Este trabalho foi apoiado pela Fundação de Pesquisa Alemã (WE 3548 / 4-1) e Roland-Ernst-Stiftung für Gesundheitswesen (1/14).

Os experimentos com animais apresentados aqui foram revisados ​​e permitidos de forma independente por um comitê institucional e governamental de Cuidados e Uso de Animais e foram conduzidos de acordo com as diretrizes da Federação de Associações de Ciências de Animais de Laboratório (FELASA). Todas as medidas possíveis foram tomadas para minimizar o sofrimento, incluindo anestesia e analgesia ou, se necessário, eutanásia prematura. 1. Preparação de células e organismos orgânicos NOTA: Use um volume de 20 μL com 100.000 células para cada injeção. Use a matriz da membrana basal (BMM) para evitar vazamentos e garantir a injeção padronizada. Para garantir resultados reprodutíveis, realize ensaios de autenticação de linha celular ( por exemplo , via perfil STR) em intervalos regulares. <ol><li> Preparação de suspensões celulares primárias de tecido fresco NOTA: sempre trabalhe em condições estéreis. Transferência imediata do recém-chegadoO tecido ressecado da sala de operação para o laboratório em gelo é necessário para assegurar uma alta viabilidade das células. O bem-estar do paciente e o tratamento ideal sempre devem ser a primeira prioridade. Portanto, amostras de tecido para pesquisa devem ser obtidas de maneira que não interfira com o subsequente tratamento patológico e o estadiamento do tumor ressecado. Na maioria dos casos, é razoável ter as amostras para pesquisas obtidas por um patologista treinado para garantir a não interferência no diagnóstico patológico. <ol><li> Imediatamente após a remoção estéril do espécime ressecado (idealmente ~ 1 cm 3 ), coloque a amostra de tecido em um tubo de 50 mL pré-cheio com 20-30 mL de solução salina tamponada com fosfato (PBS). Armazene o tubo no gelo e transfira-o imediatamente para o laboratório. </li><li> Coloque o tecido em uma placa de Petri e lave-o duas vezes com uma abundância de PBS para remover o sangue restante. </li><li> Corte o tecido em pequenos pedaços (~ 2-4 mm) com uma scaLpel ( por exemplo , com uma lâmina # 20 ou # 36). </li><li> Use o dissociador de acordo com as instruções do fabricante para dissociar ainda mais o tecido do tumor a uma suspensão de célula única. Alternativamente, use outros protocolos de digestão de tumores enzimáticos. </li><li> Contar as células da suspensão de célula única resultante ( por exemplo , em um contador de cunha ou um hemocitómetro). </li><li> Centrifugação (5 min a 1.500 xg) e lavar a suspensão celular duas vezes com PBS. </li><li> Ressuspender as células no BMM em uma concentração de 5 x 10 6 células / mL e mantê-las em gelo. </li></ol></li><li> Preparação de linhas celulares para injeção <ol><li> Cultive todas as linhas celulares de câncer colorretal sob condições de cultura padrão (37 ° C, 5% de CO 2 ) e prepará-las no dia da cirurgia. </li><li> Colhe as células de acordo com os protocolos de cultura de células padrão, conte as células ( por exemplo , em um contador de cúpula ou um hemocitómetro) e calcule a reqQuantidade estimada para todas as injeções, dependendo do número de animais a serem injetados. </li><li> Prepare 3-5x do volume realmente necessário para explicar as perdas de pipetagem e o volume morto da seringa de injeção. </li><li> Centrifugação (5 min a 1.500 g) e lavar a suspensão celular duas vezes com PBS. </li><li> Ressuspender as células no BMM em uma concentração de 5 x 10 6 células / mL e mantê-las em gelo. </li></ol></li><li> Preparação de orgóides NOTA: Todas as culturas de organoides 3D são cultivadas sob condições de cultura padrão (37 ° C, 5% de CO 2 ). Protocolos detalhados para a cultura de organoides foram publicados antes. 