Os autores reconhecem os serviços técnicos fornecidos pelo centro de citologia molecular e a facilidade de animais do Memorial Sloan Kettering Cancer Center. Este trabalho foi financiado por doações de NIH CA157686 (para R.J. Wong) e P30 CA008748 (concessão de apoio do Memorial Sloan Kettering Cancer Center).

Todos os procedimentos com animais sujeitos foram aprovados pelo Comitê de uso no Memorial Sloan Kettering Cancer Center e institucional Cuidado Animal.
1. preparação das pilhas de Cancer
<ol><li>Colheita sub Panc02-H7 confluentes com tripsina 0,25% por 5 min a 37 ° C. Colete as células em um tubo de centrífuga de 15 mL. Nota: As células são cultivadas em frasco de T-225, que contém cerca de 12 x 106 células por balão na confluência de 80% e tripsina de 4 mL / frasco é usado.</li>
	<li>Centrifugar as células no x 900 g a 4° C por 5 min. Lave as células por resuspending a célula de pelotas em 1 mL de PBS (pipetando acima e para baixo duas vezes) e a suspensão de transferência para um tubo de microcentrifugadora de 1,5 mL no gelo.</li>
	<li>Centrifugar as células novamente no x 900 g a 4° C por 5 min e desprezar o sobrenadante sem perturbar o sedimento. Manter as células peletizadas no gelo até injeção.</li>
</ol>2. preparação dos ratos e cirurgia
Nota: camundongos C57BL/6J de 8 semanas de idade, masculinos e femininos são usados neste estudo. As condições de cirurgia segue as regras IACUC da nossa instituição. Os instrumentos são esterilizados, a superfície de trabalho cirúrgico é desinfectada, o animal é desinfectado e o cirurgião usa luvas estéreis.
<ol><li>No dia antes da cirurgia, anestesia o mouse usando 2% de isoflurano e em seguida, retire a pele ao longo do comprimento do fêmur do lado dorsal com uma navalha fina ou um agente de remoção química do cabelo.</li>
	<li>No dia da cirurgia, anestesia os ratos usando 2% isofluorano em uma câmara de indução. Confirme o anesthetization por um estímulo de pitada de dedo do pé e uma falta de resposta.</li>
	<li>Aplica o vet pomada nos olhos para evitar a secura sob anestesia.</li>
	<li>Coloque o mouse anestesiado de lado ventral e aperte suavemente cada membro com fita hipoalergênica para criar suave tensão no membro a ser injetado. Anestesia é mantida utilizando isoflurano entregado através de um vaporizador de precisão e cone de nariz.</li>
	<li>Limpe o local da injeção com Betadine, então novamente com álcool a 70%. Repita este processo mais duas vezes. Certifique-se de que nenhum cabelo solto permanece no campo cirúrgico.</li>
	<li>Faça uma incisão de 1 cm com uma tesoura pequena aproximadamente 2mm abaixo e paralelo ao fêmur. Retrai a pele com pinças lateralmente para expor os músculos por baixo.</li>
	<li>O nervo ciático é executado profundo para os músculos glúteos e bíceps femoral. Separar estes dois músculos ao longo de um plano fascial com tesouras pequenas e expor o nervo ciático, por baixo. Livre o nervo dos músculos ao redor usando dissecção romba.</li>
	<li>Retire 3 μL de células de câncer (cerca de 50.000 células) a pelota em uma seringa de 10 μL. Nota: Como alternativa, desenhe 3 μL de PBS como um controle.</li>
	<li>Coloque uma espátula de metal pequena debaixo do nervo no ponto de injeção para apoio. Sob visualização com um microscópio de dissecação, insira a agulha no nervo contra o metal base, mantendo a agulha como paralela ao nervo como possível mediante a inserção. Tenha cuidado para não perfurar através da parte traseira do nervo. Minimize a manipulação do nervo, tanto quanto possível em todo este processo.</li>
