Agradecemos J. e J. Cappellani Qiu para suporte técnico. Este trabalho foi financiado por fundos do Instituto Nacional do Câncer (U01 CA141451), o Starr Cancer Consortium, Susan G. Komen for the Cure, Long Island Caminhada dia 2 de Combate ao Câncer de Mama, Manhasset Coalizão das Mulheres Contra o Câncer de Mama para mim, e um pré-doutoramento bolsa de estudos do Programa Dirigido pelo Congresso Breast Cancer Research, dos EUA para ESNESN também é o destinatário da C. Leslie rápida e William Randolph Hearst bolsas da Fundação da Faculdade de Ciências Biológicas Watson. HAA foi apoiada por fundos do Conselho de Pesquisa da Noruega (160698/V40 e 151882), e Sudeste Autoridades Regionais de Saúde (2.007.060).

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Os autores não têm conflitos de interesse de divulgar. Todas as experiências com animais foram conduzidos de acordo com os protocolos aprovados IACUC.

As respostas dos tumores aos tratamentos sistémicos in vivo pode ser dramaticamente diferentes das células cancerosas in vitro, como o microambiente influencia tanto a resposta aguda e recaída 5. Um dos maiores obstáculos para a explicitação das vias quimiorresistência Vivo é a complexidade das interações entre as células de câncer e seu microambiente. Em particular, porque os tumores sólidos têm desenvolvido um equilíbrio entre o cancro e as células do estroma, perturbações de um componente, como a morte celular que ocorre durante o tratamento anti-cancro, pode resultar em efeitos dramáticos na organização dos tecidos. A técnica de imagem intravital fornecido aqui permite a visualização direta de câncer de células do estroma interações dentro dos tumores de ratos anestesiados ao vivo, durante o tratamento com drogas anti-câncer. Usamos microscopia confocal de disco giratório, que tem uma profundidade de penetração relativamente limitada (ver 17 </sup&gt;), mas as nossas técnicas cirúrgicas e rotulagem pode ser usado com outros tipos de microscopia. Filmes obtidos a partir destas experiências pode ser utilizada para controlar os processos, que incluem alterações na intensidade de fluorescência (por exemplo, devido à infiltração de uma população marcada ou indução da morte celular), motilidade celular, a distribuição de injectáveis ​​(por exemplo, para controlar derrame vascular), e co- localização dos sinais fluorescentes 5,12,17,20. Rotineiramente ratos de imagem para mais de 6 horas e de imagem também realizada por mais de 18 horas. Isto requer a manutenção dos ratinhos continuamente sob anestesia para estes períodos de tempo longos. Com anestesia adequada, mais de 90% dos animais sobrevivem nossos 6 horas, e cerca de 80% sobrevivem mais de 18 horas. Detalhes sobre como realizar a longo prazo da anestesia foram publicados previamente 19. Em nossa experiência, cinco fatores são fundamentais: evitar a hipotermia, evitando a desidratação, mantendo fisiológicas no sangue os níveis de saturação de oxigênio, ucantar gás transportador umidificado pelo isoflurano, e manter os animais com o menor nível de anestesia em que eles não mostram sinais de dor. Para manter a temperatura do corpo, mantemos ratos sob um cobertor aquecido (38 ° C). Para evitar a desidratação, é injectar pequenos volumes de solução salina IP em todo o procedimento de imagem inteira. Para manter níveis fisiológicos de saturação de oxigénio do sangue, que começa com 21% de oxigénio no gás de transporte (em vez de a comummente utilizado a 100%). Isso nos permite aumentar os níveis de oxigênio inalado se um rato - hora em um experimento - mostra uma diminuição na saturação de oxigênio no sangue. Em nossa experiência, aumentando os níveis de oxigênio inalado em tempo tal (por exemplo, 30-40%), quase sempre irá estabilizar o animal permitindo horas de imagem adicional. O nível ideal de anestesia é para a maioria dos mouses alcançados com 1-1,5% isoflurano e resulta em taxas de respiração de 60-65/min e taxas de pulso de 400-450/min. Em nossos experimentos de imagem, utilizamos principalmente modelo de camundongo transgênicos (MMTV-PyMT e C3 [1]-Tag), no qual os tumores são multifocal, permitindo imagens de múltiplas lesões em diferentes fases da progressão do tumor, em paralelo em múltiplos, as posições x y durante uma sessão única imagem. Isto diminui as preocupações relativas rato para rato variação no que diz respeito a experiências destinadas a identificar fase dependentes de respostas terapêuticas, e reduz o número de animais necessários para a imagiologia. O modelo de MMTV-PyMT (e outros modelos de cancro espontâneas) passam