Name: pathological analysis of lung metastasis following lateral tail-vein injection of tumor cells
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Description: Watch this Scientific Journal Video about Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells at JoVE.com

Os dados representativos foram financiados por meio do Instituto Nacional de Câncer (K22CA218549 para S.T.S). Além de sua ajuda no desenvolvimento do método de análise abrangente aqui relatado, agradecemos ao Centro Abrangente de Câncer da Universidade estadual de Ohio e ao Recurso Compartilhado de Fenotipagem de Camundongos (Director – Krista La Perle, DVM, PhD) pelos serviços de histologia e imunohistoquímica e pelo Núcleo de Imagem de Patologia para desenvolvimento e análise de algoritmos.

O uso de animais seguiu as regulamentações do University Laboratory Animal Resources (ULAR) sob o Comitê Institucional de Cuidados e Uso de Animais (IACUC) da OSU – protocolo aprovado 2007A0120-R4 (PI: Dr. Gina Sizemore).
1. Injeção de veias traseiras de células cancerígenas de mama
<ol><li>Preparação de células e seringas para injeção<ol>
<li>Placa um número apropriado de células com base no número de camundongos e concentração celular a ser usado. NOTA: O número de células injetadas e o tempo para o desenvolvimento de metástases dependerão da linha celular utilizada e precisarão ser otimizadas. Nesta demonstração, 1 x 106 células MDA-MB-231 são injetadas por via intravenosa em camundongos NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) e lesões pulmonares macroscópicas são observadas no máximo 24 dias após a injeção. Para a linha de células tumores mamárias MVT1 murina17, 3 x 106 células são injetadas em camundongos FVB/N imuno-competentes com numerosas metástases pulmonares observadas por 14 dias22,23.</li>
		<li>Aspirar mídia e enxaguar placas celulares com 1x PBS. Células de trypsinize em volume mínimo, adicione o volume apropriado de mídia e conte células usando um hematócito ou outro método preferido. O azul trypan (0,4%) ou outros corantes de células vivas/mortas podem ser usados para determinar a contagem de células viáveis.</li>
		<li>Células de pelota girando a 180 x g por 5 min.</li>
		<li>Resuspenque o número apropriado de células em 1x PBS estéril, de tal forma que um volume de 100 μL seja injetado por mouse. Mantenha a suspensão celular no gelo para manter a viabilidade.</li>
		<li>Antes da injeção, ressuspendam completamente as células com uma pipeta de 200 μL ou 1 mL para evitar a aglomeração. Desenhe 100 μL em uma seringa de insulina de 28 G (ver Tabela de Materiais).</li>
		<li>Elimine quaisquer bolhas de ar mantendo a seringa vertical, tocando na seringa e ajustando lentamente o êmbolo. A injeção de bolhas de ar na veia pode causar uma embolia de ar/gás que pode ser fatal.</li>
	</ol></li>
	<li>Injeção lateral da veia traseira NOTA: Para ensaios experimentais de metástase do câncer de mama, as injeções são realizadas em camundongos fêmeas de > 6 semanas de idade.<ol>
<li>Manuseie o mouse pela cauda e deslize animal em um tubo ranhuted/dispositivo de contenção de um tamanho apropriado (consulte a Tabela de Materiais para o dispositivo de contenção usado).</li>
		<li>Insira a porção do plugue do dispositivo de contenção e posicione o mouse de lado, de forma que sua veia traseira lateral seja fácil de visualizar. O camundongo tem uma artéria ventral alinhada com a genitália, uma veia dorsal e duas veias caudais laterais.</li>
		<li>Limpe a superfície da cauda do rato com um lenço asséptico. Segure a cauda entre o dedo indicador e o polegar com a mão não dominante e aplique uma leve tensão.</li>
		<li>Começando pela porção distal da cauda, insira a agulha paralela à veia com o bisel acabar.</li>
		<li>Se permitido, recapitula a agulha cuidadosamente e dobre para um ângulo de 20-30°. Uma abordagem individual ou dispositivo de recapeamento de agulhas é altamente recomendado. NOTA: Não é necessário aspirar, pois isso pode causar o colapso da veia. No entanto, um pequeno flash de sangue pode ser visto quando colocado pela primeira vez. A agulha avançará suavemente na veia com a colocação adequada.</li>
		<li>Distribua lentamente o volume completo na veia. Não deve haver resistência quando o êmbolo é empurrado.</li>
		<li>Se houver alguma resistência, remova prontamente a agulha da seringa. Se necessário, reinsira a agulha (idealmente não mais do que 3 tentativas) movendo-se em direção à extremidade proximal da cauda ou veia lateral oposta.</li>
		<li>Um pequeno volume de sangue provavelmente será deslocado após a injeção. Aplique pressão suave com gaze estéril e limpe com limpeza asséptica.</li>
		<li>Descarte prontamente a seringa no recipiente afiado apropriado.</li>
		<li>Devolva o mouse a uma gaiola limpa e ventilada e monitore por sinais de angústia.</li>
		<li>Monitore camundongos 2-3x/semanalmente para sinais de formação de metástase (respiração trabalhada, postura curvada, perda de peso) e sofrimento geral. O tempo para o desenvolvimento da metástase dependerá da linha celular e da tensão do rato.</li>
		<li>Se usar um dispositivo de imagem animal in vivo vivo, os ratos de imagem imediatamente após a injeção da veia traseira para confirmar a injeção bem sucedida de células e obter dados "zero" do tempo (detalhes específicos sobre imagens de bioluminescência in vivo não estão incluídos aqui, mas são descritos por Yang et al.24).</li>
	</ol></li>
</ol>2. Fixação do tecido pulmonar e análise da carga do tumor pulmonar metastático
<ol><li>Inflação do tecido pulmonar para manter o formato estrutural dos pulmões para histopatologia<ol>