	<li>Injetar lentamente no nervo mais 5 s. Uma formação de um bulbo na área de injeção indica uma boa injeção. Então, deixe a agulha no lugar por 3 s antes de retirar a agulha suavemente. Mantendo a agulha no lugar por 3 s minimiza recuo das células do nervo. Nota: A injeção pode ser realizada para o nervo distal ou da medula espinhal. É importante manter a coerência dentro de um conjunto de experiências. Se as células derramar lá fora, o animal não deve ser incluído na análise do experimento. Com experiência, esses eventos são muito raros.</li>
	<li>Retorne o nervo para sua posição original. Cubra a lata com os músculos sobrejacentes. Tratar os ratos com analgesia adequada e em seguida, feche a pele com suturas de Nylon 5-0.</li>
	<li>Coloque o mouse sozinho em uma gaiola limpa em observação durante a recuperação, até que totalmente desperta do anestesia. Nota: Demora de 5-15 min para o animal recuperar a plena consciência. O animal não for deixado desacompanhado até que recuperou a consciência suficiente para manter a prostração esternal. O animal não é retornado para a companhia de outros animais até que se recuperou totalmente. Posteriormente, avalie a recuperação de pelo menos uma vez a cada 24h por 72 h.</li>
</ol>3. o nervo ciático extração
<ol><li>No passado dia 7 #, eutanásia os ratos com CO2. Coloque o mouse de lado ventral e estabilizar os membros distais usando pinos.</li>
	<li>Retire a pele do lado dorsal do membro injetado e tronco.</li>
	<li>Usando a dissecção romba, expor o nervo ciático profundo aos músculos.</li>
	<li>Os cursos do nervo entre o ílio e Sacro. Para ter acesso ao nervo na região da medula espinhal, separe os dois ossos. Primeiro, insira uma tesoura fechada na área estreita, onde se localiza o nervo ciático e então abrir a tesoura, mantendo o mouse. Ser delicado e manter a integridade do nervo ciático durante a dissecção.</li>
	<li>Cuidadosamente, disse o nervo ciático distalmente à extremidade do fêmur e proximal da medula espinhal.</li>
	<li>Nota: Invadiu os nervos são extremamente frágeis e propensos à quebra sob tensão ou manipulação enérgica.</li>
	<li>Colheita do nervo por um primeiro corte sua extremidade distal. Com cuidado, levante o nervo enquanto libertando-o do tecido adjacente. Cortou o nervo na extremidade proximal, tão próximo quanto possível para sua saída da coluna vertebral.</li>
	<li>Registro, em seguida, comprimento bruto de invasão usando um paquímetro. Esta estimativa macroscópica é apenas indicativa.</li>
</ol>4. processamento e quantificação de nervo
<ol><li>Incorpore os nervos dissecados em OCT composto. Faça certeza coloc os nervos longitudinalmente e tão plana na parte inferior do molde quanto possível.</li>
	<li>Indicar o IEF lado proximal e distal do nervo, marcando a letra P e D.</li>
	<li>Coloque os nervos incorporados em cima do gelo seco, se eles não são seccionados imediatamente. Amostra pode ser preservado a-80 ° C durante várias semanas.</li>
	<li>Amostras de seção usando um micrótomo criostato em 5 seções de espessura e lugar μm em lâminas de vidro. Se possível, coloque 2 seções de nervo por slide. Indica o lado proximal do nervo.</li>
	<li>Mancha de slides usando H & E mancha12.</li>
	<li>Digitalmente Digitalize secções nervosas manchada com um scanner de slides que fornece dados digitais de alta resolução.</li>
	<li>Usando o software de imagem, quantificar o comprimento da invasão clicando no botão de distância medida, área de invasão, ou de outros parâmetros de desejado. Para uma boa estimativa do comprimento da invasão, use várias seções (2 a 4) do mesmo nervo.</li>
</ol>

