The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

NOTA: colheita de tecidos do paciente, autorizado por conselho de revisão institucional (IRB) -aprovado Biospecimen e Protocolos Clínicos (números de protocolo 09-121 e 11-041, respectivamente) no Memorial Sloan Kettering Cancer Center. Procurement 1 Tissue <ol><li> Aquisição de paciente primário tumor / metástase NOTA: Até à data, este protocolo foi realizado em pancreáticos, gástricos e tipos de tumor da mama removidos cirurgicamente, bem como, as metástases de linfoma. <ol><li> Direcionar a equipe cirúrgica para entregar a amostra por correio ou sistema de tubos pneumáticos para o departamento de Patologia em hermeticamente fechado e estéril saco plástico espécime à prova de vazamentos dentro de recipiente à prova de derrame estéril. Direcionar a equipe cirúrgica para transportar a amostra no estado fresco, sem formol ou fixador. </li><li> Dirigir o patologista ou patologista assistente para colher a amostra frescos utilizando uma técnica estéril, que inclui uso de luvas e instrumentos esterilizados sob uma capela de fluxo laminar. </li><li> Anote o tempo de colheita das amostras adquiridas, que é mantido rigorosamente menos de 30 min a partir da conclusão do procedimento cirúrgico. Levando-se em conta os efeitos de isquemia fria, não tome amostras de espécimes ressecados, com um tempo de isquemia fria de&gt; 30 min 18. </li><li> Dirigir o pessoal do Departamento de Patologia para remover uma amostra do tumor primário de aproximadamente 0,5 cm3 a 1,0 cm 3 de uma câmara de fluxo laminar, para manter um ambiente estéril. Quando possível, escolher o tecido do tumor a partir da periferia da lesão, para evitar potenciais índice de necrose central (morte celular) franca. NOTA: O tecido necrótico pode ser grosseiramente reconhecível por qualquer um dos seguintes critérios: a perda de cor ou palidez do tecido; perda de força em que o tecido necrosado é macio e friável; uma demarcação clara entre o tecido necrosado e viável. </li><li> Dirigir o pathologiST ou assistente patologista para fornecer tecido periférico que é em excesso (descarte cirúrgico), após a recolha imediata do tumor para avaliação diagnóstica. Colocar a amostra num tubo de centrífuga cónico de 15 ml estéril contendo cerca de 5 ml de meio essencial mínimo (MEM) contendo 1% de antibióticos (penicilina e estreptomicina). </li><li> Se estiver disponível (por exemplo, linfonodo axilar de cauda de espécime mastectomia), adquirir um grosseiramente positivos espécime gânglio linfático do mesmo tamanho (0,5 cm 3 a 1,0 cm 3) e comparar a resposta à droga do tumor primário. Como as amostras de tumor primário, colocar as amostras de nódulos linfáticos associados em um tubo de centrífuga cónico de 15 ml estéril contendo cerca de 5 ml de meio essencial mínimo (MEM) contendo 1% de antibióticos (penicilina e estreptomicina). </li><li> Se o tamanho de licenças peça cirúrgica (por exemplo, espécime mastectomia), remova (distante do tumor primário) de uma amostra de tecido normal (ou seja,fibrosa parênquima normal densa / peito) e colocar em um tubo de centrífuga cônico de 15 ml estéril contendo aproximadamente 5 ml de meio essencial mínimo (MEM) contendo 1% de antibióticos apenas para fins de transferência. Após a transferência para as instalações de laboratório, transferir a amostra &#39;normal&#39; para um, congelar e armazenar cryovial &#39;estalo&#39; em um freezer -80 ° C para análises moleculares futuras. </li><li> Coloque todas as amostras em gelo molhado e transportar imediatamente para o espaço de laboratório para ex vivo de corte de tecido fresco. Não secção do espécime normal, mas sim armazenar em um freezer -80 ° C para análises moleculares futuras. Transfira uma porção do tumor primário e de metástases associadas a um, congelar e armazenar criotubo &quot;pressão&quot; de uma temperatura de -80 ° C congelador para futuras análises moleculares. </li></ol></li><li> Aquisição de tecido a partir de estudos clínicos (por exemplo, biópsias com agulha grossa opcional (CNB)) ATENÇÃO: Nós obter tecidos tumorais de pacientesmatriculados em protocolos clínicos que dão o seu consentimento se submeter a uma CNB de uma metástase de tumor acessíveis antes do início da terapia. CNBS, até à data, têm sido realizados em pacientes com câncer de mama, linfoma da zona marginal, linfoma de células do manto, paraganglioma metastático, colo do útero (de células escamosas) e ovário. <ol><li> Ter pessoal clínico de Radiologia Intervencionista ou departamento competente realizar uma biópsia com agulha pré-tratamento e colocar a amostra em um tubo de centrífuga de 15 ml cônico estéril contendo aproximadamente 5 ml de meio essencial mínimo (MEM) contendo 1% de antibióticos (penicilina e estreptomicina). </li><li> Coloque a amostra de biópsia no gelo molhado e transportar imediatamente para o espaço de laboratório para ex vivo de corte de tecido fresco. </li></ol></li></ol> 2 Preparação de materiais para corte e Vibratome Configurações <ol><li> Elaboração de Materiais para Seccionamento <ol><li> Adicione uma solução de agarose a 4% por adição de 4 g de agasubiu para 100 ml de PBS. Autoclave (configurações de ciclo: Líquido, 121 ° C, 30 min, 15 libras por polegada quadrada), a solução antes do primeiro uso. Para utilizações seguintes, coloque a agarose 4% em um micro-ondas para liquefazer (fogo alto por intervalos de 30 segundos) antes de cada utilização e resfriar adequadamente para ~ 25 ° C (ou seja, quente ao toque) consciente de que agarose solidifica à temperatura ambiente. </li><li> Use a agarose 4% como uma mídia tecido incorporação não permanente. Quando a agarose 4% esfriou o suficiente e ainda está em estado líquido, com uma pinça estéril, colocar as amostras de tecido em um molde de incorporação e despeje a agarose 4%. </li><li> Posicione a amostra de tecido estreitas (~ mm distância 2), mas não diretamente na parede superfície de corte do molde de incorporação. A agarose a 4% começa a solidificar rapidamente, por conseguinte, a posição apropriada do espécime de tecido rápida. Durante o corte, manter a agarose 4% em um banho de água a 55 ° C para se manter em solução. </li><li> Para uma placa de 24 poços para análise secção, adicionar 1 ml de of meio completo (ou seja, de MEM + 10% de soro fetal de bovino 1% + antibióticos) a cada poço. Manter a placa de 24 cavidades à temperatura ambiente (~ 25 ° C) durante toda a duração do seccionamento. </li></ol></li><li> Preparação de Configurações Vibratome <ol><li> Usando o micrótomo vibratório, definir a velocidade a que a lâmina se aproxima da amostra [0 (0,00 mm / seg) a 10 (2,5 mm / seg)] e a frequência da lâmina [0 (0 Hz) a 10 (100 Hz) ]. Ajuste as configurações de acordo com a textura do tecido (ou seja, a densidade / firmeza). </li><li> Para um tumor de mama primário de carcinoma ductal invasivo, ajuste as configurações de 6 e 3, velocidade e freqüência, respectivamente. Para o tecido do tumor menos denso / firme, diminuir a velocidade e aumentar a frequência em conformidade. </li><li> Defina a espessura de corte dentro dos limites do instrumento (1-999 mm). Para estas análises, definir a espessura de corte de 200 m. </li><li> Montar o espécime embebidas em agarose utilizando uma fina camada de cola líquida sobre o disco de amostra that está submerso em um reservatório de mídia que é envolto em gelo molhado. </li></ol></li></ol> 3. Tissue Seccionamento, Tratamento e Análise <ol><li> Remova os cortes cortado com precisão imediatamente após corte usando uma pinça estéril e coloque em um prato multi-bem com 1 ml de meio completo. Quando as secções são adquiridos suficientes para resposta e análises de correlação, colocar a placa de multi-poços de resposta ao tratamento em uma incubadora de cultura de tecido fixado em condições de cultura de tecidos padrão. </li><li> Tire várias secções de controlo que bookend as secções tratadas em um esforço para confirmar a resposta dependente da dose, em oposição aos artefactos cultivadas. Realizar tecido seccionamento rapidamente (~ 30 - 40 min), bem dentro de quaisquer efeitos prejudiciais de tempo de isquemia fria (~ 2-4 hr) 18. Realizar o tratamento de secções só depois de um período de aclimatação de 1 hora. </li><li> Após o período de aclimatação de 1 hora, adicionar (doses terapeuticamente relevante aumentando; <em.&gt; Por exemplo, 0-10 mM concentrações) de drogas para as seções e casa em condições de cultura de tecidos para o período de tratamento. Para este estudo, utilizar períodos de avaliação de 24 horas e / ou 48. No entanto, cortes de tecidos de controle (veículo) só vai permanecer viável, como testado neste estudo, por períodos de até e incluindo os períodos de avaliação de 96 horas. </li><li> Após o tratamento, remover todas as secções em condições estéreis e formalina-fix, colocando a secção de tecido em um tubo contendo quer 10% de formalina tamponada e lugar à TA S / N ou em um tubo contendo paraformaldeído a 4% (16% de paraformaldeído diluir a partir de um frasco com PBS para uma concentração final de 4%) e lugar à temperatura ambiente durante 2 horas. <ol><li> Fixar as secções de tecido maiores (por exemplo,., ~ De 6 mm x 4 mm) de 200 um de espessura em 10% de formalina tamponada O / N à temperatura ambiente. Fix secções menores (por exemplo,. 1 mm x 1 mm) de 200 um de espessura, em 4% de paraformaldeído durante duas horas à temperatura ambiente. NOTA: Para a penetração eficiente de fixador o volume de fixative deve ser 20x o volume do tecido. Dado que este protocolo resulta em tamanhos extremamente pequenos de tecido (por exemplo, de 6 mm de tecido 3 = 6 ul de volume), para garantia de utilização adequada fixação 1 ml de fixador para todos os tamanhos de tecido. </li><li> Após a fixação ter as amostras de tecido embebidas em parafina pelo pessoal do Departamento de Patologia. Seção espécime blocos de parafina em lâminas carregadas ser hematoxilina e eosina e avaliadas por imunohistoquímica (por exemplo, apoptose). </li></ol></li></ol>

