A pesquisa relatada nesta publicação foi apoiada pelo Centro Nacional de Promoção de Ciências Translacionais sob o prêmio número UL1TR003096 (MDE), National Heart, Lung, and Blood Institute Predoctoral Fellowship in Lung Diseases Training Program 5T32HL134640 (MLD).

Todos os métodos descritos aqui foram aprovados pelo Comitê Institucional de Cuidados e Uso de Animais (IACUC) da Universidade do Alabama em Birmingham.
1. Protocolo experimental
<ol><li>Sacrifique o mouse usando um método IACUC aprovado. Aqui, usamos luxação cervical de um camundongo anestesiado com 5% de isoflurane. Use um mouse apropriado para o estudo; aqui, usamos um mouse FVB de 8 semanas de idade</li>
	<li>Utilizando uma tesoura cirúrgica, faça uma incisão horizontal de 3,5-5 mm no meio do abdômen inferior. Em seguida, insira uma tesoura cirúrgica no pequeno orifício criado a partir da incisão e corte verticalmente até a linha média central até um pouco abaixo do pescoço do mouse.</li>
	<li>Puxe a pele para trás com os dedos e inspecione os linfonodos axilares.</li>
	<li>Usando uma tesoura cirúrgica, faça uma incisão lateral de 3,5 mm para abrir a cavidade abdominal e, em seguida, corte na direção anterior até o fundo do tórax. Inspecione os órgãos na cavidade abdominal: fígado, baço, rins, etc.</li>
	<li>Com o plano da tesoura cirúrgica, ou usando fórceps, mova o fígado para expor o diafragma. Inspecione o diafragma para crescimento do tumor ou metástases. Em seguida, corte suavemente o diafragma no lado direito do operador, permitindo que ele se expanda. Corte suavemente o diafragma da direita para a esquerda para expor a cavidade torácica e os pulmões. Tenha cuidado para não cortar os pulmões.</li>
	<li>Corte através do extremo lateral da caixa torácica esquerda (à direita do operador) para inspecionar o lobo esquerdo dos pulmões.</li>
	<li>Mova suavemente os lóbulos direito do pulmão para fora do caminho e corte o extremo lateral da caixa torácica direita e remova a caixa torácica. NOTA: A remoção da caixa torácica é opcional, embora a remoção permita uma visão mais clara da inflação pulmonar posterior.<ol>
<li>Se for necessário tecido pulmonar fresco ou congelado, use fórceps hemostat para fixar os brônquios do lobo esquerdo e ressecular o pulmão esquerdo usando uma tesoura cirúrgica antes da perfusão.</li>
	</ol>
</li>
	<li>Usando os fórceps para levantar o tecido que cobre a traqueia, corte qualquer excesso de tecido. Em seguida, corte suavemente o tecido fino que reveste a traqueia para expor as vias aéreas.</li>
	<li>Corte a artéria renal com uma tesoura cirúrgica.</li>
	<li>Para permear os pulmões, use uma seringa de 3 mL com uma agulha de 22 G para injetar 1x PBS com heparina de 10 U/mL no ventrículo direito do coração. Perfundir lentamente os pulmões a aproximadamente 300 μL/s com PBS/heparina. Os pulmões ficam brancos. 2,5 mL de PBS são geralmente usados nesta etapa.</li>
	<li>Para a inflação pulmonar, use uma seringa de 3 mL com uma agulha de 22 G, desta vez mantida paralela à traqueia. Insira a agulha na traqueia e injete 10% de formalina com taxa de fluxo não superior a ~200 μL/s até que os pulmões tenham inflado completamente. Uma vez que os pulmões são inflados, formalina vai recuar para fora da traqueia. Segure a agulha no lugar por mais alguns segundos e depois retire-se.<ol>
<li>(Opcional) Antes de retirar a agulha e a inflação pulmonar, use linha de sutura para amarrar a traqueia. Para isso, use 4 polegadas de rosca de sutura segurando o ponto da rosca com um pequeno par de fórceps. Coloque a rosca no lado dorsal da traqueia e puxe para fazer um loop em torno da agulha. Em seguida, faça um nó de mão em torno da agulha. Puxe o nó apertado, retire a agulha da traqueia, feche o nó.</li>
	</ol>
</li>
	<li>Use fórceps para levantar o coração, inserir tesouras cirúrgicas diretamente atrás dos pulmões e cortar tecido conjuntivo enquanto o lifting do coração para ressecção dos pulmões.</li>
	<li>Corte o coração para removê-lo dos pulmões.</li>
	<li>Coloque os pulmões com um rotulado com o ID do mouse ou estude o ID. Coloque o em formalina 10% tamponada e fixe por 24-48 h. Os pulmões podem ser deixados em fixação por mais de um ano, se desejar.</li>
	<li>Transfira o contendo os pulmões para 70% de etanol e prossiga para o processamento para histologia.</li>
</ol>