por fases progressivas histopatológicos que podem ser reconhecidos durante imagiologia intravital, embora o estadiamento patológico das lesões tumorais que podem ser feitas durante a imagiologia intravital é diferente de, e menos detalhado, do que a de cortes histológicos 5,17. Em contraste, os modelos de transplante de tumores tendem a reflectir melhor fase tardia, como as células cancerosas normalmente são isolados a partir de tumores avançados. Tais modelos são, assim, mais susceptíveis de estudar a tumores do estromainterações de tumores em estágio final que diferenças nessas interações entre diferentes etapas. Nós mostramos exemplos de como a imagem nucleares alterações estruturais que ocorrem após a morte celular induzida por terapia. Na ausência de tratamento anti-cancro, esta estratégia de etiquetagem pode ser utilizado em vez da divisão celular de imagem in situ, elucidar, por exemplo, como proliferação celular e a dormência é influenciada pelo microambiente. No futuro, a marcação fluorescente de componentes mitocondriais pode tornar possível a mudança de imagem mitocondriais que ocorrem durante a morte celular por apoptose. Neste protocolo, mostrámos também exemplos de como a imagem de cancro interacções célula-célula do sistema imunológico após a terapia, mas outros componentes do microambiente pode também ser visualizada e impressa. As linhas repórter transgênicos para os componentes do estroma incluem os de fibroblastos (por exemplo., FSP1-EGFP 17, αSMA-RFP 21, COL1A1-EGFP 21), ou células da vasculatura (por exemplo, Tie2-GFP 22; VEGF-GFP 23). Intravital de imagem permite a visualização em tempo real das interacções e processos que ocorrem na administração in vivo a seguir a quimioterapia. Com a tecnologia atualmente disponível, temos feito grandes progressos na compreensão de como as células mielóide influenciar a resposta terapêutica, no entanto, porque o microambiente do tumor é tão complexo, grandes avanços ainda são necessários para começar a mergulhar em mecanicamente os processos subjacentes ambiente mediado por resistência às drogas. Um dos avanços mais importantes ainda necessário é a identificação de melhores marcadores de populações de células específicas. A importância dos melhores marcadores é bem ilustrada com dois dos componentes mais importantes das células estromais do microambiente do tumor da mama, fibroblastos e células do sistema imune. α-actina de músculo liso (αSMA) é freqüentemente usado para identificarfibroblastos, mas a proteína também é expressa por células musculares lisas vasculares e células mioepiteliais 24. Assim, embora ratos αSMA-GFP repórter existir, determinar o tipo específico de célula estar abaixo durante um experimento de imagens intravital é desafiador. De modo semelhante, um único marcador fluorescente tal como expressa EGFP sob o promotor de c-fms, muitas vezes identificar múltiplas subpopulações de células imunes 12,17. Assim, embora um Prefere utilizar um único marcador para identificar um tipo específico de célula a complexidade da biologia subjacente representa um grande desafio na utilização desta abordagem. Uma solução parcial para este problema é a utilização de etiquetas de injectáveis ​​para diferenciar ainda mais sub-populações de células que expressam o mesmo repórter fluorescente. Por exemplo, os dextranos fluorescentes são captados por macrófagos e podem ser usados ​​para marcar as subpopulações de macrófagos que são etiquetados pelo c-fms-EGFP e Fsp1-EGFP ratinhos transgénicos repórter <sup&gt; 17. Os anticorpos conjugados de sondas fluorescentes também têm sido utilizados para identificar subpopulações específicas (por exemplo, a população Gr1-positivo de células mielóides rotulados pelo repórter c-fms-EGFP 17). Diferentemente das curvas de resposta do tumor gerados pelo compasso de calibre de medição, o qual fornece informações mecanicista pouco sobre como ou por tumores respondem à terapêutica administrada, ou a série histológica derivadas de tecidos colhidos em momentos diferentes, que necessitam de grandes grupos de animais, a nossa técnica fornece informação directa quantificável sobre a dinâmica da morte celular e as interações de células do estroma com células cancerosas em pequenas coortes. Assim, a vantagem de utilizar o processo descrito aqui, é que ela fornece uma informação dinâmica sobre as respostas de drogas no contexto de um microambiente do tumor intacto, em tempo real. Tal informação pode levar a importantes insights sobre os processos subjacentes que impulsionam respostas terapêuticas 5 &lt;/ Sup&gt;.

