Fontes de financiamento da pesquisa: um fundo comum de empresa da Universidade de Tel Aviv transferência de tecnologia, RAMOT eo Centro-Sheba Medical (M. CA e SI.), ICRF - Câncer israelense fundação de pesquisa (M. CA.) E Fundação Israel ciência ( SI).

1. Cultura celular Preparação MDA-MB-231 de células foram adquiridos a partir da ATCC (American Type Culture Collection) e armazenada em azoto líquido. <ol><li> Semente 10 6 MDA-MB-231 células em 92 mm de diâmetro, uma placa de Petri em 10 ml de meio completo contendo Dulbeco Modified Eagle Médium (DMEM), 10% de soro de cavalo, 1% de L-glutamina, e 1% de Penstrep-Anfotericina B. Permitir células a proliferar até cerca de 80-100% de confluência. </li><li> Remover meio de cultura de prato e descarte. </li><li> Lava-se a camada de células brevemente com 0,25% (w / v) de solução de tripsina-EDTA para remover todos os vestígios de soro. </li><li> Adicionar 2,0 ml de solução de tripsina-EDTA para prato e observar as células por microscópio invertido até a camada de células é disperso (geralmente dentro de 5 a 15 min.) </li><li> Adicionar 18 ml de meio de crescimento completo e aspirar suavemente as células com uma pipeta. Transferir para um tubo. </li><li> Centrifugar a suspensão de células a 1.200 rpm. </li><li> Re-suspender o sedimento celular em 24 ml de cuLTURE médio. </li><li> Adicionar 2 ml de suspensão de células a 35 pratos de vidro de fundo mm (cerca de 25% de confluência) e colocar em incubadora (5% CO2, 37 ° C). </li><li> Soluções: <ol><li> Meio Completo para a proliferação de células: DMEM com 10% de FBS, 1% de antibióticos (100 unidades / ml de penicilina G, 100 ug / ml de estreptomicina, a solução de Pen-Strep-ampho) e 2 mM de L-glutamina. </li><li> Solução de tripsina-EDTA, com 0,25% de tripsina-EDTA. </li></ol></li><li> Pratos: 92 milímetros de diâmetro placas de Petri. 35 mm de diâmetro com poli-D-lisina de vidro revestida de placas de cultura de fundo. </li></ol> 2. Preparação de células para imagem confocal ao vivo <ol><li> Semente de 2 x 10 5 MDA-MB-231 células em placas de cultura de vidro de fundo em 2 ml de meio completo, como mencionado na seção 1. Quando a cultura de células atinge confluência de 60-70% (cerca de 3-4 x 10 5 células por prato), prossiga com a transfecção. </li><li> Transfecção as células com dois plasmídeos Encodção das proteínas de fusão γ-tubulina-GFP (para detecção de fluorescência de centrossomas) e histona-RED (H2b-RED, para a detecção de fluorescência de cromossomas), utilizando o reagente de transfecção lipossómica Jet-PI, seguindo o protocolo de fabricação. Resumidamente, mistura de 2 ug de cada plasmídeo em um tubo com 100 ul de NaCl (150 mM). Misture o reagente de transfecção (100 ul) com 100 ul de NaCl (150 mM) em um segundo tubo, e incubar 5 minutos à temperatura ambiente (RT). Em seguida, combinar as duas soluções, misture (usando vórtice leve) e spin-down. Incubar durante 30 minutos à temperatura ambiente. </li><li> Durante a incubação da mistura de transfecção, lave as células uma vez com PBS e substituir o meio de células com 2 ml de DMEM morno sem suplementos (37 ° C). </li><li> Adicionar cuidadosamente a mistura de transfecção às células em DMEM e em seguida as células voltem para a incubadora (37 ° C, 5% de CO 2) durante 8 horas. </li><li> Após 8 horas de incubação, substituir o DMEM com 2 ml de meio completo e incubar as células na incubadora durante 24hr. </li><li> 24 horas após a transfecção, o meio de substituir as células com 2 ml de meio completo contendo 20 uM PJ-34. </li><li> Incubar as células durante mais 18 horas (37 ° C, 5% de CO 2). </li><li> Sujeitar as células a viver imagem confocal por pelo menos 16 horas em câmara de imagem mantendo as células em 5% de CO 2 e 37 ° C. </li><li> Paralelamente, examinar a eficácia de transfecção 36 horas após a transfecção por microscopia de fluorescência como segue: <ol><li> Semente de 2 x 10 5 MDA-MB-231 células em placas de 6 poços contendo uma lamela por poço em 2 ml de meio completo. </li><li> Transfectar as células, como mencionado em secções de 2,2-2,5. </li><li> 36 horas após a transfecção as células transfectadas fixar montados numa lamela por incubação em metanol: acetona solução fria (1:1), 7 min, -20 ° C. </li><li> Aspirar a solução de fixação e deixar a lamela com as células montadas para secar em uma capa química. </li><li> Aplicar Prolongar reagente antifade ouro com DAPI e deixe ªlamela e secar no escuro durante 6 horas. </li><li> Examine a lâmina sob o microscópio fluorescente e calcular a porcentagem das células transfectadas (sinais vermelho e verde) da população total de células (coloração DNA por DAPI). A percentagem de transfecção