18 , 19 , 20 Semelhante às linhas celulares convencionais, recomendamos um volume de 20 μL de BMM com 100.000 células por injeção. Os organoides são preparados no dia da cirurgia da seguinte forma: <ol><li> Prepare o seguinte meio (no seguintePara DMEM / F12 +++): Advanced Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) / F12 + 1% HEPES (1M) + 1% de glutamina (200 mM) + 1% de penicilina / estreptomicina. </li><li> Preencha os tubos de 15 mL com 1 mL de DMEM / F12 +++. </li><li> Elimine cuidadosamente os organoides da superfície da placa de cultura e transfira os conteúdos de 3 a 5 poços para um tubo de 15 mL. </li><li> Corte cuidadosamente os organoids em pedaços menores, pipetando-os para cima e para baixo com uma pipeta de vidro estendida. </li><li> Adicionar 5 mL de DMEM / F12 +++ ao tubo, seguido de centrifugação a 1000 xg durante 5 min. </li><li> Após centrifugação, remova o sobrenadante, ressuspenda o sedimento em 600 μL de tampão de dissociação enzimática e transfira a suspensão para um poço de uma placa de 6 poços. </li><li> Incubar a 37 ° C durante 1-5 minutos e, em seguida, cuidadosamente pipetar a suspensão para cima e para baixo para dissolver os organoides. NOTA: Verifique a digestão através do microscópio; Está completo quando todos os clusters celulares foram dissociadosD para células individuais. </li><li> Após a digestão bem sucedida, adicione 1,4 mL de DMEM / F12 +++ para parar a digestão. Em seguida, transferir todos os poços de organoides digeridos para um tubo de 50 mL. </li><li> Contar as células e calcular a quantidade necessária para todas as injeções. </li><li> Prepare 3 - 5x do volume realmente necessário para contabilizar as perdas de pipetagem e o volume morto da seringa de injeção </li><li> Centrifugação (5 min a 1.500 xg) e lavar a suspensão celular duas vezes com PBS. </li><li> Ressuspendeu as células em BMM para uma concentração de 5 x 10 6 células / mL e mantê-las em gelo. </li></ol></li></ol> 2. Orthotopic Mouse Model <ol><li> Preparação de animais receptores para cirurgia NOTA: Use ratos NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NOD scid gamma, NSG) de 6-8 semanas de idade como destinatários. 21 NSG estão entre os camundongos mais imunocomprometidos, sem células maduras B, T e NK, juntamente com múltiplos outros imunes deFects. 21 Eles crescem de forma confiável, mesmo que sejam baixados números de células e sejam altamente propensos a metástases à distância. Os ratos NSG são criadores excelentes e podem ser mantidos em unidades convencionais de patógenos livres (SPF). <ol><li> Antes da primeira incisão, injete 0,05 mg / kg de buprenorfina por via subcutânea. </li><li> Use sevoflurano em 3-3,5 vol% para anestesia geral. Uma perda do reflexo de pinça do dedo do pé indica anestesia suficiente. </li><li> Cubra os olhos dos ratos anestesiados com pomada oftálmica para evitar a dessecação da córnea. </li><li> Conduza os camundongos em posição supina numa pequena mesa. </li><li> Raspar o abdômen com uma máquina de barbear elétrica (o creme depilatório pode ser usado alternativamente) e desinfectar com pelo menos 3 vezes clorhexidina / iodo e 70% de álcool alternativamente. </li><li> Cubra o campo cirúrgico com cortinas estéreis. NOTA: O uso de antibióticos perioperatórios é opcional e está sujeito a diretrizes institucionais. </li></ol></li><li> Laparotomia da linha média e exposição do ceco <ol><li> Use tesoura (os escalpelos podem ser usados ​​alternativamente) para fazer uma pequena incisão da linha média (3 - 5 mm) da pele na parte inferior do abdômen. Pegue a musculatura da parede abdominal com fórceps e cuidadosamente o incise com uma tesoura, abrindo assim a cavidade abdominal. </li><li> Identificar e externalizar cuidadosamente o ceco com fórceps atraumáticos. Posicione a bolsa final cega do ceco no abdômen apontando de forma cranial. </li><li> Uma vez exteriorizado, mantenha o cecum úmido usando esfregões salinos quentes em todos os momentos. </li></ol></li><li> Injeção ortotópica, fechamento do abdômen e recuperação pós-operatória <ol><li> Para injeção intracecal, use uma seringa padrão de 1 mL com uma cânula de 30 G. Monte esta seringa em uma bomba de microinjeção, que por sua vez é montada em um micromanipulador. </li><li> Cuide cuidadosamente a ponta do ceco com fórceps atraumáticos e suavemente alise-a acariciando-aD com um segundo conjunto de fórceps umedecido com solução salina quente. </li><li> Posicione a cânula diretamente acima do ceco. </li><li> Execute as seguintes etapas sob controle visual com um microscópio cirúrgico binocular. </li><li> Cuide cuidadosamente o ceco com duas pinças atraumáticas em ambas as extremidades da parte exteriorizada do ceco, estique-a ligeiramente e depois puxe-a lentamente sobre a cânula que está posicionada em paralelo e diretamente acima. É crucial não perfurar toda a parede do intestino (assim, injetar as células no lúmen), bem como não perfurar a serosa além do ponto inicial de penetração, pois isso levaria a vazamento e disseminação peritoneal. <ol><li> Mova