Os autores receberam apoio da Avner Pancreatic Cancer Foundation.

Todos os procedimentos foram aprovados pelo Comitê de Cuidado e Ética Animal da Universidade de Nova Gales do Sul (17/109A). Camundongos nus atímicos fêmeas, com idade entre 8 e 10 semanas pesando 16-19 g, foram usados para este protocolo. Os ratos estavam alojados em gaiolas micro-isoladores e alimentados comercialmente com alimentos e ad libitumde água.
1. Implantação de câncer pancreático ortotópico
<ol><li>Prepare as células para implantação. Primeiro, calcule o número de células necessárias para o procedimento (1 x 106 células AsPC-1 com marca de luciferase e 1 x 106 células estelares pancreáticas humanas associadas ao câncer [CAhPSCs] são necessárias para cada animal).<ol>
<li>Mantenha essas células em uma incubadora de CO2 controlada pela temperatura umidificada e realize testes de mycoplasma rotineiros. O meio de cultura utilizado para AsPC-1 e CAhPSCs são RPMI 1640 (com 300 mg/L-glutamina, 20% v/v soro bovino fetal, 1% v/v penicilina/estreptomicina) e IMDM (com 4 mM L-glutamina, 10% v/v soro bovino fetal, 1% v/v penicilina/estreptomicina).</li>
		<li>Use técnicas padrão de cultura celular para tentarpsinizar as células em uma suspensão celular. Neutralizar a trippsina usando o respectivo meio de cultura completa em um volume duas vezes maior do que a solução de trippsina utilizada.</li>
		<li>Lave essas células duas vezes com soro fisiológico tamponado de fosfato (PBS) e resuspend em uma mistura contendo 1 x 106 células AsPC-1 e 1 x 106 CAhPSCs em uma suspensão celular de 50 μL.</li>
		<li>Mantenha esta suspensão no gelo até usar.</li>
	</ol></li>
	<li>Prepare um armário de biossegurança classe II para o procedimento. Use um tapete de aquecimento sobreposto por uma cortina de plástico estéril. Para ampliação durante o procedimento, utilize um par de lupas cirúrgicas de ampliação de 2,5x a 3,5x.</li>
	<li>Prepare os cotonetes de corda da bolsa cortando um orifício de 1 cm de diâmetro, em um cotonete de gaze. Segure este orifício com uma sutura de corda de bolsa. Qualquer sutura trançada fina pode ser usada para isso (por exemplo, sutura de ácido poliglicóclico 5/0). O material de sutura trançada é recomendado, pois permite que o nó solto permaneça no lugar após o aperto. Isto é ilustrado na Figura 1a.</li>
	<li>Anestesiar o rato com 80 mg/kg de cetamina e 10 mg/kg de xilazina por injeção intraperitoneal.</li>
	<li>Administre 5 mg/kg de profilaxia antibiótico enrofloxacina, 2,5 mg/kg de analgesia flunixina e 1 mL de 0,9% de soro fisiológico subcutâneamente.</li>
	<li>Uma vez anestesiado, coloque o rato no campo estéril em uma posição supina e aplique povidone-iodo seguido de 70% de etanol para preparação da pele.</li>
	<li>Faça uma incisão longitudinal na pele do quadrante craniano esquerdo do abdômen, e depois entre no abdômen incisando a camada muscular entre fórceps.</li>
	<li>Carregue uma seringa de insulina de 29 G com 50 μL de suspensão celular – isso equivale a 1 x 106 CAhPSCs e 1 x 106 células AsPC-1 com marca de luciferase por injeção. Monte-o no dispositivo de injeção. O design e a função deste dispositivo de injeção são explicados em detalhes na Figura 1b e sua legenda.</li>