<ol>
	<li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R., Structure, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Vasilescu, D. M., Knudsen, L., Ochs, M., Weibel, E. R., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography.">Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.</a> Journal of Applied Physiology. 112 (1), 159-166 (2012).</li><li>Blumler, P., Acosta, R. H., Thomas-Semm, A., Reuss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+for+the+preservation+of+air+spaces.">Lung fixation for the preservation of air spaces.</a> Experimental Lung Research. 30 (1), 73-82 (2004).</li><li>Oldmixon, E. H., Suzuki, S., Butler, J. P., Hoppin, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions.">Perfusion dehydration fixes elastin and preserves lung air-space dimensions.</a> Journal of Applied Physiology. 58 (1), 105-113 (1985).</li><li>Braber, S., Verheijden, K. A., Henricks, P. A., Kraneveld, A. D., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 299 (6), 843-851 (2010).</li><li>Renne, R., 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=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Limjunyawong, N., Mock, J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.+A+comparison+of+eleven+laboratory+species.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage. A comparison of eleven laboratory species.</a> American Review of Respiratory Disease. 132 (5), 1078-1083 (1985).</li><li>Edmonds, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer.">MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer.</a> Journal of Clinical Investigation. 126 (1), 349-364 (2016).</li><li>Zhao, 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=Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways.">Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.</a> PLoS One. 9 (9), 106458 (2014).</li></ol>

Os autores não têm nada a revelar.

O procedimento descrito acima para a perfusão, inflação e fixação de pulmões de camundongos é ideal para uma preparação rápida e eficiente dos pulmões do camundongo para histologia de tumores pulmonares e análise patologia. O procedimento não requer nenhum equipamento especial e pode ser realizado em menos de 6 minutos por mouse. O procedimento não requer um volume fixo para inflação nem pressão constante do fluido. Como este procedimento não é padronizado, não é recomendado para aqueles que desejam ...

Uma variedade de modelos de camundongos de oncogênese pulmonar e metástase de câncer para o pulmão existem que vão desde GEMMs complexos até modelos induzidos por cancerígenos até modelos sinéticos e xenoenxertos, onde as células cancerígenas são injetadas via intracardiac, intrathoracic, a veia da cauda ou outros métodos para estabelecer tumores dentro do pulmão. Todos esses modelos compartilham a necessidade comum de avaliação histológica da histologia pulmonar e da patologia. Assim, é necessário ter um método robusto, porém rápido, para realizar necropsias de camundongos enquanto perfumia os pulmões para remover o excesso de sangue, e inflar e fixar os pulmões para visualizar claramente a arquitetura pulmonar. A velocidade é um componente crítico deste procedimento, pois pode ser necessário coletar os pulmões de dezenas de ratos em um único ponto de tempo. Este procedimento pode ser realizado em menos de 6 minutos por mouse.
Embora este procedimento seja mais do que suficiente para avaliar a histologia tumoral, não é recomendado para aqueles que desejam realizar medições estereométricas ou morfômicas dos pulmões. Tais medidas exigem que a inflação pulmonar seja padronizada, assim como o cálculo da área de superfície absoluta do pulmão, volume absoluto e tamanho alveolar e número1. Este método também não é ideal para algumas abordagens de imagem. Por exemplo, a imagem dos pulmões via μCT para análise morfométrica ex vivo requer que os pulmões permaneçam preenchidos com ar2. Quando a preservação dos espaços e dimensões do ar são a principal preocupação, recomenda-se fixar os pulmões por técnicas de desidratação por perfusão3,4. Uma das maiores preocupações desse modelo é o potencial de ruptura das paredes alveolares, diminuindo seu uso em estudos de enfisema; no entanto, o procedimento recomendado para fixação de pulmões para o estudo do enfisema ainda é bastante semelhante, pois recomenda-se fixar os pulmões por instilação intratraqueal de 10% de formalina (semelhante ao protocolo descrito aqui) sob pressão constante do fluido ou por fixação in situ5.
A vantagem do procedimento descrito aqui é que ele não requer pressão constante do fluido, em vez de inflar os pulmões até que eles tenham se expandido totalmente, diminuindo assim o tempo necessário para o procedimento. O procedimento aqui descrito se assemelha muito aos métodos recomendados por um armamento da Sociedade de Patologia Toxicológica, onde um subcomitê foi formado para recomendar os melhores métodos de fixação pulmonar para estudos toxicológicos. A maioria dos cientistas deste subcomitê recomendou a fixação dos pulmões por instilação intratraqueal com uma seringa, embora houvesse recomendações variadas sobre o tempo que o pulmão foi deixado na fixação6. Assim, enquanto existe uma variedade de métodos de inflação e fixação pulmonar, o método descrito aqui é proposto para ser o método ideal para inflar rapidamente e fixar os pulmões para avaliação histológica do tumor a jusante.

