Agradecemos ao SF 4206 Icore (Université de Caen) para o uso da facilidade de citometria. JC recebeu bolsas de pós-doutorado do Conseil de Radioproteção d&#39;EDF e Conseil Régional de Basse-Normandie. Esses dados fazem parte de projetos financiados pelo Ligue Nationale contre le Cancer - Comités de l&#39;Orne et du Calvados.

1. Preparação de C12FDG, bafilomicina A1 e Cultura de Células <ol><li> Dissolve-se o pó C 12 FDG em dimetil sulfóxido (DMSO) a uma concentração final de 20 mM. Alíquota e armazenar a -20 ° C. DMSO deve ser utilizado com condições de segurança apropriadas. </li><li> Dissolve-se o pó A1 bafilomicina em DMSO para uma concentração final de 0,1 mM. Alíquota e armazenar a - 20 ° C. Bafilomicina deve ser utilizado com condições de segurança apropriadas. </li><li> Cultivar a linha de células RPMI 8226, ou outras linhas de células aderentes ou não, em meios contendo 10% de soro fetal bovino e 1% de antibióticos, a 37 ° C, 5% de CO 2. Estimar o número de células necessárias para o experimento. A fim de registar um número suficiente de eventos durante a análise de citometria de fluxo, 5 x 10 5 células são necessários. Para determinar a porcentagem de células senescentes, são necessárias três amostras de células: Amostra sem rótulo, as células não tratadas marcadas com C 12 FDG, células tratadas corados comC 12 FDG. </li><li> Sementes das células 24 h antes do experimento de realização de modo a que as células estão na fase de log da curva de proliferação celular. </li></ol> 2. A indução da senescência <ol><li> Induzir a senescência das células cancerosas, adicionando um medicamento quimioterápico. Concentração da droga e da duração do tratamento deve ser adaptado ao tipo de célula testada. Comece com um tratamento de 48 horas com a concentração final de 50 nM para a doxorrubicina numa concentração de células de cerca de 10 6 células / ml. Não se esqueça de incluir amostra não tratada (controle negativo). </li></ol> 3. Bafilomicina A1 Tratamento <ol><li> Verificar a viabilidade celular em células de mistura com azul de tripano (v / v), com o medicamento quimioterápico utilizado no passo anterior pode levar à apoptose celular. Encher uma câmara de Malassez e contar o número de células azuis (células mortas) e de glóbulos brancos (células vivas). Se a percentagem de viabilidade for inferior a 70%, as células mortas pode ser removed usando um kit adequado para assegurar que as células mortas não influenciará a análise subsequente. </li><li> Remova os meios de cultura e substituir por novos meios de cultura pré-aquecido. Tratar as células com uma concentração final de 100 nM de bafilomicina A1. Bafilomicina A1 é utilizado para neutralizar o pH ácido dos lisossomos. Incubar 1 hora a 37 ° C, 5% de CO 2. </li></ol> 4. C 12 FDG Coloração <ol><li> Dissolve-se 12 C FDG em meios de cultura fresco pré-aquecido, a uma concentração final de 2 mM. </li><li> Adicionar o composto às células na concentração final de 33 uM e incubar 2 h a 37 ° C, 5% de CO 2. Se utilizar as células não aderentes, centrifugar as células a 300 xg por 5 min a 4 ° C (a velocidade pode ter que ser ajustada para o tipo de célula utilizado), lavar 2x com PBS. Ressuspender o sedimento celular em 500 uL de PBS frio. Se estiver usando células aderentes, remova a mídia e lavar 2x com PBS. Colher as células aderentes utilizando uma solução de tripsina e centrifugaras células a 300 xg durante 5 min a 4 ° C (a velocidade pode ter que ser ajustada para o tipo de célula utilizado). Ressuspender o sedimento celular em 500 uL de PBS frio. Em ambos os casos, a concentração de células deve ser de cerca de 10 6 células / ml. </li><li> Realizar um co-imunomarcação para caracterizar ainda mais a população de células senescentes. </li></ol> 5. Análise de Citometria de Fluxo <ol><li> Calibrar o citómetro de fluxo, utilizando os grânulos de controlo recomendadas pelo fabricante do citómetro de fluxo. Para todas as etapas de, pelo menos, 10 eventos de 4 têm de ser registadas. </li><li> Executar a primeira amostra contendo células não marcadas. Prepare a dois parâmetros gráfico exibindo Canal Forward Scatter (FSC) versus Side Canal Scatter (SSC). Defina os tubos fotomultiplicadores (PMT) e definir uma região de interesse excluindo as células mortas e restos celulares que poderiam ter aparecido durante a preparação da amostra. Portão esta região de interesse de um histograma de um parâmetro FL1 exibindo na escala de logdo eixo x e o número de eventos no eixo y. O número total de eventos representa o número de células analisadas. Definir a PMT de modo que as células não marcadas aparecem na primeira década na escala de log do eixo x. Determinar a autofluorescência das células, se necessário. </li><li> Executar a segunda amostra contendo células não tratadas. No histograma um parâmetro exibindo FL1 (fluorescência de C 12 FDG), coloque o cursor após a população mal rotulados. Este limite vai permitir a discriminação entre células não senescentes (células mal rotulados) e células senescentes (células vivas rotulados). </li><li> Executar a terceira amostra contendo células tratadas. Brilhantemente células rotuladas, ou seja, células senescentes aparecem acima do limite determinado na etapa anterior. Avaliar a percentagem de células senescentes, dividindo-se o número de eventos brilhantes por o número total de eventos. Se necessário, a actividade de a-galactosidase β também pode ser estimada utilizando a média de fluorescênciaintensidade. </li></ol>