<li>Após os procedimentos aprovados de eutanásia (por exemplo, dióxido de carbono a 30 - 70% de deslocamento do volume da câmara/min) são seguidos, proteja a carcaça do rato em uma placa de dissecação com pinos. Pulverizar ou aplicar 70% de etanol para manter a pele do rato fora do caminho durante a dissecação. NOTA: Asfixia por dióxido de carbono pode causar hemorragia pulmonar como lesão de fundo esperada, especialmente em taxas de fluxo mais lentas.</li>
		<li>Abra o tórax com uma incisão midline, estenda a incisão cranially/caudally através do peritônio, e corte o diafragma agarrando o processo xifoide.</li>
		<li>Usando um conjunto separado de tesouras para não entorpecer as lâminas, corte as costelas ao longo de cada lado do esterno e remova cuidadosamente a caixa torácica deixando espaço para os pulmões se expandirem.</li>
		<li>Isole a traqueia removendo glândulas salivares submandibulares e musculatura infra-ióide. Colocar pinos em ambos os lados da traqueia pode evitar movimentos indesejados durante a inserção da agulha.</li>
		<li>Encha uma seringa de 26 G com 2-3 mL de formalina tamponada neutra de 10% e insira na traqueia.</li>
		<li>Injete lentamente formalina e observe que os pulmões se expandam (geralmente requer ~1,5 mL de formalina).</li>
		<li>Uma vez que a formalina começa a vazar dos pulmões (evite a inflação), belisque a traqueia com um par de fórceps, remova a agulha da seringa e desprende todo o aparelho respiratório. Coloque pulmões, coração, etc. diretamente em formalina, pois o corte adicional de tecido pode ser feito após a fixação.</li>
		<li>Processamento completo, incorporação, secção de tecido e hematoxilina e eosina (H&E) de coloração usando métodos padrão.</li>
	</ol></li>
	<li>Análise da carga do tumor pulmonar metastático<ol>
<li>Escaneie seções pulmonares manchadas de H&E em um scanner de slides de alta resolução a 40x de ampliação (Figura 3).</li>
		<li>Importar imagens em software de análise de imagens (por exemplo, Análise de Imagem visiopharm) para quantificação de metástases pulmonares. NOTA: Recomendamos que novos usuários obtenham treinamento no local ou online para usar o software de análise de imagens. Inúmeros webinars também estão disponíveis através da página comercial.</li>
		<li>Selecione o Aplicativo de Metástase pulmonar do Visiopharm 10118 H&E Na biblioteca de aplicativos do software. NOTA: O objetivo deste aplicativo é rotular e quantificar a metástase pulmonar em slides manchados de H&E. Como parte do Aplicativo de Metástase Pulmonar 10118, a primeira etapa de processamento de imagem segmenta o tecido pulmonar com o Aplicativo de Detecção de Tecidos. A segunda etapa de processamento de imagem usa o Aplicativo de Detecção de Metástase que identifica as metástases dentro do tecido pulmonar. Metástases são identificadas via forma juntamente com regiões que são muito desacompusadas, muito vermelhas ou muito esparsas para serem identificadas como metástases.</li>
		<li>Ajuste os parâmetros que definem a forma e a escassez para melhor se encaixar em imagens representativas. Áreas segmentadas de metástases tumorais e tecido pulmonar normal podem ser exibidas usando diferentes rótulos de cor para cada tipo de tecido. NOTA: No caso de o aplicativo não conseguir separar com precisão metástases do tecido pulmonar normal, um aplicativo personalizado usando o programa Visiopharm Decision Forest pode precisar ser escrito como foi feito para os experimentos (ver Figura 2 e Figura 3). Os detalhes para escrever um algoritmo personalizado seguem abaixo. Caso contrário, prossiga para a etapa 2.2.9.</li>
		<li>Abra o programa Floresta de Decisão, que funciona treinando várias classes [ou seja, tecido pulmonar (não neoplásico), metástases, glóbulos vermelhos, epitélio e/ou espaço branco] em uma imagem desejada. Na Figura 2, as metástases tumorais são azuis, o tecido normal é verde, e o epitélio bronquiolar é amarelo. Além disso, os glóbulos vermelhos estão em espaços vermelhos e de ar em rosa.</li>
		<li>Siga a série solicitada de perguntas sim ou não para treinar adequadamente cada Classe para uma imagem. A precisão do algoritmo determinará o número de perguntas sim/não. Para a análise, o algoritmo/aplicativo personalizado foi escrito com precisão definida para 50 (faixa 0-100).</li>
		<li>Ajuste recursos para cada classe aplicando filtros para afiar, desfocar, classificar por forma, etc. para melhorar a precisão do algoritmo/App. Visiopharm visualiza cada Classe através de uma ou várias lentes conhecidas como Recursos. As características mudam a forma como a Classe vê a imagem para trazer certas cores ou intensidades. NOTA: Para o algoritmo personalizado, metástases medindo 8500 μm2 ou mais são rotuladas e medidas como metástases. Isso explica a variância de tamanho e metástases muito pequenas para detectar. Pequenas áreas desalinhadas e pequenas áreas metastáticas abaixo de 8500 μm2 foram incluídas na quantificação normal do tecido.</li>
		<li>Salve as configurações modificadas do aplicativo ou do algoritmo personalizado e, em seguida, aplique o algoritmo/aplicativo a um conjunto inteiro ou série de tecidos manchados de H&E.</li>
		<li>Por fim, exporte todas as variáveis de produção, que incluem as listadas na Tabela 1. A área em mícrons ao quadrado (μm2) pode ser quantificada para cada tipo de tecido e as porcentagens são derivadas da área total do tecido líquido da amostra (ou seja, tecido total menos espaço aéreo).</li>
		<li>Ao criar um algoritmo personalizado, revise as marcações teciduais em consulta com um conselho de patologista veterinário certificado pelo American College of Veterinary Pathologists para garantir medidas precisas e diferenciar entre os tipos de tecidos.</li>
	</ol></li>
</ol>