o intestino em direção à cânula, não a cânula em direção ao intestino. Segure e estique o intestino entre duas fórceps. As mãos do cirurgião devem descansar sobre uma superfície para reduzir o tremor. </li></ol></li><li> Injetar as células entre a serosa (vista como revestimento muito fino e translúcido acima daE vasos sanguíneos intramurais) e muscularis. Por conseguinte, a cânula deve ser colocada visualmente acima dos vasos sanguíneos e embaixo da fina membrana translúcida. </li><li> Use um interruptor de pé para iniciar a injeção para reduzir o tremor enquanto a cânula está dentro da parede do intestino. </li><li> Uma vez que a cânula está em posição, comece a injeção. Use uma duração de 20 s, resultando em 1 μL / s de configuração na unidade de controle da bomba. NOTA: A injeção dentro ou perto de um vaso sanguíneo danificado conduz a disseminação intravascular direta e metástase à distância e, portanto, deve ser evitada. </li><li> Após a conclusão da injeção, remova cuidadosamente a cânula puxando-a para trás. </li><li> Coloque um cotonete seco sob o ceco e, em seguida, enxágue completamente o ceco com água destilada para liar células vazias e, assim, evite a disseminação peritoneal artificial. </li></ol></li><li> Encerramento do abdômen e recuperação pós-operatória <ol><li> Depois deInsistente, gentilmente retorna o ceco à cavidade abdominal. </li><li> Feche a parede abdominal com suturas de roda rapidamente absorvíveis de 6-0. </li><li> Feche a pele com os clipes da ferida cirúrgica. </li><li> Coloque o mouse em um mapa de aquecimento configurado para 38 ° C até se recuperar completamente da anestesia. </li><li> Observe de perto a condição pós-operatória dos ratos nas 48 horas seguintes. Em caso de angústia, tratar com 0,05 mg / kg de buprenorfina a cada 12 h. </li><li> Monitore os ratos pelo menos uma vez por dia para detectar sinais de angústia devido ao crescimento do tumor. </li></ol></li></ol> 3. Isolamento de células tumorais circulantes de amostras de sangue inteiro NOTA: Obtenha sangue por punção cardíaca transcutânea em ratos anestesiados, seguido de eutanásia. Idealmente, 1000 μL de sangue são empurrados para uma seringa pré-cheia com 100 μL de um anticoagulante (ácido etilenodiaminotetraacético (EDTA) ou heparina). Use um anti-humano-EpCAM (adesão de células epiteliaisMolécula) anticorpo para identificar os CTCs. Isso funciona muito bem se as linhas de células epiteliais humanas forem usadas no modelo do mouse. Para outros aparelhos, diferentes anticorpos podem ser necessários. <ol><li> Enriquecimento de CTC: <ol><li> Preencha os tubos de 15 mL com meio de gradiente de densidade de 5 mL e transfira cuidadosamente o sangue para o tubo de 15 mL. </li><li> Centrifugação (30 min a 300 xg sem freio) e remova cuidadosamente o sobrenadante superior. </li><li> Despeje o resto em um novo tubo de 15 mL e centrifugue (15 min, 300 xg com freio). </li><li> Recupere a interfase que contém as células mononucleares (descarte o resto) e lave as células duas vezes com PBS. </li><li> Ressuspender o sedimento em 200 μL de PBS / 1% de EDTA e adicionar 4 μL de anticorpo EpCAM. Incube 20 min no gelo no escuro. </li></ol></li><li> Triagem e escolha <ol><li> Prepare o seguinte buffer (a seguir denominado buffer de seleção): PBS + 10% de soro de vitelo fetal (FCS) + 1% de penicilina / STreptomicina (1%) + 0,8% de EDTA. </li><li> Use uma caneta PAP para desenhar um círculo de ~ 1 cm em uma placa de Petri estéril de 6 cm (evita que o fluido se disperse no prato) e pipetar 700 μL de tampão de colheita para este círculo. </li><li> Adicione 50 μL de suspensão celular ao tampão de picking de 700 μL dentro do círculo. Verifique a densidade das células com o microscópio. Divida a amostra em pratos diferentes se for muito denso. </li><li> Aguarde as células se acalmarem (~ 5 min). </li><li> Teste a queda da suspensão celular para células coradas (= EpCAM-positivas). </li><li> Uma vez que uma célula é encontrada, escolha a célula com o micromanipulador e coloque-a em tampão de 50 μL, dependendo da análise a jusante pretendida ( p . Ex. , Tampão de extração de RNA ou meio de cultura). </li><li> Dependendo das análises a jusante pretendidas, isole células e / ou medidores negativos para EpCAM como controles negativos. </li><li> Proceda com análises a jusante ( por exemplo , cultura celular, PCR). </li></ol></li></ol>