<ol>
	<li>Liebig, C., Ayala, G., Wilks, J. A., Berger, D. H., Albo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+in+cancer.">Perineural invasion in cancer.</a> Cancer. 115 (15), 3379-3391 (2009).</li><li>Bapat, A. A., Hostetter, G., Von Hoff, D. D., Han, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perineural+invasion+and+associated+pain+in+pancreatic+cancer.">Perineural invasion and associated pain in pancreatic cancer.</a> Nat Rev Cancer. 11 (10), 695-707 (2011).</li><li>Deborde, S., Wong, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Schwann+cells+facilitate+cancer+progression+in+nerves.">How Schwann cells facilitate cancer progression in nerves.</a> Cell Mol Life Sci. 341 (177-186), 1236361-1236416 (2017).</li><li>Abiatari, I., 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=Consensus+transcriptome+signature+of+perineural+invasion+in+pancreatic+carcinoma.">Consensus transcriptome signature of perineural invasion in pancreatic carcinoma.</a> Mol Cancer Ther. 8 (6), 1494-1504 (2009).</li><li>Ayala, G. 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=In+vitro+dorsal+root+ganglia+and+human+prostate+cell+line+interaction:+redefining+perineural+invasion+in+prostate+cancer.">In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.</a> Prostate. 49 (3), 213-223 (2001).</li><li>Gil, Z., Cavel, O., 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=Paracrine+regulation+of+pancreatic+cancer+cell+invasion+by+peripheral+nerves.">Paracrine regulation of pancreatic cancer cell invasion by peripheral nerves.</a> J Natl Cancer Inst. 102 (2), 107-118 (2010).</li><li>Deborde, S. T., 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=Schwann+cells+induce+cancer+cell+dispersion+and+invasion.">Schwann cells induce cancer cell dispersion and invasion.</a> J Clin Invest. 126 (4), 1538-1554 (2016).</li><li>Pour, P. M., Egami, H., Takiyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+growth+and+metastases+of+induced+pancreatic+cancer+in+relation+to+the+prognosis+and+its+clinical+implications.">Patterns of growth and metastases of induced pancreatic cancer in relation to the prognosis and its clinical implications.</a> Gastroenterology. 100 (2), 529-536 (1991).</li><li>Eibl, G., Reber, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+xenograft+nude+mouse+model+for+perineural+invasion+and+recurrence+in+pancreatic+cancer.">A xenograft nude mouse model for perineural invasion and recurrence in pancreatic cancer.</a> Pancreas. 31 (3), 258-262 (2005).</li><li>Stopczynski, R. 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=Neuroplastic+changes+occur+early+in+the+development+of+pancreatic+ductal+adenocarcinoma.">Neuroplastic changes occur early in the development of pancreatic ductal adenocarcinoma.</a> Cancer Res. 74 (6), 1718-1727 (2014).</li><li>Gil, 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=Nerve-sparing+therapy+with+oncolytic+herpes+virus+for+cancers+with+neural+invasion.">Nerve-sparing therapy with oncolytic herpes virus for cancers with neural invasion.</a> Clin Cancer Res. 13 (21), 6479-6485 (2007).</li><li>Cardiff, R. D., Miller, C. H., Munn, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manual+hematoxylin+and+eosin+staining+of+mouse+tissue+sections.">Manual hematoxylin and eosin staining of mouse tissue sections.</a> Cold Spring Harb Protoc. 2014 (6), 655-658 (2014).</li><li>Cremer, 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=Inactivation+of+the+N-CAM+gene+in+mice+results+in+size+reduction+of+the+olfactory+bulb+and+deficits+in+spatial+learning.">Inactivation of the N-CAM gene in mice results in size reduction of the olfactory bulb and deficits in spatial learning.</a> Nature. 367 (6462), 455-459 (1994).</li><li>He, S., 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=The+chemokine+(CCL2-CCR2)+signaling+axis+mediates+perineural+invasion.">The chemokine (CCL2-CCR2) signaling axis mediates perineural invasion.</a> Mol Cancer Res. 13 (2), 380-390 (2015).</li><li>He, S., 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=GFR&#945;1+released+by+nerves+enhances+cancer+cell+perineural+invasion+through+GDNF-RET+signaling.">GFR&#945;1 released by nerves enhances cancer cell perineural invasion through GDNF-RET signaling.</a> P Natl Acad Sci USA. , 02944 (2014).</li></ol>