<ol>
	<li>Hayflick, L., Moorhead, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+serial+cultivation+of+human+diploid+cell+strains.">The serial cultivation of human diploid cell strains.</a> Experimental Cell Research. 25, 585-621 (1961).</li><li>Campisi, J., d'Adda di Fagagna, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+senescence:+when+bad+things+happen+to+good+cells.">Cellular senescence: when bad things happen to good cells.</a> Nature Review Molecular Cell Biology. 8 (9), 729-740 (2007).</li><li>Shay, J. W., Roninson, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+senescence+in+carcinogenesis+and+cancer+therapy.">Hallmarks of senescence in carcinogenesis and cancer therapy.</a> Oncogene. 23 (16), 2919-2933 (2004).</li><li>Bartkova, J., Rezaei, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogene-induced+senescence+is+part+of+the+tumorigenesis+barrier+imposed+by+DNA+damage+checkpoints.">Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.</a> Nature. 444 (7119), 633-637 (2006).</li><li>Takacova, S., Slany, R., Bartkova, J., Stranecky, V., Dolezel, P., Luzna, P., 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=DNA+damage+response+and+inflammatory+signaling+limit+the+MLL-ENL-induced+leukemogenesis+in+vivo.">DNA damage response and inflammatory signaling limit the MLL-ENL-induced leukemogenesis in vivo.</a> Cancer Cell. 21, 517-531 (2012).</li><li>Krtolica, A., Parrinello, S., Lockett, S., Desprez, P. Y., Campisi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescent+fibroblasts+promote+epithelial+cell+growth+and+tumorigenesis:+a+link+between+cancer+and+aging.">Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging.</a> Proceedings of the National Academy of Sciences. 98, 12072-12077 (2001).</li><li>Coppe, J. P., Patil, C. K., Rodier, F., Sun, Y., Munoz, D. P., Goldstein, J., 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=Senescence-associated+secretory+phenotypes+reveal+cell-nonautonomous+functions+of+oncogenic+RAS+and+the+p53+tumor+suppressor.">Senescence-associated secretory phenotypes reveal cell-nonautonomous functions of oncogenic RAS and the p53 tumor suppressor.</a> PLoS Biology. 6, 2853-2868 (2008).</li><li>Roninson, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+senescence+in+cancer+treatment.">Tumor cell senescence in cancer treatment.</a> Cancer Research. 63 (11), 2705-2715 (2003).</li><li>Dimri, G. P., Lee, X., 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=A+biomarker+that+identifies+senescent+human+cells+in+culture+and+in+aging+skin+in+vivo.">A biomarker that identifies senescent human cells in culture and in aging skin in vivo.</a> Proceedings of the National Academy of Sciences. 92 (20), 9363-937 (1995).</li><li>Kurz, D. J., Decary, S., Hong, Y., Erusalimsky, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+(beta)-galactosidase+reflects+an+increase+in+lysosomal+mass+during+replicative+ageing+of+human+endothelial+cells.">Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.</a> Journal of Cell Science. 113, 3613-3622 (2000).</li><li>Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., Toussaint, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+to+detect+senescence-associated+beta-galactosidase+(SA-betagal)+activity,+a+biomarker+of+senescent+cells+in+culture+and+in+vivo.">Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo.</a> Nature Protocols. 4 (12), 1798-1806 (2009).</li><li>Cahu, J., Bustany, S., Sola, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Senescence-associated+secretory+phenotype+favors+the+emergence+of+cancer+stem-like+cells.">Senescence-associated secretory phenotype favors the emergence of cancer stem-like cells.</a> Cell Death and Disease. Dec 20, 3-e446 (2012).</li></ol>

Não há conflitos de interesse declarados.