Os tumores sólidos são constituídos por dois compartimentos principais: células de cancro e os componentes do estroma (tanto celulares e não celulares) que ajudam na manutenção de um ambiente adequado para o crescimento do tumor 1. Estes componentes do estroma desempenham papéis críticos no desenvolvimento, progressão e metástase de vários tipos de cancros, incluindo os da mama 2-5. O ambiente também influencia estromal respostas terapêuticas 2,4,5. Assim, determinar como as células cancerosas responder às terapias convencionais e novel dentro do contexto do microambiente intacto é importante para favorecer a nossa compreensão da biologia do cancro e na melhoria actuais estratégias terapêuticas. Além disso, a definição de como cancro das células do estroma interacções mudar após a administração da terapia é crítica para a compreensão da biologia de recidiva tumoral. Organotípicas co-cultura de sistemas têm sido úteis para o estudo de tumores do estroma interacções <sse&gt; 6, mas é evidente que os métodos actualmente disponíveis não podem com sucesso recapitular o microambiente do tumor intacto in vitro, em particular no que diz respeito à função vascular e recrutamento de células imunitárias. Com efeito, as respostas celulares de cancro para terapias que são observadas in vitro muitas vezes são significativamente diferentes dos observados in vivo, onde o microambiente está intacta 5. Assim, em modelos in vivo para o estudo de tumores do estroma interações e seu impacto sobre a resposta terapêutica pode refletir melhor os mecanismos clinicamente relevantes 7. O principal meio de estudar as respostas para a terapia anti-cancro in vivo tem sido através de medições do tamanho do tumor e exame histológico de tecidos derivados de animais ou de pacientes tratados. No entanto, os avanços na microscopia e repórteres fluorescentes mais nas últimas duas décadas permitiram a visualização dos processos inter e intracelularem alta resolução em animal vivo, anestesiados (intravital imagem) 8. Estas tecnologias de microscopia intravital provaram ser especialmente valioso para espacialmente e temporalmente dissecando a dinâmica de tumor in vivo de interacções a-estroma resolução celular e subcelular 8,9. Os processos que foram desvendados por imagiologia intravital incluem anti-tumor a citotoxicidade das células T 10,11, as interacções entre as células T e as células mielóides 12, a dinâmica das modificações na composição de colagénio e organização após o tratamento terapêutico 13, macrófagos dependente intravasation célula cancerosa e metástase 14, e permeabilidade vascular 15. Existem três principais requisitos para imagiologia intravital. Estes incluem a preparação adequada exposição dos tecidos e para geração de imagens, marcação fluorescente dos componentes do tecido de interesse, e um par de microscopia câmara-sistema capaz de adquirir imagens de 16. As estratégias mais comuns utilizadas para atender a esses requisitos incluem: 1) os preparativos heterotópicos (por exemplo, a inoculação do orbital orelha ou olho), permanente Windows Imaging, ou preparações exteriorizado (por exemplo, retalho de pele dorsal), 2) transgênicos fluorescentes e repórteres.. injetáveis ​​fluorescentes para identificar os componentes do tecido, e 3) sistemas de microscopia confocal multifotônica ou emparelhado com tubos fotomultiplicadores ou charge-coupled device (CCD) câmeras, respectivamente 9. Usamos um modelo de retalho cirurgicamente preparado pele para expor a glândula mamaria inguinal para imagens em animais anestesiados que expressam transgenes que codificam os repórteres fluorescentes. As imagens são obtidas ao longo de um período de 12 a 40 horas utilizando um CCD intensificada (ICCD) câmara emparelhado com um sistema de fiação de disco de quatro a laser microscópio confocal 17. Imagiologia desta forma, permitiu-nos estudar os processos, tais como in vivo de distribuição de droga, dependente da fase chrespostas emotherapeutic, tipo de quimioterapia induzida morte celular, e o comportamento de células mielóides 5. Nós fornecemos um protocolo para a criação de imagens intravital de respostas de células cancerosas para anti-câncer de terapia e de tumor do estroma interações em modelos do rato do câncer de mama. Este protocolo pode ser usado para controlar os comportamentos e morte de tanto as células cancerosas e componentes do estroma com uma grande variedade de transgênicos e injetáveis ​​etiquetas fluorescentes em ambos os modelos transgênicos e transplante por períodos de até 40 horas em uma sessão de imagem única.