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M., 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=Is+tail+vein+injection+a+relevant+breast+cancer+lung+metastasis+model.">Is tail vein injection a relevant breast cancer lung metastasis model.</a> Journal of Thoracic Disease. 5 (4), 385-392 (2013).</li><li>Goodale, D., Phay, C., Postenka, C. O., Keeney, M., Allan, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+tumor+cell+dissemination+patterns+in+preclinical+models+of+cancer+metastasis+using+flow+cytometry+and+laser+scanning+cytometry.">Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry.</a> Cytometry Part A. 75 (4), 344-355 (2009).</li><li>Goddard, E. T., Fischer, J., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Portal+Vein+Injection+Model+to+Study+Liver+Metastasis+of+Breast+Cancer.">A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.</a> Journal of Visualized Experiments. (118), (2016).</li><li>Wright, L. E., 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=Murine+models+of+breast+cancer+bone+metastasis.">Murine models of breast cancer bone metastasis.</a> BoneKEy Reports. 5, 804 (2016).</li><li>Simmons, J. K., 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=Animal+Models+of+Bone+Metastasis.">Animal Models of Bone Metastasis.</a> Veterinary Pathology. 52 (5), 827-841 (2015).</li><li>Liu, Z., 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=Improving+orthotopic+mouse+models+of+patient-derived+breast+cancer+brain+metastases+by+a+modified+intracarotid+injection+method.">Improving orthotopic mouse models of patient-derived breast cancer brain metastases by a modified intracarotid injection method.</a> Scientific Reports. 9 (1), 622 (2019).</li><li>Kodack, D. P., Askoxylakis, V., Ferraro, G. B., Fukumura, D., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+strategies+for+treating+brain+metastases+from+breast+cancer.">Emerging strategies for treating brain metastases from breast cancer.</a> Cancer Cell. 27 (2), 163-175 (2015).</li><li>Brown, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+Stereology+Applications+for+the+Pathologist.">Practical Stereology Applications for the Pathologist.</a> Veterinary Pathology. 54 (3), 358-368 (2017).</li><li>Aeffner, F., 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=Digital+Microscopy,+Image+Analysis,+and+Virtual+Slide+Repository.">Digital Microscopy, Image Analysis, and Virtual Slide Repository.</a> Institute for Laboratory Animal Research Journal. 59 (1), 66-79 (2018).</li></ol>