desejada é de cerca de 20-40% quando 100-200 células são contadas. </li></ol></li></ol> 3. Parâmetros técnicos do confocal Configurações do scanner imagens ao vivo <ol><li> ScanMode XYZT; Pinhole [arejado] 1,00; Zoom 3,5; Resolução de 8 bits; DPSS 561 nm; argônio, laser visível 488 nm; laser He / Ne visível 633 nm; Objetivo HCX PL APO CS 63x 1,40 OIL UV; Abertura numérica 1,4; Velocidade de digitalização de 700 Hz, índice de refração 1,52. </li><li> Imagem apresentação 3-D foram preparadas por Imaris imaging software 7.0. </li></ol> 4. Imagem confocal de mitose em células fixas <ol><li> Semente de 2 x 10 5 MDA-MB-231 células de câncer de mama (ATCC), fibroblastos embrionárias de camundongos normais (MEF), ou PARP1 deficiente MEF (PARP - / -, preparado pelo Dr. Françoise Dantzer) em lamelas de vidro em placas de 6 cavidades em 2 ml de meio completo. As lamelas foram lavadas com etanol a 96%, seguido de lavagem com água estéril DD, secou-se durante 2 horas e colocada em cada poço do prato de 6 poços. </li><li> Adicionar PJ-34 (10-30 uM) ao meio e incubar as células durante o tempo necessário (normalmente até 96 horas). </li><li> Lavar as lamelas uma vez com PBS (tampão fosfato salino), e fixar as células usando a incubação em metanol arrefecido em gelo: solução de acetona (1:1), 7 min, -20 ° C. </li><li> Aspirar a solução fixadora e deixar as lamelas secar ao capuz química (nesta fase, as lamelas podem ser mantidos em -20 ° C durante várias semanas). </li><li> Lavar as lamelas uma vez com PBST (PBS suplementado com 0,1% de Tween-20), para permeabilizar as membranas celulares e bloquear as células com 10% de NDS (soro normal de burro) em PBST (&#39;solução de bloqueio &quot;) durante 1 hora à temperatura ambiente. </li><li> Incubar as células fixas permeabilizadas com Antibodi primárioes durante 2 horas à temperatura ambiente (para fusos e centrossomas coloração). Os anticorpos são diluídas em solução de bloqueio, como segue: anti-tubulina α (diluição 1:250) e anti-γ tubulina (diluição 1:200). Os anticorpos primários são aplicados como se segue: 100 ul de aplicar (em uma gota) de uma mistura dos anticorpos presentes na solução de bloqueio para cada uma lamela sobre a cobertura da placa de 6 poços (a tampa é de cabeça para baixo). Com cuidado, coloque a lamela sobre a queda de anticorpos, as células semeadas enfrentando a queda. Incubar as lamelas que enfrentam os anticorpos durante 2 horas à temperatura ambiente. </li><li> Coloque as lamelas em volta dos poços e lavam as células 3 vezes com PBST. Em seguida, usar o mesmo processo descrito no ponto 4.6 para a marcação de células sobre as lamelas com os anticorpos secundários fluorescentes. Incubar as células nas lamelas de cobertura, com os anticorpos secundários durante 1 hora, temperatura ambiente, no escuro. Os anticorpos são diluídos em solução de bloqueamento como a seguir: Alexa Fluor 488 (diluição 1:1000; verde) e Alexa Fluor 568 (diluição 1:1000;vermelho). </li><li> Monte as lamelas utilizando Prolongar reagente antifade ouro com DAPI (para coloração cromossomos) e incubar durante a RT no escuro para secar. </li><li> Examine as lamínulas por microscopia confocal. </li></ol> 5. A viabilidade celular medido pela produção de ATP Produção de ATP é medida por um kit de ensaio de detecção de ATP luminescente. <ol><li> Semente as células na placa de 96 poços, a aproximadamente 20.000 células em 800 ul de meio, em cada poço. Três poços do branco deve ser utilizado para a determinação da luminescência do meio do fundo. </li><li> Prepare a série de diluições padrão de ATP a partir de cerca de 10 uM a 100 uM e manter em gelo. </li><li> Adicionar 50 ul de detergente a cada poço e agitar a placa durante 5 minutos, em agitador orbital, a 700 rpm. </li><li> Reconstituir cada frasco do &quot;substrato liofilizado &#39;com 5 ml de&quot; tampão de substrato de&#39; no kit. </li><li> Adicionar 50 ul da solução de substrato reconstituído aos poços, e agitara placa por 5 min em agitador orbital, a 700 rpm. </li><li> Manter a placa na obscuridade durante 10 min. </li><li> Meça luminiscence de cada poço por ELISA leitor de microplacas. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Moro imagem confocal fornecida uma documentação em tempo real do efeito citotóxico do PJ-34 em células multi-centrosomal vivo durante a mitose (Figura 3 e da informação suplementar). Esta foi a primeira documentação vivo atribuindo a citotoxicidade de PJ-34 em células de cancro humano para centrossomas extras de-agrupamento e da morte celular, o que sugere a indução de morte celular catástrofe mitótica por PJ-34 5-9. Em contraste, clustering bi-focal de centrossomas super-numerá...