	<li>Coloque o cotonete de corda de bolsa sobre a incisão laparotomia e, em seguida, exteriorize o baço e a cauda pancreática através da abertura deste cotonete. Aperte a corda da bolsa para cercar suavemente o corpo do pâncreas, expondo a cauda pancreática para injeção. É importante estar apertado o suficiente para que a gaze entre em contato com o pâncreas circunferencialmente enquanto ao mesmo tempo não a constrita.</li>
	<li>Usando um par de fórceps, segure a cauda do pâncreas e coloque suavemente a tensão lateral sobre ele. Puna a superfície peritoneal ventral com a agulha em um ângulo raso e, em seguida, injete a suspensão celular no pâncreas de forma lenta e controlada (mais de 10-15 s) com o dispositivo de injeção.</li>
	<li>Durante o processo de injeção, observe cuidadosamente o vazamento — tanto ao redor do local da injeção (do refluxo) quanto do outro lado do lobule pancreático (em caso de penetração através e através). Se ocorrer vazamento visível, pare a injeção e observe o volume de vazamento verificando o volume de injeção restante na seringa. Se o vazamento for de pequeno volume (<10 μL), e então absorver qualquer vazamento com gaze e reposicionar a agulha em um lobule pancreático diferente para completar a injeção.</li>
	<li>Substitua o baço e o pâncreas e feche a parede abdominal com sutura de ácido poliglicólico 5/0 de forma contínua. Feche a pele com clipes.</li>
	<li>Monitore o rato em uma gaiola aquecida até se recuperar do anestésico. Uma vez acordado e alerta, mova o rato de volta para sua gaiola.</li>
</ol>2. Cirurgia de ressecção do câncer: Pancreatectomia distal e esplenectomia
<ol><li>O tempo de ressecção em relação à implantação pode variar dependendo do protocolo experimental. Em geral, permitem que os tumores cresçam pelo menos por 3 semanas antes da ressecção, mas otimizem isso empiricamente para a linha de células cancerígenas implantadas em particular.</li>
	<li>No dia anterior à cirurgia de ressecção, realize imagens de bioluminescência nos animais para confirmar a presença de um tumor primário localizado. Observe que este estudo de imagem é simplesmente usado para excluir camundongos com doença extra-pancreática óbvia da ressecção. Nem o tamanho nem o fluxo radiante devem ser usados como limiares para determinar a elegibilidade para a ressecção.<ol>
<li>Pese camundongos e injete com D-luciferin intraperitoneally (150 mg/kg).</li>
		<li>Determine o tempo da etapa de imagem em relação à injeção de luciferina para cada experimento pelo desempenho de uma curva cinética de luciferina. O período em que o fluxo radiante está acima de 90% do seu máximo representa o tempo ideal para a imagem de bioluminescência (neste experimento, 18 a 26 minutos após a injeção)</li>
		<li>Induzir anestesia e manter o uso de isoflurane (4% e 3% com oxigênio, respectivamente) e realizar imagens usando um dispositivo de imagem bioluminescente (por exemplo, IVIS Lumina II). Use configurações automáticas de exposição e binning (isso pode, no entanto, ser otimizado para o fluxo radiante esperado).</li>
	</ol>
</li>
	<li>Prepare o armário de biossegurança classe II para o procedimento. Use um tapete de aquecimento sobreposto por uma cortina de plástico estéril. Para ampliação durante a dissecção, use um par de lupas cirúrgicas de ampliação de 2,5x a 3,5x.</li>
	<li>Anestesiar o rato com 80 mg/kg de cetamina e 10 mg/kg de xilazina por injeção intraperitoneal.</li>
	<li>Administre 5 mg/kg de profilaxia antibiótico enrofloxacina, 2,5 mg/kg de analgesia flunixina e 1 mL de 0,9% de soro fisiológico subcutâneamente.</li>