Os autores não têm nada a revelar.

À medida que os pesquisadores continuam a usar a injeção intravenosa de células tumorais como modelo experimental para metástase, faltam práticas padrão para analisar a carga tumoral metastática resultante. Em alguns casos, diferenças significativas na carga tumoral metastática após a manipulação de linhas celulares específicas e/ou uso de compostos químicos podem ser observadas macroscopicamente. No entanto, em outros casos, diferenças sutis na semeadura metastática e no crescimento podem ser negligenci...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61270/pathological-analysis-lung-metastasis-following-lateral-tail-vein">Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells</a>. The author list was updated.
The author list was updated from:
Katie A. Thies1,&#160;Sue E. Knoblaugh2, and Steven T. Sizemore1 
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 
2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University
to:
Katie A. Thies1, Sarah Steck1, Sue E. Knoblaugh2, and Steven T. Sizemore1 
1Department of Radiation Oncology, Arthur G. James Comprehensive Cancer Center and Richard L. Solove Research Institute, The Ohio State University Medical Center 
2Department of Veterinary Biosciences, Comparative Pathology and Digital Imaging Shared Resource, The Ohio State University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61270/pathological-analysis-lung-metastasis-following-lateral-tail-vein">Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells</a>. The author list was updated.

Erratum: Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Apesar de ser um processo altamente complexo e ineficiente1, a metástase é um contribuinte significativo para a morbidade e mortalidade de pacientes com câncer2. De fato, a maioria das mortes relacionadas ao câncer é atribuída à disseminação metastática da doença3,4. Para que as células tumorais se metástasizem com sucesso, elas devem se desprender do local primário, invadir através do estroma adjacente, intravasar em circulação sanguínea ou linfáticos, viajar para o leito capilar de um local secundário, extravasar no tecido secundário e proliferar ou crescer para formar lesões metastáticas5. O uso de modelos de camundongos tem sido fundamental para promover a compreensão dos mecanismos moleculares responsáveis pela semeadura metastática e crescimento6,7. Aqui, focamos na metástase do câncer de mama, para a qual tanto modelos de camundongos geneticamente modificados quanto métodos de transplante são frequentemente utilizados – cada um com seu próprio conjunto de vantagens e limitações.
Modelos de tumor mamário geneticamente modificados fazem uso de promotores específicos da glândula mamária, incluindo MMTV-LTR (vírus tumoramático de camundongos longa repetição terminal) e WAP (Whey Acidic Protein), para impulsionar a expressão de transgenes no epitélio mamário8. Oncogenes incluindo antígeno T médio de polioma (PyMT), ErbB2/Neu, c-Myc, Wnt-1 e vírus símio 40 (SV40) foram expressos desta forma9,10,11,12,13, e embora esses modelos genéticos sejam úteis para estudar a iniciação e progressão do tumor primário, poucos prontamente metástases para órgãos distantes. Além disso, esses modelos genéticos de camundongos são muitas vezes mais tempo e custo proibitivos do que modelos de metástase espontânea ou experimental. Dada a limitação da maioria dos modelos de tumores mamários geneticamente modificados para estudar a metástase, as técnicas de transplante tornaram-se métodos atraentes para estudar esse processo complexo. Isso inclui ortotópica, veia traseira, intracardiac e injeção intracraniana de linhas celulares adequadas.
Embora várias linhas de células cancerígenas de mama prontamente metástases após a injeção ortotópica na almofada de gordura mamária14,15, a consistência e a reprodutibilidade da carga tumoral metastática podem ser um desafio, e a duração desses estudos pode ser na ordem de vários meses. Para avaliar a metástase pulmonar, em particular, a injeção intravenosa na veia traseira é muitas vezes um método mais reprodutível e eficaz com propagação metastática tipicamente ocorrendo dentro do período de algumas semanas. No entanto, uma vez que o modelo de injeção intravenosa contorna as etapas iniciais da cascata metastática, deve-se tomar cuidado na interpretação dos resultados desses estudos. Nesta demonstração, mostramos a injeção de veias traseiras de células tumorais mamárias, juntamente com um método de análise preciso e abrangente.
Embora a comunidade de pesquisa tenha feito progressos significativos na compreensão do complexo processo de metástase do câncer de mama, estima-se que mais de 150.000 mulheres tenham atualmente câncer de mama metastático16. Das pessoas com câncer de mama estágio IV, >36% das pacientes têm metástase pulmonar17; no entanto, o padrão específico do local e a incidência de metástases podem variar de acordo com o subtipo molecular18,19,20,21. Pacientes com metástases pulmonares associadas ao câncer de mama têm uma sobrevida mediana de apenas 21 meses, destacando a necessidade de identificar tratamentos eficazes e novos biomarcadores para esta doença17. O uso de modelos experimentais de metástase, incluindo a injeção intravenosa de células tumorais, continuará avançando nosso conhecimento sobre este importante desafio clínico. Quando combinadas com a patologia da imagem digital e o método de análise da carga de tumores pulmonares metastáticos descritos neste protocolo, as injeções de veias traseiras são uma ferramenta valiosa para a pesquisa de metástase do câncer de mama.

