Name: pathological analysis of lung metastasis following lateral tail-vein injection of tumor cells
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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 c...

<ol>
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A., 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=Prognostic+impact+of+metastatic+pattern+in+stage+IV+breast+cancer+at+initial+diagnosis.">Prognostic impact of metastatic pattern in stage IV breast cancer at initial diagnosis.</a> Breast Cancer Research and Treatment. 161 (3), 537-548 (2017).</li><li>Pei, X. 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=Explant-cell+culture+of+primary+mammary+tumors+from+MMTV-c-Myc+transgenic+mice.">Explant-cell culture of primary mammary tumors from MMTV-c-Myc transgenic mice.</a> In Vitro Cellular and Developmental Biology: Animal. 40 (1-2), 14-21 (2004).</li><li>Mathsyaraja, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CSF1-ETS2-induced+microRNA+in+myeloid+cells+promote+metastatic+tumor+growth.">CSF1-ETS2-induced microRNA in myeloid cells promote metastatic tumor growth.</a> Oncogene. 34 (28), 3651-3661 (2015).</li><li>Yang, S., Zhang, J. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+tumor+metastasis.">Mouse models for tumor metastasis.</a> Methods in Molecular Biology. 928, 221-228 (2012).</li><li>La Perle, K. M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Pathologists:+Ultimate+Control+Freaks+Seeking+Validation.">Comparative Pathologists: Ultimate Control Freaks Seeking Validation.</a> Veterinary Pathology. 56 (1), 19-23 (2019).</li><li>Blomberg, O. S., Spagnuolo, L., de Visser, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+regulation+of+metastasis:+mechanistic+insights+and+therapeutic+opportunities.">Immune regulation of metastasis: mechanistic insights and therapeutic opportunities.</a> Disease Models &amp; Mechanisms. 11 (10), (2018).</li><li>Gonzalez, H., Hagerling, C., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+the+immune+system+in+cancer:+from+tumor+initiation+to+metastatic+progression.">Roles of the immune system in cancer: from tumor initiation to metastatic progression.</a> Genes and Development. 32 (19-20), 1267-1284 (2018).</li><li>Borowsky, A. 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=Syngeneic+mouse+mammary+carcinoma+cell+lines:+two+closely+related+cell+lines+with+divergent+metastatic+behavior.">Syngeneic mouse mammary carcinoma cell lines: two closely related cell lines with divergent metastatic behavior.</a> Clinical and Experimental Metastasis. 22 (1), 47-59 (2005).</li><li>Yang, Y., 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=Immunocompetent+mouse+allograft+models+for+development+of+therapies+to+target+breast+cancer+metastasis.">Immunocompetent mouse allograft models for development of therapies to target breast cancer metastasis.</a> Oncotarget. 8 (19), 30621-30643 (2017).</li><li>Resch, M., Neels, T., Tichy, A., Palme, R., Rulicke, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+assessment+of+tail-vein+injection+in+mice+using+a+modified+anaesthesia+induction+chamber+versus+a+common+restrainer+without+anaesthesia.">Impact assessment of tail-vein injection in mice using a modified anaesthesia induction chamber versus a common restrainer without anaesthesia.</a> Laboratory Animals. 53 (2), 190-201 (2019).</li><li>Rashid, O. 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. 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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>for cleaning tail prior to injection</td>
		</tr>
		<tr>
			<td>dissection scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-5</td>
			<td>for mouse dissection and lung tissue inflation</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&#39;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&#39;s fur during dissection; use caution - flammable</td>
		</tr>
		<tr>
			<td>Evan&#39;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&#39;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&#39;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 Video at JoVE.com

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells Video (Video) | JoVE

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.

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JoVE Journal

Cancer Research

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

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