<ol>
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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=Resistance+to+dual+blockade+of+the+kinases+PI3K+and+mTOR+in+KRAS-mutant+colorectal+cancer+models+results+in+combined+sensitivity+to+inhibition+of+the+receptor+tyrosine+kinase+EGFR.">Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR.</a> Sci Signal. 7 (351), ra107 (2014).</li><li>Hung, K. 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=Development+of+a+mouse+model+for+sporadic+and+metastatic+colon+tumors+and+its+use+in+assessing+drug+treatment.">Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment.</a> Proc Natl Acad Sci USA. 107 (4), 1565-1570 (2010).</li><li>Wang, F., Johnson, R. L., Snyder, P. W., DeSmet, M. L., Fleet, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Inducible,+Large-Intestine-Specific+Transgenic+Mouse+Model+for+Colitis+and+Colitis-Induced+Colon+Cancer+Research.">An Inducible, Large-Intestine-Specific Transgenic Mouse Model for Colitis and Colitis-Induced Colon Cancer Research.</a> Dig Dis Sci. 61 (4), 1069-1079 (2016).</li><li>Xue, Y., Johnson, R., Desmet, M., Snyder, P. W., Fleet, J. 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Apesar de sua atividade pré-clinicamente comprovada em modelos de ratos subcutâneos, a grande maioria dos novos compostos falha em ensaios clínicos e nunca atinge a clínica. 11 Esta óbvia insuficiência de modelos subcutâneos de ratos para simular com precisão a biologia e os padrões de crescimento dos tumores levou ao desenvolvimento de modelos de ratos ortotópicos baseados na injeção de células tumorais diretamente no órgão original. Os modelos de rato...