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No conflicts of interest disclosed.

Biólogos câncer enfrentam desafios significativos ao tentar desenvolver estratégias terapêuticas eficazes. Testar medicamentos em desenvolvimento em linhas celulares de cancro estabelecidas não pode refletir com precisão em resposta vivo e in vivo em modelos PDX são trabalhoso e muito caro. Dado o acima, a aplicação de técnicas ex vivo de tumores primários do paciente 14, 15 está agora posicionada ao lado da análise molecular das linhas celulares de cancro e xenoenxerto...

O desenvolvimento de cancro terapêutica eficazes provou ser extremamente desafiador. Linhas de câncer de células tumorais e explantes - bem como enxertos foram usados ​​em pesquisa sobre o câncer há mais de meio século 1,2,3. Até à data, a análise molecular de sensibilidade à droga e resistência em ambas as linhas celulares de cancro e xenoenxertos estabelecidas derivadas de pacientes (PDX) é indispensável. No entanto, o teste de compostos em linhas celulares de cancro estabelecidas muitas vezes não é de previsão de eficácia in vivo, e estudos in vivo correspondente em animais, especialmente em modelos de PDX, é muito cara e demorada. As limitações destes sistemas modelo, ou seja, a incapacidade para informar sobre a influência do microambiente nativo em progressão do tumor e a resposta de estratégias terapêuticas, levou o campo de investigação para o desenvolvimento de métodos adicionais para complementar estas análises. De recente, maior atenção está sendo pago para ex vivo de análise de tum pacienteou explantes 4, 5, devido à maior compreensão de que a resposta terapêutica do câncer não é exclusivo para a composição molecular inerente das células cancerosas mas é muito influenciada pelo microambiente celular tumor 6, 7 um recurso que não pode ser reproduzido por meio de métodos e de cultivo tradicionais / ou PDX. análise ex vivo, no contexto acima (ie., influência do microambiente adjacente célula tumoral circundante) implica a avaliação de seções tumor / metástase de primário viáveis, ao invés de ex vivo de análise de isolados celulares 8, 9. Registramos aqui uma técnica ex vivo (ie., Seções cortado com precisão frescas de tecidos) de ambos os tumores primários de pacientes e metástases associadas (ou seja, os gânglios linfáticos), que informa fielmente na resposta (IC50), efeitos fora do alvo e permite molecular análise dos mecanismos de resistência e de feedback. Além disso, uma análise correlativa de therapeUTIC sensibilidade / resistência contra biomarcador e perfil de expressão do gene pode ser realizada em um esforço para identificar os pacientes com maior probabilidade de responder à droga experimental de interesse (ou seja., resposta de droga elevada corresponde paciente com particular perfil biológico). Aplicação da técnica ex vivo e avaliação de uma forma multi-parâmetro é movimento para a seleção dos pacientes e melhoria global dos resultados clínicos. Análise ex vivo da resposta ao tratamento pode se tornar uma ferramenta padrão no desenvolvimento pré-clínico e clínico da terapêutica do câncer e é visto como um passo em direção a uma abordagem da medicina personalizada em estratégias de desenvolvimento de terapias.