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> Reagente </td></tr><tr><td> MMTV-PyMT ratos </td><td> Jackson Laboratory </td><td> 2374 </td><td></td></tr><tr><td> C3 (1)-Tag ratos </td><td> Jackson Laboratory </td><td> 13591 </td><td></td></tr><tr><td> ACTB-ECFP ratos </td><td> Jackson Laboratory </td><td> 3773 </td><td></td></tr><tr><td> ACTB-H2B-EGFP ratos </td><td> Jackson Laboratory </td><td> 5418 </td><td></td></tr><tr><td> 1 x PBS </td><td> Feita em casa </td><td></td><td></td></tr><tr><td> 2 MD Dextran, fluoresceína conjugada </td><td> Invitrogen </td><td> D-7137 </td><td> Reconstituir em dH 2 O (4 loja mg / ml, à temperatura de -20 ° C); diluído a 1 mg / ml em 1 x PBS </td></tr><tr><td> Dextran 10 kD, Alexa Fluor 647-conjugados </td><td&gt; Invitrogen </td><td> D-22914 </td><td> Reconstituir em dH 2 O (4 loja mg / ml, à temperatura de -20 ° C); diluído a 1 mg / ml em 1 x PBS </td></tr><tr><td> Cloridrato de doxorrubicina </td><td> Sigma-Aldrich </td><td> 44583 </td><td> Reconstituir em dH2O e armazenar a 4 ° C; diluir em 1 x PBS </td></tr><tr><td> Isothesia (isoflurano) </td><td> Butler Abastecimento Saúde Animal </td><td> 029450 </td><td> 250 ml </td></tr><tr><td> Iodeto de propídio </td><td> Invitrogen </td><td> P3566 </td><td> 1 mg / ml; diluído 1:15 em 1 x PBS </td></tr><tr><td> Nitrogênio </td><td></td><td></td><td></td></tr><tr><td> Oxigênio </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Equipamento </td></tr><tr><td> 18G x 1 ½ &quot;agulha de bisel regulares </td><td> BD </td><td> 305196 </td><td></td></tr><tr><td> μManager </td><td> Vale Lab, UCSF </td><td> <a href="www.micro-manager.org" target="_blank">www.micro-manager.org</a> </td><td> Software de código aberto </td></tr><tr><td> Algodão embebido em álcool </td><td> BD </td><td> 326895 </td><td> Compressas com álcool isopropílico a 70% </td></tr><tr><td> Sistema de anestesia </td><td> Moleculares Imaging Products Co., </td><td></td><td></td></tr><tr><td> Tampa de vidro </td><td> Corning </td><td> 2940-245 </td><td> No. 1 ½, mm 24x50; desinfectar com 70% toalhetes isopropanol </td></tr><tr><td> Gurança esponjas de gaze (esterilizada) </td><td> Kendall </td><td> 6939 </td><td></td></tr><tr><td> Lâminas de vidro de microscópio </td><td> Corning </td><td> 2948-75x25 </td><td> Pré-limpeza, se não pré-limpeza, desinfecção com 70% toalhetes isopropanol </td></tr><tr><td> Endurecidos tesoura fina </td><td> Belas Science Tools </td><td> 14090-11 </td><td> 2 pares; aço inoxidável, afiado afiadas pontas, ponta reta, 26 vanguarda mm, 11 cm de comprimento </td></tr><tr><td> Cobertor aquecido </td><td> Gaymar Indústrias </td><td></td><td></td></tr><tr><td> Hot talão esterilizador </td><td> Belas Science Tools </td><td> 18000-45 </td><td> Transformar em cerca de 30 min antes da sua utilização; esterilizar instrumentos a&gt; 200 ° C durante 30 seg </td></tr><tr><td> Imaris </td><td> Bitplane </td><td> <a href="www.bitplane.com" target="_blank">www.bitplane.com</a> </td><td></td></tr><tr><td> Krazy Glue </td><td> Produtos Elmer </td><td> KG484 </td><td></td></tr><tr><td> Laboratório de fita (½ &quot;) </td><td></td><td></td><td></td></tr><tr><td> Fita Laboratory (1 &quot;) </td><td></td><td></td><td></td></tr><tr><td> A tampaum refrigerador de isopor transporte </td><td></td><td></td><td> Isto será utilizado como plataforma cirúrgica </td></tr><tr><td> Micro pinça de dissecação </td><td> Roboz </td><td> RS-5153 </td><td> Dentes 1x2, curva ligeira, 0,8 de largura ponta 4 mm, comprimento &quot; </td></tr><tr><td> Micro pinça de dissecação </td><td> Roboz </td><td> RS-5135 </td><td> Serrilhada, ligeira curva, 0,8 de largura ponta mm; 4 de comprimento &quot; </td></tr><tr><td> Microscópio </td><td></td><td></td><td> Spinning-disco confocal, XYZ fase Piezo, capablility epifluourescence 17,25 </td></tr><tr><td> Inserção do microscópio </td><td> Aplicada Instrumentação Científica </td><td></td><td> Personalizado fabricado, com duas portas de imagens circulares </td></tr><tr><td> MouseOx oxímetro, software e sensores </td><td> STARR Ciências da Vida </td><td> <a href="www.starrlifesciences.com" target="_blank">www.starrlifescience</a>s.com </td><td></td></tr><tr><td> Nalgene Super Versi-Dry laboratório soakers </td><td> Thermo Scientific </td><td> 74218-00 </td><td> Corte em quartos para alinhar plataforma cirúrgica </td></tr><tr><td> Nebulizador </td><td> Salter Labs </td><td> 8901 </td><td> Usado para umidificar gases; evita a irritação dos tecidos pulmonares </td></tr><tr><td> Deslizamento ponta de seringa de tuberculina descartável (1 ml) </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> Surflo conjunto infusão alado </td><td> Terumo </td><td> SV-23BLK </td><td> 23G x ¾ &quot;agulha ultrafina, 12&quot; tubo </td></tr><tr><td> Betadine spray </td><td> Purdue Pharma </td><td> BASP3H </td><td></td></tr></tbody></table>