O câncer colorretal (CRC) é uma das causas mais comuns de morte por câncer nos países ocidentais. 1 Embora o tumor primário possa ser frequentemente ressecado, a ocorrência de metástases à distância piora dramaticamente o prognóstico e muitas vezes leva à morte. 2 , 3 A correlação biológica da metástase é a circulação de células tumorais (CTCs), que se destacam do tumor, sobrevivem em circulação, se juntam ao epitélio no órgão alvo, invadem o órgão e, finalmente, superam novas lesões. 4 Embora os CTCs sejam de relevância prognóstica, 5 , 6 , 7 , 8 , 9 , sua biologia é parcialmente entendida como resultado de sua extrema raridade no CRC. 10 Os modelos de mouse são um poderoso tOol para estudar vários aspectos da biologia do câncer. Os modelos clássicos de tumor subcutâneo são produzidos por injeção subcutânea de células tumorais em camundongos receptores, que podem ser imunocompetentes (se forem utilizadas células de tumor murino singenésicas) ou imunodeficientes. Os modelos de tumor subcutâneo são baratos e produzem dados rapidamente; Seu crescimento de tumor de ponto final pode ser medido facilmente e não invasivamente. No entanto, 88% dos novos compostos que demonstraram atividade antitumoral em tais modelos falham nos ensaios clínicos. 11 Isto é em parte devido a diferenças entre espécies entre humanos e camundongos; No entanto, uma grande parte dessa falha é devido ao baixo valor preditivo de modelos subcutâneos de mouse. Os modelos de ratos ortotópicos, nos quais as células tumorais são injetadas no órgão de origem e, portanto, crescem em seu microambiente original, são, portanto, cada vez mais utilizados na pesquisa do câncer. 11 , 12 , </sUp&gt; 13 , 14 Os modelos ortotópicos não apenas simulam as condições locais de crescimento tumoral; Como resultado do sítio anatômico correto do crescimento tumoral, os modelos de ratos ortotópicos também permitem a simulação realista de metástases e, portanto, são usados ​​para estudar biologia 8 , 15 e 16 de CTC ou sua resposta a diferentes tratamentos em CRC. 13 , 17 Uma grande desvantagem dos modelos de mouse ortotópicos é a sua complexidade técnica. Dependendo do órgão em que as células devem ser injetadas, a curva de aprendizado até que o experimentador seja capaz de induzir tumores reprodutíveis é bastante longa. Isto aplica-se especialmente aos modelos de câncer colorretal, uma vez que as células tumorais precisam ser injetadas na parede intestinal, o que muitas vezes resulta em perfuração, vazamento de células tumorais ou perda de células tumorais endoluminais. Isso é umO artigo pretende descrever o método de preparação celular a partir de amostras de tecido primário, linhas celulares 2D e cultura organo 3D e sua injeção no ceco de camundongos. A técnica descrita aqui leva a tumores altamente uniformes e, dependendo da biologia do tumor da linha celular usada para injeção, a formação reprodutível de metástases à distância e CTC nos camundongos receptores. 15