	<li>Coloque o mouse no campo estéril em uma posição supina e aplique povidone-iodo seguido de 70% de etanol para preparação da pele.</li>
	<li>Faça uma incisão longitudinal na pele do quadrante craniano esquerdo do abdômen, de preferência através do local de incisão anterior.</li>
	<li>Dissecar sem rodeios a pele da parede abdominal muscular subjacente, e então coloque um retraínte auto-retentor de Alm para manter a ferida da pele aberta.</li>
	<li>Incisar a camada muscular entre fórceps apenas para um lado da linha de sutura da operação anterior, e, em seguida, estender a incisão para extirpara extirpara toda a linha de sutura anterior.</li>
	<li>Exteriorize o baço e o pâncreas distal e retraia-o cranialmente. No aspecto caudal do pâncreas, o cólon pode ser encontrado preso por aderências cinematográficas. Se isso for encontrado, disseca o cólon sem rodeios.</li>
	<li>Passe cuidadosamente um par de fórceps dorsais para o corpo do pâncreas e vasos esplênicos e abra este espaço. Isso libera um segmento de pâncreas para a ligadura subsequente.</li>
	<li>Ligate o corpo do pâncreas proximal ao tumor com um clipe de ligadura de titânio, e depois transecte o pâncreas distal para isso com cauteria. Uma maneira alternativa de controlar o toco pancreático é liga-lo em continuidade com sutura de ácido poliglicólico de 5/0 antes da transeção.</li>
	<li>Retraia o pâncreas caudalmente e cauterize os vasos gastrospínicos entre o polo craniano do baço e o estômago.</li>
	<li>Remova a amostra e confirme a haemostasis.</li>
	<li>Feche a parede abdominal com sutura de ácido poliglicólico 5/0 de forma contínua. Feche a pele com clipes.</li>
</ol>3. Gestão pós-operatória
<ol><li>No período pós-anestésico imediato (para ambos os procedimentos acima), monitore o rato em uma gaiola aquecida até ser recuperado do anestésico. Uma vez acordado e alerta, mova o rato de volta para sua gaiola. No pós-operatório, monitore os animais em busca de dor e sinais de angústia. Administre 0,05 mg/kg de buprenorfina por injeção subcutânea e observe de perto os animais por 12 horas.</li>
	<li>Posteriormente, monitore os camundongos diariamente para o peso, a ingestão de alimentos e a atividade. Examine os locais de incisão e palpato para o tamanho do tumor. Remova os clipes de pele no sétimo dia pós-operatório.</li>
	<li>Eutanize o rato se os pontos finais humanos forem alcançados. Estes pontos finais humanos incluem: perda de peso corporal >20%, características de angústia intratável (incluindo postura curvada, falta de movimento ou preparo) e tamanho do tumor superior a 1 cm3, conforme estimado pela palpação externa.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration:+a+novel+aspect+of+pancreatic+stellate+cell+biology.">Cell migration: a novel aspect of pancreatic stellate cell biology.</a> Gut. 52 (5), 677-682 (2003).</li><li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Egberts, J. 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=Superiority+of+extended+neoadjuvant+chemotherapy+with+gemcitabine+in+pancreatic+cancer:+a+comparative+analysis+in+a+clinically+adapted+orthotopic+xenotransplantation+model+in+SCID+beige+mice.">Superiority of extended neoadjuvant chemotherapy with gemcitabine in pancreatic cancer: a comparative analysis in a clinically adapted orthotopic xenotransplantation model in SCID beige mice.</a> Cancer Biology &amp; Therapy. 6 (8), 1227-1232 (2007).</li></ol>