<table>
	<tbody>
		<tr>
			<td>10% buffered formalin</td>
			<td>Fisher</td>
			<td>23-245685</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Needle</td>
			<td>BD</td>
			<td>305155</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon</td>
			<td>2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8595</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisher</td>
			<td>H19</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusion and inflation of the mouse lung for tumor histology

A capacidade de avaliar a histologia pulmonar é fundamental para os campos da pesquisa sobre câncer de pulmão e metástase do câncer. É igualmente importante realizar necropsias de forma rápida e eficiente a partir de estudos sem sacrificar a qualidade dos tecidos adquiridos. O objetivo deste protocolo é apresentar um método para perfusear rapidamente, inflar e fixar pulmões de camundongos para análise histológica a jusante. Este método não padroniza a inflação pulmonar; assim, não requer procedimentos ou equipamentos especiais e, em vez disso, simplesmente instila fixação diretamente através da traqueia após a perfusão através do coração. Isso permite uma estimativa suficiente do tamanho do tumor, histologia e pontuação. Isso também permite a coleta de tecido congelado antes da fixação do tecido pulmonar. Este método é limitado na forma de não permitir quantificação morfométrica posterior do pulmão; no entanto, é mais do que suficiente para a análise de tumores pulmonares de modelos de camundongos geneticamente modificados (GEMMs), modelos singênicos, bem como estudos de tumor xenoenxerto e metástase.

O protocolo acima permite a perfusão rápida, inflação e fixação de pulmões de camundongos. Os números abaixo representam a importância de cada etapa. A Figura 1 retrata os pulmões manchados de H&E que foram perfundidos com PBS e pulmões nos quais o passo de perfusão foi ignorado ou os pulmões não conseguiram perfumar corretamente. Como mostrado, o excesso de sangue nos pulmões mal perfundidos cria histologia menos do que ideal e pode torná-lo desafiador observar totalmente a ...

Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Perfusion and Inflation of the Mouse Lung for Tumor Histology

O objetivo deste método é apresentar um método simples e eficiente para a perfusão, inflação e fixação de pulmões de camundongos para o exame da patologia do tumor pulmonar e avaliação de metástases no pulmão.

Perfusão e Inflação do Pulmão do Rato para Histologia tumoral

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

perfusion-and-inflation-of-the-mouse-lung-for-tumor-histology

Research

JoVE Journal

Cancer Research

17.6K visualizações.  University of Alabama at Birmingham. O objetivo deste método é apresentar um método simples e eficiente para a perfusão, inflação e fixação de pulmões de camundongos para o exame da patologia do tumor pulmonar e avaliação de metástases no pulmão.

Vídeo: Perfusion and Inflation of the Mouse Lung for Tumor Histology

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

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

O Colon-26 Carcinoma Tumor-rolamento Mouse como um modelo para o estudo do câncer caquexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Amplificação de genoma pré-enriquecimento e toda célula alvo para jusante caracterização única célula

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

O estabelecimento de um ensaio de colonização de pulmão para visualização de células de Tumor nos tecidos do pulmão de circulação

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

Utilizando 18F-FDG PET-CT Imaging e histologia quantitativa para medir mudanças dinâmicas no metabolismo glicose em modelos de Mouse de câncer de pulmão

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Modelo de pulmão acelular e celular para estudar o Tumor metástase

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

Ensaio de reação em cadeia de polimerase digital para a variação genética em paciente esporádicos polipose adenomatosa familiar usando o formato de microplaqueta-em-um-tubo

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Detecção de Progressão do Tumor Pulmonar em Camundongos por ultrassom

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

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

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

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 ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Geração de Xenoenxertos Larval de Zebrafish e Análise do Comportamento do Tumor

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...