<table border="0" cellpadding="0" cellspacing="0" width="1294">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Cell culture Media and Components</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td style="width:457px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Advanced DMEM F12</td>
			<td style="width:297px;">Invitrogen</td>
			<td style="width:119px;">12634010</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">HEPES (1 M)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15630056</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Glutamax-I Supplement (200 mM)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">35050038</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Penicillin/Streptomycin (PenStrep)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15140122</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">DMEM</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">61965026</td>
			<td>basic medium of 2D cell lines (DMEM/10%FCS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal Calf Serum (FCS)</td>
			<td style="width:297px;">BIOCHROM AG</td>
			<td style="width:119px;">S 0115</td>
			<td>basic medium of 2D cell lines (DMEM/10%FCS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">TrypLE Express enzymatic dissociation buffer</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">12604021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Matrigel basement membrane matrix (BMM, phenol red free)</td>
			<td style="width:297px;">CORNING B.V. Life Sciences</td>
			<td style="width:119px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">14190169</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Trypsin-EDTA (0,25%, Phenol-Red)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">6-/48-well plates with lid</td>
			<td style="width:297px;">CORNING</td>
			<td style="width:119px;">3516/3548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cell culture flask 75cm&#178;, 250 mL</td>
			<td style="width:297px;">VWR International GmbH</td>
			<td style="width:119px;">734-2066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cell culture flask 150cm&#178;, 600 mL</td>
			<td style="width:297px;">Corning B.V. Life Sciences</td>
			<td style="width:119px;">355001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Eppendorf tubes 1,5 mL / 2 mL</td>
			<td style="width:297px;">Sarstedt AG &#38; Co.</td>
			<td style="width:119px;">72.706.400/ 72.695.400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">15 ml, 50 ml centrifuge tubes</td>
			<td style="width:297px;">Greiner-Bio-One GmbH</td>
			<td style="width:119px;">188271/227270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">TC10 Counting Slides (for TC20 Counting Machine)</td>
			<td style="width:297px;">Bio-Rad Laboratories GmbH</td>
			<td style="width:119px;">1450016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Pasteur pipettes (glass, 150 mm)</td>
			<td style="width:297px;">Fisher Scientific GmbH</td>
			<td style="width:119px;">11546963/ FB50251</td>
			<td>thinly pulled by using a bunsen burner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS Dissociator</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSmix Tube Rotator</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C Tubes</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Tumor Dissociation Kit</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-095-929</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 70&#181;m Cell Strainer</td>
			<td style="width:297px;">Corning B.V. Life Sciences</td>
			<td>352350</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Surgical Equipment</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Sevoflurane</td>
			<td style="width:297px;">AbbVie Germany GmbH &#38; Co. KG</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Medical oxygen</td>
			<td style="width:297px;">Air Liquide Medical GmbH</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Buprenorphine</td>
			<td style="width:297px;">Temgesic</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Bepanthen - opthalmic ointment</td>
			<td style="width:297px;">Bayer Vital GmbH</td>
			<td style="width:119px;">10047757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Normal saline 0.9% (E154)</td>
			<td style="width:297px;">Serumwerk Bernburg AG</td>
			<td style="width:119px;">10013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Aqua ad injectabilia</td>
			<td style="width:297px;">Braun</td>
			<td style="width:119px;">235144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">1 mL Syringe (without dead volume) - Injekt-F SOLO</td>
			<td style="width:297px;">Braun/neoLab</td>
			<td style="width:119px;">194291661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">30G injection needle</td>
			<td style="width:297px;">BECTON DICKINSON</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cellulose swabs</td>
			<td style="width:297px;">Lohmann &#38; Rauscher Deutschland</td>
			<td style="width:119px;">13356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Micro-Adson Forceps</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">11018-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Iris Scissor - ToughCut</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">14058-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Olsen-Hegar Needle Holder</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">12002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">AutoClip Kit</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">12020-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PDS Z1012H 6/0 C1 (surgical suture)</td>
			<td style="width:297px;">Johnson &#38; Johnson Medical GmbH</td>
			<td style="width:119px;">Z1012H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Table Top Research Anesthesia Machine w/O2 Flush and a Sevoflurane Vaporizer</td>
			<td style="width:297px;">Parkland Scientific</td>
			<td style="width:119px;">V3000PS/PK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">UltraMicro Pump with Micro4 Controller</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">UMP3-4</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Footswitch for SYS-Micro4 Controller</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">15867</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Three-axis Manual Micromanipulator</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">M325</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Magnetic Stand for Micromanipulator</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">M10</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Steel Base Plate for M10 Magnetic Stand</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">5479</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Hot Plate 062</td>
			<td style="width:297px;">Labotect</td>
			<td style="width:119px;">13854</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Isis - Hair shaver</td>
			<td style="width:297px;">AESCULAP - Braun</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Binocular Surgical Microscope</td>
			<td style="width:297px;">Parkland Scientific</td>
			<td style="width:119px;">VS-2Z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">CTC isolation</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">EDTA</td>
			<td style="width:297px;">Roth</td>
			<td>8040.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Density gradient medium &#8211; Ficoll</td>
			<td>StemCell - Lymphoprep</td>
			<td>7801</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4</td>
			<td>BioLegend</td>
			<td>324210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8</td>
			<td>BioLegend</td>
			<td style="width:119px;">118210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish, &#248; 60 x 15 mm, 21 cm&#178;, Vent</td>
			<td>Greiner bio-one</td>
			<td>628102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Cell Culture Microscope</td>
			<td style="width:297px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transferman 4r Micromanipulator</td>
			<td style="width:297px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTram Air</td>
			<td style="width:297px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:457px;">aspiration pump connected to the micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dmz Universal Microelectrode Puller</td>
			<td style="width:297px;">Dagan Corporation</td>
			<td style="width:119px;"></td>
			<td>required for the manufacturing of micro capillaries for single cell aspiration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism Glass Capillaries</td>
			<td style="width:297px;">Dagan Corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PAP pen</td>
			<td style="width:297px;">Abcam</td>
			<td style="width:119px;">ab2601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">14190169</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal Calf Serum (FCS)</td>
			<td style="width:297px;">BIOCHROM AG</td>
			<td style="width:119px;">S 0115</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Penicillin/Streptomycin (PenStrep)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15140122</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">EDTA</td>
			<td style="width:297px;">Roth</td>
			<td>8040.1</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Immunohistochemistry</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Purified anti-human CD326 (EpCAM) antibody clone 9C4</td>
			<td>BioLegend</td>
			<td style="width:119px;">324201</td>
			<td>EpCAM immunohistochemistry (cf, fig 2C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">HRP rabbit anti-mouse IgG</td>
			<td style="width:297px;">Abcam</td>
			<td style="width:119px;">ab97046</td>
			<td>EpCAM immunohistochemistry (cf, fig 2C)</td>
		</tr>
	</tbody>
</table>