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Um modelo de camundongo ortotópico resectional de câncer de pâncreas é importante porque permite o teste de tratamentos adjuvantes e neoadjuvantes. Isso é particularmente importante no câncer de pâncreas, onde a cirurgia continua sendo o tratamento mais eficaz, mas está associada ao alto risco de recidiva. Este artigo descreve um método que produzirá de forma confiável um câncer pancreático potencialmente curável com ressecção, replicando o cenário clínico onde a terapia neoadjuvante/adjuvante é necess...

O adenocarcinoma ductal pancreático (câncer de pâncreas [PC]) está associado a um prognóstico ruim1. A ressecção cirúrgica continua sendo o único tratamento potencialmente curativo para PC e deve ser considerada para pacientes que apresentam doença em estágio inicial. Infelizmente, mesmo com ressecção de R0 (ou seja, margens de ressecção livres de tumor), a taxa de recidiva (doença metastática local ou não detectada) é alta2,3. Portanto, a terapia adjuvante sistêmica é indicada em quase todos os pacientes submetidos à ressecção4. Além disso, enquanto a terapia neoadjuvante é agora recomendada apenas para cânceres limítrofes ressecáveis, suas indicações estão se expandindo de modo que seu uso rotineiro é o foco de muita pesquisa clínica5,6,7,8. Para desenvolver novas abordagens terapêuticas para PC envolvendo ressecção, essas abordagens precisam ser primeiramente avaliadas em modelos pré-clínicos que recapitulem com precisão os cenários clínicos.
Modelos ortotópicos de camundongos de PC têm sido frequentemente usados no passado para testar tratamentos medicamentosos9,10. Muitas delas foram produzidas apenas pela injeção de células cancerígenas no pâncreas do rato, resultando em tumores que não tinham o estroma proeminente característico do PC. Mais recentemente, modelos ortotópicos co-injetores, como o que desenvolvemos pela primeira vez injetando uma mistura de PC humano e células estelares pancreáticas humanas (PSCs, os principais produtores do estroma colágeno em PC), entraram em uso regular11,12. Os tumores produzidos por essa co-injeção de câncer e células estrômicas exibem (i) tanto os elementos cancerígenos quanto o componente escrogórico característico (desmoplástico) do PC, e (ii) maior proliferação celular cancerígeno e metástase11. Assim, este modelo se assemelha muito ao PC humano. Embora uma série de modelos resseccionais de PC ortotópico tenham sido descritos13,14,15,16, nenhum refletiu as realidades clínicas da ressecção pancreática em humanos tão precisa quanto este modelo, e, portanto, têm sido subótimais para testar tratamentos adjuvante ou neoadjuvante.
Os objetivos do modelo de camundongos apresentados foram demonstrar como: (i) implantar com sucesso câncer de pâncreas ortotópico, minimizando a disseminação peritoneal inadvertida e (ii) posteriormente resseccionar completamente o câncer. O trabalho destaca dicas e potenciais armadilhas dessa técnica.