live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer

O microambiente do tumor desempenham um papel crucial na iniciação do tumor, progressão, metástase, e a resposta a terapias anti-cancro. Tridimensionais co-cultura de sistemas são frequentemente usados ​​para explicar a tumores do estroma interacções, incluindo o seu papel na resposta de drogas. No entanto, muitas das interacções que ocorrem in vivo no microambiente intacta não pode ser completamente reproduzido nestas configurações em in vitro. Assim, a visualização direta destes processos em tempo real tornou-se uma ferramenta importante na compreensão das respostas tumorais às terapias e identificar as interações entre as células cancerosas e do estroma que pode influenciar essas respostas. Aqui nós fornecemos um método para a utilização de microscopia confocal de disco giratório ao vivo, ratos anestesiados para observar directamente de distribuição de medicamentos, as respostas celulares de cancro e alterações no tumor do estroma interacções após a administração de terapêutica sistémica em modelos de cancro da mama. Descrevemos os procedimentos para labeling diferentes componentes do tumor, tratamento de animais para a observação de respostas terapêuticas, bem como o procedimento cirúrgico para expor tecidos tumorais para imagiologia de até 40 horas. Os resultados obtidos com este protocolo são lapso de tempo em que os filmes, os processos tais como a infiltração de drogas, a morte de células cancerosas e a migração das células do estroma pode ser avaliada utilizando o software de análise de imagem.

Watch this Scientific Journal Video about Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer at JoVE.com

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Descreve-se um método para a resposta de imagem para tratamento anti-cancro<em&gt; In vivo</em&gt; E a resolução de células simples.

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 ...

live-imaging-drug-responses-tumor-microenvironment-mouse-models

Research

JoVE Journal

Medicine

24.6K visualizações.  Watson School of Biological Sciences. Descreve-se um método para a resposta de imagem para tratamento anti-cancro In vivo E a resolução de células simples.

Vídeo: Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

Método para Desenvolvimento de Drogas Anti-Câncer Novel utilizando explantes Tumor de espécimes cirúrgicos


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Medicina

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 ...

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 ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Modelos MAME para formação de imagens 4D Live-célula do tumor: Interações microambiente que a progressão de Impacto maligno

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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 ...

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 ...

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 ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Profiling Molecular do microambiente do tumor invasivo em um modelo 3-Dimensional de células de câncer colorretal e<em&gt; Ex vivo</em&gt; Os fibroblastos

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

De alta frequência Ecografia de mouse linfonodos cervicais

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In Vivo e Ex Vivo Abordagens para o Estudo do cancro do ovário metastático Colonização de Estruturas Láctea natural em Peritoneal adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...