Fenantreno derivados inibidores PARP1, incluindo PJ-34, foram projetados para proteger as células quiescentes de morte celular por apoptose induzida pela energia consumindo PARP1 mediada de reparo do DNA sob condições de estresse (acidente vascular cerebral ou infarto do miocárdio) 1. No entanto, recentemente descobrimos que PJ-34, em duas vezes maior concentração do que PARP1 inibição da indução, pode causar exclusivamente morte celular em células cancerosas humanas 2,3. Quanto mais rápida for a proliferação da célula era mais eficiente na erradicação das células era. A atividade citotóxica do PJ-34 foi atribuída a extra-centrosomes de-agrupamento na mitose 2. Muitos humano células cancerosas porto multicentrosomes 4,5. A incubação de células humanas de câncer de mama MDA-MB-231, que abrigam centrosomes supranumerários, com 20 mM PJ-34 eficientemente erradicadas destas células dentro de 72-96 horas, sem prejudicar as células quiescentes ou algumas células em proliferação benigna abrigar dois centrossomas em mitose <sup&gt; 2,3. Células benignas incluídas células epiteliais mamárias humanas MCF-10, as células endoteliais humanas (HUVEC) primárias e células mesenquimais preparadas a partir de timo humano. Estas células eram resistentes à actividade citotóxica de PJ-34. PJ-34 não interferiu com o ciclo celular durante 96 horas de incubação ou afetar seus centrossomas e formação eixo bi-focal 2,3. Montagem centrosome Bipolar é crucial para a formação do fuso bipolar na mitose 4,5. Portanto, células com mais de dois centrossomas desenvolveram um mecanismo molecular mal compreendida, agrupando os centrossomas extras em dois pólos 4-9. Falha de montagem bipolar dos seus centrossomas pode causar multipolar distorcida fusos e aberrante cromossomas segregação que prende o ciclo celular em G2 / M de detenção, e leva à morte das células por falha mitótico 4,5. Os mecanismos moleculares subjacentes extra-centrosomes de-agrupamento são intensamente investigadas &lt;sup&gt; 10. Compreender este mecanismo de morte permitirá erradicação exclusivos das células cancerosas, poupando os tecidos saudáveis ​​5,10. Assim, os compostos que activam a morte celular catástrofe mitótica oferecer um novo modo de tratamento de um cancro selectiva, o que pode ser eficaz numa larga gama de resultados cancers.Our sólidos humanos sugerem que a imagem confocal pode ser utilizado para identificar moléculas que afectam extra-centrossomas no agrupamento mitose 2,3, tornando estes compostos câncer alvo candidatos a fármacos. Nós documentamos a atividade citotóxica do fenantridina PJ-34, verificando células cancerosas humanas fixas e ao vivo (com alta ocorrência de extra-centrossomas na mitose) contra as células normais. Um passo a passo descrição dos procedimentos de imagiologia utilizadas para identificar a actividade citotóxica de PJ-34 em células de cancro humanas estão incluídas a seguir.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (GIBCO)</td>
			<td>41965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine Serum)</td>
			<td>Invitrogen (GIBCO)</td>
			<td>12657</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep-Ampho solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-033-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Tripsin-EDTA</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92 mm Petri dishes</td>
			<td>Nunc,Thermo scientific</td>
			<td>150350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-D-lysine coated glass bottom culture dishes</td>
			<td>MatTek Corporation, USA</td>
			<td>P35GC-0-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminescent ATP detection assay kit</td>
			<td>Abcam</td>
			<td>ab113849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NDS (Normal Donkey Serum)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti &alpha;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>1:250 dilution (IF)</td>
		</tr>
		<tr>
			<td>Anti &gamma;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5192</td>
			<td>1:200 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-10042</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI (mounting)</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JetPEI (liposomal transfection reagent )</td>
			<td>Polyplus</td>
			<td>101-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica (Mannheim, Germany)</td>
			<td>TCS SP5II</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