isolation of circulating tumor cells in an orthotopic mouse model of colorectal cancer

Apesar das vantagens de uma fácil aplicabilidade e custo-eficácia, os modelos de ratos subcutâneos têm limitações severas e não simulam com precisão a biologia do tumor e a disseminação de células tumorais. Modelos de mouse ortotópicos foram introduzidos para superar essas limitações; No entanto, esses modelos são tecnicamente exigentes, especialmente em órgãos vazios, como o intestino grosso. Para produzir tumores uniformes que crescem e metastatizam de forma confiável, são críticas técnicas padronizadas de preparação e injeção de células tumorais. Desenvolvemos um modelo de câncer colorretal ortopsico (CRC) que desenvolve tumores altamente uniformes e pode ser utilizado para estudos de biologia tumoral, bem como ensaios terapêuticos. As células tumorais de ambos os tumores primários, linhas celulares bidimensionais (2D) ou organoides tridimensionais (3D) são injetadas no ceco e, dependendo do potencial metastático das células tumorais injetadas, formam tumores altamente metastáticos. Além do que, além do mais,Os CTCs podem ser encontrados regularmente. Aqui descrevemos a técnica de preparação de células tumorais a partir de linhas celulares 2D e organoídeos 3D, bem como tecido tumoral primário, técnicas cirúrgicas e de injeção, bem como o isolamento de CTCs dos camundongos portadores de tumor e dicas atuais para solução de problemas.

A geração bem sucedida e reprodutível de tumores colorretais neste modelo depende criticamente da injeção precisa das células sem derramamento ou vazamento. Se isso for alcançado, este modelo é extremamente confiável e muito raramente resulta em disseminação peritoneal artificial. A cinética de crescimento dos tumores, bem como os seus padrões de disseminação, dependem da biologia dos organoídeos e células utilizados. 15 Enquanto as células HCT11...

Watch this Scientific Journal Video about Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer at JoVE.com

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Descrevemos o estabelecimento de tumores colorretais ortotópicos através da injeção de células tumorais ou organoides no ceco de camundongos e o subsequente isolamento de células tumorais circulantes (CTCs) deste modelo.

Isolamento de células tumorais circulantes em um modelo de rato ortotópico de câncer colorretal

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

isolation-circulating-tumor-cells-an-orthotopic-mouse-model

Research

JoVE Journal

Cancer Research

9.9K visualizações.  Technische Universität Dresden.  Descrevemos o estabelecimento de tumores colorretais ortotópicos através da injeção de células tumorais ou organoides no ceco de camundongos e o subsequente isolamento de células tumorais circulantes (CTCs) deste modelo. 

Vídeo: Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Fluorescente modelo de camundongo ortotópico de câncer de pâncreas

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Pesquisa do câncer

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Murino Experimental Modelo de desenvolvimento do tumor original e Peritoneal Metástase<em&gt; via</em&gt; Ortotópico injetar células do ovário Carcinoma

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

Captura e libertação de viáveis ​​circulantes células tumorais do sangue

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

Um modelo de tumor de murino de bexiga ortotópico e Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Um modelo de mouse de engenharia genética de câncer colorretal esporádico

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Isolamento de células endoteliais humanas do cólon Normal e Carcinoma colorrectal - um protocolo melhorado

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

Deteção de um painel personalizado de MicroRNA circulantes em pacientes com câncer colorretal metastático

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Micromanipulação de células tumorais circulantes para análise molecular a jusante e avaliação do potencial metastático

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

Cultura, Manipulação e Transplante Ortotópico de Organoides tumorais da bexiga de camundongo

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

Um modelo de rato resemcional ortotópico de câncer pancreático

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

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