<table><tbody><tr><td>Vibratome Leica VT1000s</td><td>Leica</td><td>14047235613</td><td></td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 4% and 6% before use</td></tr><tr><td>Injector blade</td><td>Ted Pella</td><td>121-4</td><td></td></tr><tr><td>MEM with Penicillin&amp;nbsp; + Streptomycin</td><td>Media Core Facilities (MSKCC)</td><td></td><td>The media is prepared at Memorial Sloan Kettering Cancer Center&amp;nbsp;</td></tr><tr><td>Scalpel no. 10</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Disposable forceps</td><td>Cole-Parmer</td><td>84011182</td><td></td></tr><tr><td>Embedding mold</td><td>Electron Microscopy Science</td><td>70181</td><td></td></tr><tr><td>FBS (heat inactivated)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>24 well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Formalin (10%)</td><td>Sigma Diagnostics</td><td>SDHT501128&amp;nbsp;</td><td></td></tr><tr><td>16% Formaldehyde solution</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Embedding microsettes</td><td>Simport</td><td>M503-2</td><td></td></tr><tr><td>Ethanol (70%)</td><td>Fisher Scientific</td><td>A405P-4</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td>15-462-2SQ</td><td></td></tr><tr><td>Microwave</td><td>General Electric</td><td>ModelJES2051DNBB</td><td></td></tr><tr><td>Adhesive (Ethyl Cyanoacrylate)</td><td>Sigma-Aldrich</td><td>E1505-5G</td><td></td></tr><tr><td>10 mm dishes</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td></td></tr></tbody></table>

ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Para este estudo, a técnica ex vivo foi utilizado na análise de um correlativo de terapêutica sensibilidade / resistência de um inibidor da proteína de choque térmico 90 (Hsp90i). Em uma avaliação pré-clínica deste Hsp90i, o tumor primário de câncer de mama, um ER + ductal carcinoma invasivo (IDC), e associado metástases em linfonodos foram analisados ​​ex vivo para a resposta ao tratamento &#160; (Figura 1). Vários 200 mM cortes seriados foram tratad...

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Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Linhas celulares de cancro estabelecidos e xenotransplantes têm sido o esteio da pesquisa do câncer para as últimas décadas. No entanto, evidências recentes sugerem que a resposta terapêutica é muito influenciada pelo microambiente de células tumorais. Portanto, foi desenvolvida uma análise ex vivo de amostras de tumor primário para fins de desenvolvimento de drogas.

Ex Vivo resposta ao tratamento de tumores primários e / ou Associated Metástases para pré-clínica e clínica de Desenvolvimento Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

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Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

14.7K visualizações.  Memorial Sloan Kettering Cancer Center.  Linhas celulares de cancro estabelecidos e xenotransplantes têm sido o esteio da pesquisa do câncer para as últimas décadas. No entanto, evidências recentes sugerem que a resposta terapêutica é muito influenciada pelo microambiente de células tumorais. Portanto, foi desenvolvida uma análise ex vivo de amostras de tumor primário para fins de desenvolvimento de drogas. 

Vídeo: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

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

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

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

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Medicina

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Geração de tumores pancreáticos ortotópicos e caracterização ex vivo da citotoxicidade de células T infiltrada tumoral

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Pesquisa do câncer

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

Testando terapias-alvo em câncer usando análise estrutural de alteração de DNA e xenoenxertos derivados do paciente

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

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 ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Medição da resposta de drogas em tempo real em fatias de tecido tumoral organotípico

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Transplante Ortotópico de Tumores mamários como Modelos Pré-Clínicos para o Câncer de Mama

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

Explantas tumorais derivadas do paciente como uma plataforma pré-clínico "viva" para prever a resistência a medicamentos em pacientes

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Sensibilidade de perfil a terapias-alvo em organoides derivados do paciente NSCLC EGFR-Mutant

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Triagem de Drogas Organoides Tumorais Multiparamétricas Usando Imagem de Células Vivas de Campo Amplo para Análise a Granel e Organoide Único

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Avaliar a eficácia do Cancer Drogas Sensibilização<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Ex Vivo resposta ao tratamento de tumores primários e / ou Associated Metástases para pré-clínica e clínica de Desenvolvimento Therapeutics

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