Derivados fenantreno que actuam como inibidores potentes PARP1 impedido o agrupamento bi-focal de centrossomas excedentários em células de cancro humano multi-centrosomal na mitose. O fenantridina PJ-34 foi a molécula mais potente. Declustering de extra-centrosomes provoca insuficiência mitótico e morte celular em células multi-centrosomal. Cânceres humanos mais sólidos têm alta ocorrência de extra-centrossomas. A actividade de PJ-34 foi documentada em tempo real por imagem confocal de cancro da mama humano vivo MDA-MB-231 transfectadas com vectores que codificam para fluorescente γ-tubulina, que é altamente abundante nos centrossomas e para histona H2B fluorescente presente na os cromossomos. Aberrantes arranjos cromossomos e focos γ-tubulina de-cluster representando centrosomes declustered foram detectados nas transfectadas MDA-MB-231 células após tratamento com PJ-34. Un-cluster extra-centrossomas nos dois pólos do fuso precedeu sua morte celular. Estes resultados ligados for a primeira vez que a atividade citotóxica exclusivo recentemente detectada de PJ-34 em células cancerosas humanas com extra-centrosomes de-agrupamento na mitose e insuficiência mitótico, levando à morte celular. De acordo com descobertas anteriores observados por imagem confocal de células fixas, PJ-34 células cancerosas erradicada exclusivamente com multi-centrosomes sem prejudicar as células normais submetidos a mitose com os dois centrossomas e fusos bi-focal. Esta atividade citotóxica de PJ-34 não foi compartilhada por outros inibidores potentes PARP1, e foi observada em PARP1 deficientes MEF extracentrosomes abrigam, sugerindo a sua independência de PARP1 inibição. Viver de imagem confocal ofereceu uma ferramenta útil para a identificação de novas moléculas células erradicar durante a mitose.

PJ-34 é uma água solúvel estável fenantridina 1 (Figura 1). Os nossos resultados precedentes revelaram a morte celular e de agrupado extra-centrossomas em vários tipos de células cancerosas multi-centrosomal fixas que tenham sido tratados com o PJ-34. Em contraste, a proliferação de células normais não foram prejudicados 2,3. Centrossomas foram identificados por dupla marcação com anticorpos dirigidos contra centrine1 e γ-tubulina nas células extra-centrosomal fixos <...

Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

A actividade citotóxica do fenantridina PJ-34 em células de cancro sujeitos a mitose foi documentada em tempo real por meio de imagem confocal vivo. PJ-34 erradicou o câncer de mama humano MDA-MB-231 células abrigam extra-centrossomas na mitose. Ao contrário de mitose bi-focal normal, o Extra-centrossomas não foram agrupados nos dois pólos do fuso na presença de PJ-34.

A morte celular associada com mitose anormal observado por imagem confocal em células vivas Câncer

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

cell-death-associated-with-abnormal-mitosis-observed-confocal-imaging

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JoVE Journal

Medicine

14.9K visualizações.  Sheba Medical Center. A actividade citotóxica do fenantridina PJ-34 em células de cancro sujeitos a mitose foi documentada em tempo real por meio de imagem confocal vivo. PJ-34 erradicou o câncer de mama humano MDA-MB-231 células abrigam extra-centrossomas na mitose. Ao contrário de mitose bi-focal normal, o Extra-centrossomas não foram agrupados nos dois pólos do fuso na presença de PJ-34.

Vídeo: Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

De Alta Eficiência Transdução de células de câncer de fígado por recombinante sorotipo de vírus adeno-associado 3 Vetores

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Medicina

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotão imagem de invasão das células tumorais em um modelo de camundongo ortotópico de carcinoma epidermóide oral

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

Avaliação do Câncer de Migração Stem Cell Usando compartimentar dispositivos microfluídicos e imagens de células vivas

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Modelos MAME para formação de imagens 4D Live-célula do tumor: Interações microambiente que a progressão de Impacto maligno

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Câncer de modelagem Colite Associada com Azoximetano (OMA) e sulfato de sódio Dextran (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Isolamento, caracterização e comparativa Diferenciação de células-tronco da polpa dentária Derivado de dentes permanentes usando dois métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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

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

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

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

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

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

A plataforma de triagem baseada luciferase diversificadas para interrogar associados ao câncer transdução de sinal em células cultivadas

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.

Avaliando Clearance fagocítica de morte celular em Curso Experimental de Ligatable fluorescentes Sondas

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling de MicroRNAs estrogênio regulados em células do cancro da mama

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...