<table><tbody><tr><td>alcohol prep pads</td><td>Fisher Scientific</td><td>22-363-750</td><td>&lt;strong&gt;for cleaning tail prior to injection&lt;/strong&gt;</td></tr><tr><td>dissection scissors</td><td>Fisher Scientific</td><td>08-951-5</td><td>&lt;strong&gt;for mouse dissection and lung tissue inflation&lt;/strong&gt;</td></tr><tr><td>DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td><td>Fisher Scientific</td><td>MT10013CV</td><td>cell culture media base for MDA-MB-231 and MVT1 cell lines</td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Salt Solution 1x</td><td>Fisher Scientific</td><td>MT21030CV</td><td>used for resuspending tumor cells for injection</td></tr><tr><td>ethanol (70 % solution)</td><td>OSU</td><td></td><td>used to minimize mouse&#039;s fur during dissection; use caution - flammable</td></tr><tr><td>Evan&#039;s blue dye</td><td>Millipore Sigma</td><td>E2129</td><td>used at 1 % in sterile PBS for practice with tail-vein injection method; use caution - dangerous reagent</td></tr><tr><td>Fetal Bovine Serum</td><td>Millipore Sigma</td><td>F4135</td><td>cell culture media additive; used at 10% in DMEM</td></tr><tr><td>forceps</td><td>Fisher Scientific</td><td>10-270</td><td>for dissection and lung tissue inflation</td></tr><tr><td>FVB/NJ mice</td><td>The Jackson Laboratory</td><td>001800</td><td>syngeneic mouse strain for MVT1 cells</td></tr><tr><td>hemacytometer (Bright-Line)</td><td>Millipore Sigma</td><td>Z359629</td><td>for use in cell culture to obtain cell counts</td></tr><tr><td>insulin syringe (28 G)</td><td>Fisher Scientific</td><td>14-829-1B</td><td>for tail-vein injections (BD 329424)</td></tr><tr><td>MDA-MB-231 cells</td><td>ATCC</td><td></td><td>human breast cancer cell line</td></tr><tr><td>MVT1 cells</td><td></td><td></td><td>mouse mammary tumor cells</td></tr><tr><td>needles (26 G)</td><td>Fisher Scientific</td><td>14-826-15</td><td>used to inflate the mouse&#039;s lungs</td></tr><tr><td>neutral buffered formalin (10%)</td><td>Fisher Scientific</td><td>245685</td><td>used as a tissue fixative and to inflate lung tissue; use caution - dangerous reagent</td></tr><tr><td>NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice</td><td>The Jackson Laboratory</td><td>005557</td><td>maintained by OSUCCC Target Validation Shared Resource</td></tr><tr><td>Penicillin Streptomycin 100x</td><td>ThermoFisher</td><td>15140163</td><td>cell culture media additive</td></tr><tr><td>sterile gauze</td><td>Fisher Scientific</td><td>NC9379092</td><td>for applying pressue to mouse&#039;s tail if bleeding occurs</td></tr><tr><td>syringe (5 mL)</td><td>Fisher Scientific</td><td>14-955-458</td><td>used to inflate mouse lung tissue</td></tr><tr><td>tail-vein restrainer</td><td>Braintree Scientific, Inc.</td><td>TV-150 STD</td><td>used to restrain mouse for tail-vein injections</td></tr><tr><td>Trypan blue (0.4 %)</td><td>ThermoFisher</td><td>15250061</td><td>used in cell culture to assess viability</td></tr><tr><td>Trypsin-EDTA 0.25 %</td><td>ThermoFisher</td><td>25200-114</td><td>used in cell culture to detach tumor cells from plate</td></tr></tbody></table>

