeção intracardíaca em nossos estudos anteriores. No entanto, apenas 50% taxa de sucesso foi obtido com o manualinjecção e uma fracção de ratinhos morreram devido aos procedimentos invasivos repetidos se os ensaios anteriores falharam. Aqui, estamos introduzindo o uso de um procedimento guiado por imagem para garantir a injeção de células de câncer de mama em busca do cérebro para o ventrículo esquerdo do rato atímico. Longitudinal alta resolução de ressonância magnética é aplicada a acompanhar o desenvolvimento intracraniana de metástases cerebrais.

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			<td height="21" style="height:21px;width:413px;">DMEM</td>
			<td style="width:136px;">HyClone</td>
			<td style="width:87px;">SH30022.01</td>
			<td style="width:147px;"> </td>
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			<td height="21" style="height:21px;width:413px;">Trypsin</td>
			<td style="width:136px;">Mediatech</td>
			<td style="width:87px;">25-051-C1</td>
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			<td height="21" style="height:21px;width:413px;">Isoflurane</td>
			<td style="width:136px;">Baxter International Inc.</td>
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			<td height="21" style="height:21px;width:413px;">VisualSonics Vevo 770 High-Resolution Imaging System </td>
			<td>Visual Sonics Inc.</td>
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			<td height="21" style="height:21px;">9.4T horizontal bore magnet with a Varian INOVA Unity system </td>
			<td>Agilent</td>
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			<td height="21" style="height:21px;">O.C.T Compound</td>
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ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

Metástases cerebrais de câncer de mama, que ocorre em 30% dos pacientes com câncer de mama em estágio IV, está associada com alta mortalidade. A sobrevida média é de apenas 6 meses. É crítico para ter modelos animais adequados para imitar a propagação hemodinâmico das células metastáticas no cenário clínico. Aqui, estamos introduzindo o uso de ultra-sonografia pequeno animal para guiar uma injeção precisa do cérebro células de câncer de mama tropical para o ventrículo esquerdo de ratos pelados atímicos. RM longitudinal é usado para avaliar a iniciação intracraniana e crescimento de metástases cerebrais. Injeção intracardíaca de ultra-som-guiada garante não só uma injeção precisa e vem uma taxa de sucesso mais elevada, mas também diminuiu significativamente a taxa de mortalidade, em comparação com o nosso procedimento manual anterior. In vivo alta resolução de ressonância magnética permite a visualização de lesões multifocais hiperintensas, tão pequeno quanto 310 mM de diâmetro em T imagens 2-ponderadas em 3 semanas após a injecção. Follow-se a RM revela crescimento do tumor intracraniano e aumento do número de metástases que distribuem ao longo de todo o cérebro.

Com a elevada resolução espacial de ressonância magnética (78 mM de resolução do plano), lesões de hiper-intensas podem ser identificados tão pequeno quanto 310 um de diâmetro (Figura 2). Desde as metástases neste estudo são muito pequenos e desenvolvimento de necrose e edema é mínima, a lesão hiperintensas em T imagens 2-ponderados realmente representava a massa tumoral. Exames de ressonância magnética permitem longitudinais na avaliaçã...

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

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

Um modelo do rato de mama câncer no cérebro metástase é estabelecida com ultra-som guiada por imagem injeção intracardíaca de células MDA-MB231/Br-GFP. Desenvolvimento de metástases intracranianas multifocais tem sido monitorada longitudinalmente usando alta resolução de 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 ...

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Research

JoVE Journal

Medicine

18.6K visualizações.  University of Texas Southwestern Medical Center.  Um modelo do rato de mama câncer no cérebro metástase é estabelecida com ultra-som guiada por imagem injeção intracardíaca de células MDA-MB231/Br-GFP. Desenvolvimento de metástases intracranianas multifocais tem sido monitorada longitudinalmente usando alta resolução de 9.4 T MRI. 

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

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.

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 ...

Medicina

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Guiada por MRI rompimento da barreira hemato-encefálica utilizando Ultrassom Focalizado transcraniana em um modelo de rato

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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 ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantificação de cancro da mama invasiva das células usando um modelo (3D) tridimensional

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Aguda Cérebro Trauma em camundongos seguiu por Longitudinal dois fótons de imagem

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Um Modelo murino de metástases hepáticas pré-clínica

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Injeção Orthotopic de células cancerosas da mama na mamária Fat Pad de Ratos para o Estudo de crescimento tumoral.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilme Desenvolvimento on Foreign Bodies medicamente relevantes numa subcutânea modelo do rato Seguido por bioluminescência Imagem