Os autores não têm nada para divulgar.

Neste protocolo, descrevemos no vivo modelo murino de invasão perineural que permite a quantificação da invasão do nervo ciático por células de câncer pancreático. Este modelo permite o estudo dos mecanismos moleculares da invasão de nervo. Usando esta técnica de experiências bem sucedidas requerem uma abordagem cuidadosa para três passos críticos no processo: 1) a injeção de células cancerosas (etapas 2.7, 2.8), 2) a extração dos nervos invadiram (passo 3.4) e 3) processamento dos nervos colhi...

Os nervos formam um microambiente do tumor específico que estimula o câncer crescimento e migração1,2,3. Invasão perineural (PNI) é o processo através do qual câncer células invadem em e ao redor dos nervos. Pode ser considerado como uma única rota de metástase desde a invasão do câncer estende longe dos locais de origem, ao longo de nervos. PNI é encontrado em vários tipos de câncer, incluindo pâncreas, próstata, cabeça & pescoço, salivar, cervical e cancros colo-rectais com uma incidência variando de 22% a 100%1,2. PNI está associado com dor e se correlaciona com o prognóstico pobre e pior sobrevivência taxas1,2.
Desenvolvimento de modelos de invasão perineural é essencial para elucidar os mecanismos celulares e moleculares deste processo e para testar agentes terapêuticos candidato para diminuir o PNI. Métodos in vitro de estudar as interações entre cânceres e nervos incluem a co-cultura de células de câncer com nervo explantes4, com raízes dorsais gânglios5,6,7ou com células específicas do nervo microambiente como Schwann células7. Abordagens na vivo , no entanto, são mais fisiologicamente relevantes, incluem o uso de modelos de rato de câncer no qual câncer foi induzido ou transplantado e têm a vantagem de contabilidade para o microambiente todo nervo. Em ortotópico modelos de câncer pancreático ou da próstata, PNI tem sido relatado8,9,10 e a incidência de PNI pode ser gravada, mas por causa do pequeno tamanho dos nervos nesses órgãos, é difícil ver a lata inteira e, por conseguinte, para quantificar a extensão do PNI. O modelo que nós descrevemos aqui é um modelo em vivo do PNI no qual câncer células são injetadas para o nervo ciático de ratos através de um procedimento cirúrgico simples11. O transplante heterotópico invade dentro do nervo em direção a medula espinhal. O comprimento da invasão nervo do local da injeção da medula espinhal pode ser medido, bem como o volume do câncer dentro do nervo. Importante, o nervo invadiram também pode ser coletado para uma variedade de ensaios, incluindo análises microscópicas e moleculares. Uma variedade de células de câncer pode ser testada, e os ratos de anfitrião que tenham sido geneticamente modificados ou tratados com compostos específicos podem ser usados também. Este ensaio poderoso permite que as células cancerosas e o microambiente de acolhimento ser modificado para a investigação sobre os mecanismos de PNI.

<table border="0" cellpadding="0" cellspacing="0" style="width:973px;" width="973">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:340px;">Mouse</td>
			<td style="width:123px;"></td>
			<td style="width:344px;"></td>
			<td style="width:167px;">Number and age variable depending on experimental needs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cell culture media (PBS, Trypsin, and DMEM+10% FBS)</td>
			<td>Any</td>
			<td></td>
			<td>Steps 1.1, 1.2, 1.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Conical centrifuge tube, 50 mL</td>
			<td>Falcon</td>
			<td>352098</td>
			<td>Step 1.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microcentrifuge tube 1.5 mL</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td>Step 1.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electric razor</td>
			<td>WAHL</td>
			<td>9962</td>
			<td>Step 2.1. Can be substituted with commercial hair removal agent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Isoflurane, 250 mL</td>
			<td>Baxter</td>
			<td>1001936060</td>
			<td>Step 2.2</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Hypoallergenic surgical tape</td>
			<td>3M Blenderm</td>
			<td>70200419342</td>
			<td>Step 2.3</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Betadine Swapsticks</td>
			<td>PDI</td>
			<td>SKU 41350</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Webcol Alcohol Preps</td>
			<td>Covidien</td>
			<td>5110</td>
			<td>Step 2.4</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sterile surgical tools (scissors and forceps)</td>
			<td></td>
			<td></td>
			<td>Steps 2.4, 2.5, 3.3, 3.4, 3.5</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">10 &#956;L Hamilton syringe</td>
			<td>Hamilton</td>
			<td>80308</td>
			<td>Steps 2.7, 2.8</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Micro spatula</td>
			<td>Fisher Scientific</td>
			<td>S50823</td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissecting microscope</td>
			<td></td>
			<td></td>
			<td>Step 2.7</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bupivacine, 1 g</td>
			<td>Enzo Life Sciences</td>
			<td>BML-NA139-0001</td>
			<td>Step 2.9. Reconstitute to 0.5%</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">5-0 Nylon suture</td>
			<td>Ethicon</td>
			<td>698H</td>
			<td>Step 2.9</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek O.C.T. Compound</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Step 4.1</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue-Tek Cryomold Molds</td>
			<td>VWR</td>
			<td>25608-916</td>
			<td>Step 4.1</td>
		</tr>
	</tbody>
</table>