<table>
	<tbody>
		<tr>
			<td>Animals, Materials and Equipment for Implantation Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AsPC-1 human pancreatic cancer cell line, luciferase tagged (luc+ gene from Promega PGL3 Basic plasmid)</td>
			<td>American Type Culture Collection, Manassas, VA, USA</td>
			<td></td>
			<td>supplied by Professor Takashi Murakami, Saitama Medical University, Saitama, Japan</td>
		</tr>
		<tr>
			<td>Autoclip wound clips, 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW, Australia</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cancer associated human pancreatic stellate cells</td>
			<td>Pancreatic Research Group cell bank</td>
			<td></td>
			<td>In house cell bank</td>
		</tr>
		<tr>
			<td>Cryogenic tubes, 1.0 mL</td>
			<td>Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia</td>
			<td>366656</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal bovine serum (FBS)</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>16000044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homozygous athymic nude mice: Strain BALB/c-Fox1nu/Ausb, female</td>
			<td>Australian Bioresources, Moss Vale, NSW, Australia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM) with 4mM L-glutamine and no phenol red</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>21056023</td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Penicillin 10,000 U/mL, streptomycin 10,000 &#956;g/mL</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 with phenol red and 300 mg/L Lglutamine</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>11875085</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Trypsin 0.05%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25300054</td>
			<td>For pancreatic stellate cells</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%, EDTA 0.02%</td>
			<td>Life Technologies Corporation, Tullamarine, VIC, Australia</td>
			<td>25200056</td>
			<td>For ASPC-1 cells</td>
		</tr>
		<tr>
			<td>U-100 insulin syringes, 0.5 mL with 29 G (0.33 mm) &#215; 13 mm needle</td>
			<td>Terumo Medical Corporation, Elkton, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment for Resection Procedure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alm self-retaining retractor</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Autoclip wound clips 9 mm</td>
			<td>Becton Dickson Pty Ltd, North Ryde, NSW</td>
			<td>500346</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Dressing Pack</td>
			<td>Multigate Medical Products Pty Ltd, Villawood, NSW, Australia</td>
			<td>08-559NP</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable stainless-steel scalpel blade with handle, size 15</td>
			<td>Livingstone International, Mascot, NSW,</td>
			<td>SCP15</td>
			<td></td>
		</tr>
		<tr>
			<td>Gilles fine tooth forceps 12 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Hand-held high temperature fine tip cautery</td>
			<td>Bovie Medical Corporation, Melville, NY, USA</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated mats to maintain body temperature during surgery and postoperative recovery</td>
			<td>Generic</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina II Bioluminescent Imaging Device</td>
			<td>Caliper Life Sciences, Hopkinton, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Jewellers forceps 11.5 cm</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro needle holder (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Micro scissors (round handle) 15 cm straight</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>C1049407</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable weighing scale</td>
			<td>Precision balances, Bradford, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Reflex wound clip applier and clip remover</td>
			<td>World Precision Instruments, Sarasota, FL, USA</td>
			<td>500345</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bodied vessel dilator 15 cm, 0.1 mm tip</td>
			<td></td>
			<td></td>
			<td>Generic stainless steel microsurgical instrument set</td>
		</tr>
		<tr>
			<td>Titanium &#8220;Weck style&#8221; Ligaclip, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Titanium Ligaclip applier for open surgery, small</td>
			<td>HZMIM, Hangzhou, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Volatile anaesthetic machine, including vapouriser and induction chamber</td>
			<td>Generic</td>
			<td></td>
			<td>Generic vapouriser and induction chamber</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs for Procedures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>70% w/w ethanol solution</td>
			<td>Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia</td>
			<td></td>
			<td>Applied topically as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Buprenorphine 0.3 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 0.05 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>D-Luciferin (1 U/g)</td>
			<td>PerkinElmer, Inc., Waltham, MA, USA</td>
			<td>122799</td>
			<td>diluted in PBS to 15 mg/mL. Dose: 150 mg/kg i.p</td>
		</tr>
		<tr>
			<td>Enrofloxacin 50 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Flunixin 50 mg/mL</td>
			<td>Norbrook Laboratories Australia, Tullamarine, VIC, Australia</td>
			<td></td>
			<td>Dose: 2.5 mg/kg s.c.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis Australia Pty Ltd., Rhodes, NSW, Australia</td>
			<td></td>
			<td>Dose (vapourised with oxygen): 4% induction, 3% maintenance</td>
		</tr>
		<tr>
			<td>Ketamine 100 mg/mL</td>
			<td>Maylab, Slacks Creek, QLD, Australia</td>
			<td></td>
			<td>Dose: 80 mg/kg i.p.</td>
		</tr>
		<tr>
			<td>Povidone-Iodine 10% w/v solution</td>
			<td>Perrigo Australia, Balcatta, WA, Australia</td>
			<td>RIO00802F</td>
			<td>Applied topically to the anterior abdomen as surgical skin preparation</td>
		</tr>
		<tr>
			<td>Refresh eye ointment (liquid paraffin 42.5% w/w, soft white paraffin 57.3% w/w)</td>
			<td>Allergan Australia Pty Ltd, Gordon, NSW, Australia</td>
			<td></td>
			<td>Applied to both eyes</td>
		</tr>
		<tr>
			<td>Sodium chloride 0.9% w/v</td>
			<td>Braun Australia Pty Ltd, Bella Vista, NSW, Australia</td>
			<td>9481P</td>
			<td>Dose: 900 &#956;L s.c.</td>
		</tr>
		<tr>
			<td>Water for injections BP</td>
			<td>Pfizer Australia, Sydney, NSW, Australia</td>
			<td></td>
			<td>For dilution of drugs</td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Troy Laboratories Pty Ltd, Glendenning, NSW, Australia</td>
			<td></td>
			<td>Dose: 10 mg/kg i.p.</td>
		</tr>
	</tbody>
</table>