pathological analysis of lung metastasis following lateral tail-vein injection of tumor cells

A metástase, a principal causa de morbidade e mortalidade para a maioria dos pacientes com câncer, pode ser desafiadora para modelar preclinicamente em camundongos. Poucos modelos de metástase espontânea estão disponíveis. Assim, o modelo experimental de metástase envolvendo injeção de veias traseiras de linhas celulares adequadas é um pilar da pesquisa de metástase. Quando as células cancerígenas são injetadas na veia traseira lateral, o pulmão é seu local preferido de colonização. Uma limitação potencial dessa técnica é a quantificação precisa da carga do tumor pulmonar metastático. Enquanto alguns pesquisadores contam macrometastases de um tamanho pré-definido e/ou incluem micrometastases após a secção do tecido, outros determinam a área de lesões metastáticas em relação à área normal do tecido. Ambos os métodos de quantificação podem ser extremamente difíceis quando a carga metastática é alta. Aqui, demonstramos um modelo de injeção intravenosa de metástase pulmonar seguido de um método avançado para quantificar a carga do tumor metastático usando o software de análise de imagem. Esse processo permite a investigação de múltiplos parâmetros de ponto final, incluindo tamanho médio de metástase, número total de metástases e área total de metástase, para fornecer uma análise abrangente. Além disso, este método foi revisado por um conselho veterinário certificado pelo American College of Veterinary Pathologists (SEK) para garantir a precisão.

Se usar células não rotuladas para injeção de veias traseiras, pode ser difícil confirmar a colonização pulmonar até (1) o tempo de necropsia se macrometastases podem ser observadas ou (2) após análise histológica se existem metástases microscópicas. Com extensa carga de tumores pulmonares metastáticos, os camundongos terão a respiração trabalhada. Como em qualquer estudo de tumor, os camundongos devem ser cuidadosamente monitorados durante toda a duração do estudo. O uso de células rotuladas é uma m...

Watch this Scientific Journal Video about Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells at JoVE.com

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

A injeção intravenosa de células cancerígenas é frequentemente usada em pesquisas de metástase, mas a carga tumoral metastática pode ser difícil de analisar. Aqui, demonstramos um modelo de injeção de veias traseiras de metástase e incluímos uma nova abordagem para analisar a carga do tumor pulmonar metastático resultante.

Análise patológica da metástase pulmonar após injeção lateral de cauda-veia de células tumorais

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

pathological-analysis-lung-metastasis-following-lateral-tail-vein

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Cancer Research

8.8K visualizações.  The Ohio State University Medical Center. A injeção intravenosa de células cancerígenas é frequentemente usada em pesquisas de metástase, mas a carga tumoral metastática pode ser difícil de analisar. Aqui, demonstramos um modelo de injeção de veias traseiras de metástase e incluímos uma nova abordagem para analisar a carga do tumor pulmonar metastático resultante.

Vídeo: Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

Ensaio de recrutamento de células de Tumor de pulmão

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Pesquisa do câncer

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Ensaio Metástase Experimental

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Medicina

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Um modelo de injeção da veia porta para o Estudo do Fígado metástase do câncer de mama

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

Considerações práticas em estudar a colonização de pulmão metastático em osteossarcoma usando o ensaio de metástase pulmonar

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

Ortotópico injeção de células de câncer de mama para a almofada de gordura mamária de ratos

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Uso combinado de ensaios de metástase de veia de cauda e imagens in vivo em tempo real para quantificar a colonização metastática do câncer de mama e carga nos pulmões

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Modelando Metástase Cerebral Via Injeção Venosa Caudal de Células Inflamatórias de Câncer de Mama

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

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 ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Injeção de células Singênico Murino melanoma para determinar o seu potencial metastático nos pulmões

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Análise patológica da metástase pulmonar após injeção lateral de cauda-veia de células tumorais

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