an in vivo murine sciatic nerve model of perineural invasion

As células cancerosas invadem os nervos através de um processo denominado invasão perineural (PNI), na qual câncer células proliferaram em migram no microambiente do nervo. Este tipo de invasão é exibido por uma variedade de tipos de câncer e muito frequentemente é encontrado no cancer pancreatic. O tamanho microscópico das fibras nervosas dentro de pâncreas de rato processa o estudo de PNI difícil em modelos murino ortotópico. Aqui, descrevemos um modelo heterotópica na vivo do PNI, onde nós injetamos a linhagem de células de câncer pancreático syngeneic Panc02-H7 em nervo ciático murino. Neste modelo, os nervos ciático de ratos anestesiados são expostos e injetados com células cancerosas. As células cancerosas invadem nos nervos proximalmente em direção a medula espinhal, do ponto de injeção. Os nervos ciático invadiram então são extraídos e processados com OCT para secionar congelado. H & E e mancha da imunofluorescência dessas seções permitem a quantificação de ambos o grau de invasão e alterações na expressão da proteína. Este modelo pode ser aplicado a uma variedade de estudos sobre o PNI, dada a sua versatilidade. Usar ratos com modificações genéticas diferentes e/ou diferentes tipos de células cancerosas permite para investigação dos mecanismos celulares e moleculares do PNI e para tipos diferentes de câncer. Além disso, os efeitos de agentes terapêuticos na invasão do nervo podem ser estudados através da aplicação de tratamento para estes ratos.

Este método descreve a implantação cirúrgica de células de câncer pancreático em nervo ciático murino para criar um modelo in vivo da invasão de nervo quantificáveis. A Figura 1 ilustra a localização anatômica do nervo ciático e o local da injeção. A Figura 2 mostra os dois nervos ciático de um rato nude. Um nervo de PBS injetado (à esquerda) pode ser comparado a um nervo injetado com células de cânc...

Watch this Scientific Journal Video about An In Vivo Murine Sciatic Nerve Model of Perineural Invasion at JoVE.com

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Um modelo murino de nervo ciático na Vivo de invasão Perineural

Descrevemos no vivo modelo murino de invasão perineural injetando células de câncer pancreático syngeneic no nervo ciático. O modelo permite a quantificação da extensão da invasão do nervo e apoia a investigação dos mecanismos celulares e moleculares de invasão perineural.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

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Cancer Research

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

11.9K visualizações.  Memorial Sloan Kettering Cancer Center. Descrevemos no vivo modelo murino de invasão perineural injetando células de câncer pancreático syngeneic no nervo ciático. O modelo permite a quantificação da extensão da invasão do nervo e apoia a investigação dos mecanismos celulares e moleculares de invasão perineural.

Vídeo: An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Co-cultura Ensaios para estudar macrófagos e Microglia Estimulação do Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

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In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

No modelo in vivo para o ensaio do efeito de hipoxia em metástase tumoral

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

A Model for Perineural Invasion in Head and Neck Squamous Cell Carcinoma

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, radiation, and chemotherapy, locoregional recurrences and distant metastases occur at higher rates, and overall survival is decreased by 40% compared to HNSCC without PNI. In vitro studies of the pathways involved in HNSCC PNI have historically been challenging given the lack of a consistent, reproducible assay. Described here is the adaptation of the dorsal root ganglion (DRG) assay for the examination of PNI in HNSCC. In this model, DRG are harvested from the spinal column of a sacrificed nude mouse and placed within a semisolid matrix. Over the subsequent days, neurites are generated and grow in a radial pattern from the cell bodies of the DRG. HNSCC cell lines are then placed peripherally around the matrix and invade preferentially along the neurites toward the DRG. This method allows for rapid evaluation of multiple treatment conditions, with very high assay success rates and reproducibility.

Perineural invasion (PNI) is a common feature of head and neck squamous cell carcinoma (HNSCC), conferring lower survival rates. Its mechanisms are poorly understood. Utilizing neurites generated from murine dorsal root ganglia confined to a semisolid matrix, the pathways involved in the PNI of HNSCC cell lines can be investigated.

Um Modelo para Perineural Invasion em cabeça e pescoço carcinoma espinocelular

Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Um mouse modelo de fadiga induzida por irradiação periférica

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Se movendo para cima: Um simples e flexível em Vitro invasão tridimensional protocolo do ensaio

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

Na Vivo Avaliação de EPR de pH, pO2, Status Redox e as concentrações de fosfato e glutationa no microambiente do Tumor

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Immunophenotyping de ortotópico Homograft (Syngeneic) de murino KPC primário de Adenocarcinoma Ductal do pâncreas por citometria de fluxo

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Um fluxo de trabalho simples migração/invasão usando um automatizado Imager viver-pilha

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Um modelo de câncer endometrial ortotópico com linfadenopatia retroperitoneal feita a partir de in vivo propagadas e cultivadas VX2 células

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Controle espacial e temporal da iniciação de melanoma murino de células-tronco de melanócitos mutantes

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...