Apresentamos aqui um método baseado em citometria de fluxo para quantificar a senescência celular em células cancerosas vivas. Este método baseia-se na utilização do composto membrana permeável, a 12 C FDG, e pode, portanto, ser utilizado em qualquer tipo de célula e depois de vários tratamentos, desde que o composto é absorvido pelas células. Após a absorção celular, a 12 C FDG é hidrolisado pela β-galactosidase, enriquecido em lisossomas de células senescentes. Após excitação ...

Desde o início dos anos sessenta e sua descoberta por Hayflick e Moorhead, senescência ainda permanece uma curiosidade celular 1. As células humanas não indefinidamente proliferar e, por senescência, Hayflick e Moorhead cunhou o processo de envelhecimento celular. A senescência é uma paragem do ciclo celular estável em que as células permanecem metabolicamente activa em oposição à morte celular. Até à data, quatro mecanismos celulares têm sido mostrados para induzir a senescência: encurtamento dos telómeros, danos no ADN, perturbação da cromatina e a expressão anormal de oncogenes mitogénicos 2. Isto indica que não só as células normais, mas também as células cancerígenas são capazes de sofrer a senescência 3. Por uma questão de facto, as células de cancro sujeitos a senescência foram mostradas para constituir uma barreira para a progressão do tumor 4-5. No entanto, vários relatórios já apontou que, em determinadas circunstâncias, a senescência celular também pode promover malignidade 6-7. Estudar senescência de câncer cells está prestes a tornar-se um impulso na pesquisa do câncer, como atualmente utilizado medicamentos quimioterápicos podem levar a senescência das células cancerosas não apenas in vitro, mas também in vivo 8. O ensaio principal para investigar quanto à presença de células senescentes, é o ensaio de β-galactosidase a um pH ácido. Este ensaio histoquímico reflecte o aumento na biogénese lisossómica ocorrendo na maioria das células senescentes. Resumidamente, exógena de X-gal, aplicada às células, é clivada pela β-galactosidase enriquecido em lisossomas de células senescentes, dando origem a uma cor azul 9. As células senescentes azul podem então ser quantificado num microscópio de campo claro. Embora muito popular, o ensaio β-galactosidase apresenta grandes inconvenientes. Em primeiro lugar, este ensaio é efectuado em células fixadas. Em segundo lugar, é demorado porque implica para quantificar o grau de senescência por contagem de um número significativamente maior de células senescentes, sob um microscópio. Terceiro, este testeNão é tão sensível a discriminação entre as células senescentes e não senescentes inteiramente depende do observador. Discute-se aqui, um ensaio baseado em citometria rápido e sensível para avaliar inexplorado para a presença de células senescentes vivos. Este ensaio baseia-se na hidrólise de uma molécula da membrana permeável, o 5-di-dodecanoylaminofluorescein β-D-galactopiranósido (C 12 FDG), por β-galactosidase enriquecida em células senescentes. Depois da hidrólise e da excitação do laser, a 12 C FDG emite fluorescência verde e pode, portanto, ser detectada por citometria de fluxo 10, 11. A detecção de células senescentes através de citometria de fluxo tem a grande vantagem de ser rápida e sensível. Além disso, a detecção de células senescentes, por citometria de fluxo pode ser combinada com a detecção de outros marcadores celulares. Este ensaio pode ser usado para detectar células senescentes dentro de uma população de células cancerígenas tratadas com quimioterapia, e pode ser rotineiramente ajustadose em cortes de tecidos, após a dissociação celular, na clínica.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the Reagent</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>5-dodecanoylaminofluorescein di-&beta;-D-galactopyranoside </td>
			<td>Invitrogen</td>
			<td>D-2893</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dimethyl sulfoxide </td>
			<td>Sigma</td>
			<td>D2650</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bafilomycin A1</td>
			<td>Sigma</td>
			<td>B1793</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Doxorubicin</td>
			<td>Sandoz</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypan blue</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		 </tr>
		 <tr>
			<td>RPMI 1640 cell culture medium</td>
			<td>Lonza</td>
			<td>BE12-702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal calf serum</td>
			<td>PAA Laboratories GmBH</td>
			<td>A15 101</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Antibiotics</td>
			<td>Gibco</td>
			<td>5290-018</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Gallios flow cytometer</td>
			<td>Beckman Coulter</td>
			<td>A94291</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