an orthotopic resectional mouse model of pancreatic cancer

Faltam modelos animais satisfatórios para estudar terapia adjuvante e/ou neoadjuvante em pacientes que estão sendo considerados para cirurgia de câncer de pâncreas (PC). Para suprir essa deficiência, descrevemos um modelo de camundongos envolvendo implantação ortotópica de PC seguido de pancreatectomia distal e esplenectomia. O modelo demonstrou ser seguro e adequadamente flexível para o estudo de diversas abordagens terapêuticas em ambientes adjuvante e neo adjuvante.
Neste modelo, um tumor pancreático é gerado pela primeira vez pela implantação de uma mistura de células cancerígenas pancreáticas humanas (luciferase-tagged AsPC-1) e células estelares pancreáticas associadas ao câncer humano no pâncreas distal de ratos nus atímicos Balb/c. Após três semanas, o câncer é ressecado por re-laparotomia, pancreatectomia distal e esplenectomia. Neste modelo, a bioluminescência pode ser usada para acompanhar o progresso do desenvolvimento do câncer e os efeitos da ressecção/tratamentos. Após a ressecção, a terapia adjuvante pode ser dada. Alternativamente, o tratamento neoadjuvante pode ser dado antes da ressecção.
Dados representativos de 45 ratos são apresentados. Todos os camundongos foram submetidos a pancreatectomia distal bem sucedida/esplenectomia sem problemas de hemostasia. Uma margem pancreática proximal macroscópica maior que 5 mm foi alcançada em 43 (96%) mouses. A taxa de sucesso técnico da ressecção pancreática foi de 100%, com mortalidade e morbidade precoce de 0%. Nenhum dos animais morreu durante a semana após a ressecção.
Em resumo, descrevemos uma técnica robusta e reprodutível para um modelo de ressecção cirúrgica de câncer de pâncreas em camundongos que imita o cenário clínico. O modelo pode ser útil para o teste de tratamentos adjuvante e neoadjuvante.

Cinquenta e nove camundongos consecutivos foram submetidos a cirurgia de implantação. Vazamento bruto ocorreu em oito (14%) mouses. O grau de vazamento no momento da injeção é estimado como descrito acima na seção de protocolo. Após três semanas para permitir que esses tumores implantados crescessem, a imagem de bioluminescência pré-ressecção foi realizada para excluir camundongos com doença metastática bruta antes da ressecção. Quarenta e cinco (76%) camundongos submetidos à ressecção cirúrgica....

Watch this Scientific Journal Video about An Orthotopic Resectional Mouse Model of Pancreatic Cancer at JoVE.com

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

No contexto clínico, pacientes com câncer pancreático localizado serão submetidos à pancreatectomia seguida de tratamento adjuvante. Este protocolo aqui relatado visa estabelecer um método seguro e eficaz de modelagem deste cenário clínico em camundongos nus, através da implantação ortotópica de câncer pancreático seguido de pancreatectomia distal e esplenectomia.

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 ...

an-orthotopic-resectional-mouse-model-of-pancreatic-cancer

Research

JoVE Journal

Cancer Research

10.0K visualizações.  University of New South Wales. No contexto clínico, pacientes com câncer pancreático localizado serão submetidos à pancreatectomia seguida de tratamento adjuvante. Este protocolo aqui relatado visa estabelecer um método seguro e eficaz de modelagem deste cenário clínico em camundongos nus, através da implantação ortotópica de câncer pancreático seguido de pancreatectomia distal e esplenectomia.

Vídeo: An Orthotopic Resectional Mouse Model of Pancreatic 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

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

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

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 ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Um modelo de Mouse Syngeneic câncer pancreático para estudar os efeitos da eletroporação irreversível

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

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 ...

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 ...

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

Um modelo de camundongo de câncer hepatocelular induzido por hepatócitos oncogênico, decorrente do ajuste da inflamação e fibrose hepática

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Estudando o câncer de mama triplo negativo usando modelo de câncer de mama ortotópico

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Implantação ortotópica guiada por ultrassom do Adenocarcinoma Ductal Pancreático murine

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Geração de tumores pancreáticos ortotópicos e caracterização ex vivo da citotoxicidade de células T infiltrada tumoral

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Geração de um xenoenxerto ortotópico de células cancerígenas pancreáticas por injeção guiada por ultrassom

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...