a sensitive method to quantify senescent cancer cells

As células humanas não proliferar indefinidamente. Após a estímulos externos e / ou intrínseco, as células podem morrer ou entrar em uma parada do ciclo celular estável chamado de senescência. Vários mecanismos celulares, tais como o encurtamento dos telómeros e a expressão anormal de oncogenes mitogénicos, têm sido mostrados para causar a senescência. A senescência não está restrita às células normais, as células cancerígenas têm também sido relatada a senescência. Agentes quimioterápicos têm demonstrado induzir a senescência das células cancerosas. No entanto, permanece controverso se a senescência impede ou promove a carcinogênese. Como se pode, eventualmente, ser específico para cada paciente, um método rápido e sensível para avaliar a senescência celular no cancro logo será necessária. Para este fim, o ensaio padrão β-galactosidase, o método utilizado correntemente, apresenta inconvenientes importantes: é demorado e não é sensível. Propomos aqui um fluxo de ensaio baseado em citometria de estudar senescência em live células. Este ensaio tem a vantagem de ser rápido, sensível e pode ser acoplada ao imunomarcação de vários marcadores celulares.

Mieloma múltiplo, uma malignidade hematológica, foi utilizado para avaliar a senescência induzida por um procedimento quimioterápico. Para fazê-lo numa linha de células MM comercialmente disponível (RPMI 8226) foi tratado durante 2 dias com doxorubicina, um fármaco quimioterápico. As células foram então submetidas a um tratamento bafilomicina A1 e ainda incubadas com 12 C FDG. As células foram analisadas por citometria de fluxo. Figura 1 ilustra as diferentes etapas, e mostra que ...

Watch this Scientific Journal Video about A Sensitive Method to Quantify Senescent Cancer Cells at JoVE.com

A Sensitive Method to Quantify Senescent Cancer Cells

Se senescência impede ou promove tumorigenesis permanece controverso. Desde agentes quimioterápicos podem induzir as células cancerosas senesce, estudando senescência é essencial para propor novas terapias. No entanto, o ensaio de β-galactosidase padrão e amplamente utilizado apresenta grandes inconvenientes. Propomos aqui um fluxo de ensaio baseado em citometria rápido e sensível para quantificar a senescência.

Um método sensível para quantificar células senescentes Câncer

Human cells do not indefinitely proliferate. Upon external and/or intrinsic  cues, cells might die or enter a stable cell cycle arrest called senescence. ...

a-sensitive-method-to-quantify-senescent-cancer-cells

Research

JoVE Journal

Medicine

20.5K visualizações.  Université de Caen Basse-Normandie. Se senescência impede ou promove tumorigenesis permanece controverso. Desde agentes quimioterápicos podem induzir as células cancerosas senesce, estudando senescência é essencial para propor novas terapias. No entanto, o ensaio de β-galactosidase padrão e amplamente utilizado apresenta grandes inconvenientes. Propomos aqui um fluxo de ensaio baseado em citometria rápido e sensível para quantificar a senescência.

Vídeo: A Sensitive Method to Quantify Senescent Cancer Cells

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Um método para isolamento de ilhotas murino e transplante renal subcapsular

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Medicina

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

Microscopia Confocal de córnea: uma técnica não-invasiva Novel para Quantificar Pequenas Fibre Patologia em neuropatias periféricas

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

A técnica de impedância elétrica em tempo real baseado medir Invasão de monocamada de células endoteliais por células cancerosas

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

Um método simples parafuso Guia para intracraniana Estudos Xenoenxerto em Ratos

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Três Culturas dimensionais: Uma ferramenta para o estudo normal Acináceo Arquitetura vs transformação maligna das células de mama

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Método para obtenção de células do cancro do ovário primárias de amostras sólidas

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Um<em&gt; In Vitro</em&gt; Dormência Modelo de câncer de mama estrogênio-sensíveis na medula óssea: Uma Ferramenta para Estudos Moleculares Mecanismo e Hipótese Geração

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilização do ensaio de formação de colónia agar mole para identificar inibidores de tumorigenicidade em células de cancro da mama

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.