Um modelo de rato resemcional ortotópico de câncer pancreático
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Os autores receberam apoio da Avner Pancreatic Cancer Foundation.
Materials
| Animals, Materials and Equipment for Implantation Procedure | |||
| AsPC-1 human pancreatic cancer cell line, luciferase tagged (luc+ gene from Promega PGL3 Basic plasmid) | American Type Culture Collection, Manassas, VA, USA | supplied by Professor Takashi Murakami, Saitama Medical University, Saitama, Japan | |
| Autoclip wound clips, 9 mm | Becton Dickson Pty Ltd, North Ryde, NSW, Australia | 500346 | |
| Basic Dressing Pack | Multigate Medical Products Pty Ltd, Villawood, NSW, Australia | ||
| Cancer associated human pancreatic stellate cells | Pancreatic Research Group cell bank | In house cell bank | |
| Cryogenic tubes, 1.0 mL | Thermo Fisher Scientific Australia Pty Ltd, Scoresby, VIC, Australia | 366656 | |
| Disposable stainless-steel scalpel blade with handle, size 15 | Livingstone International, Mascot, NSW, | SCP15 | |
| Foetal bovine serum (FBS) | Life Technologies Corporation, Tullamarine, VIC, Australia | 16000044 | |
| Gilles fine tooth forceps 12 cm | Generic stainless steel microsurgical instrument set | ||
| Heated mats to maintain body temperature during surgery and postoperative recovery | Generic | ||
| Homozygous athymic nude mice: Strain BALB/c-Fox1nu/Ausb, female | Australian Bioresources, Moss Vale, NSW, Australia | ||
| Iscove's modified Dulbecco's medium (IMDM) with 4mM L-glutamine and no phenol red | Life Technologies Corporation, Tullamarine, VIC, Australia | 21056023 | |
| Jewellers forceps 11.5 cm | Generic stainless steel microsurgical instrument set | ||
| Micro needle holder (round handle) 15 cm straight | Generic stainless steel microsurgical instrument set | ||
| Micro scissors (round handle) 15 cm straight | Generic stainless steel microsurgical instrument set | ||
| Penicillin 10,000 U/mL, streptomycin 10,000 μg/mL | Life Technologies Corporation, Tullamarine, VIC, Australia | 15140122 | |
| Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle | Braun Australia Pty Ltd, Bella Vista, NSW, Australia | C1049407 | |
| Portable weighing scale | Precision balances, Bradford, MA, USA | ||
| Reflex clip applier and clip remover | World Precision Instruments, Sarasota, FL, USA | 500345 | |
| Roswell Park Memorial Institute (RPMI) 1640 with phenol red and 300 mg/L Lglutamine | Life Technologies Corporation, Tullamarine, VIC, Australia | 11875085 | |
| Round bodied vessel dilator 15 cm, 0.1 mm tip | Generic stainless steel microsurgical instrument set | ||
| Trypsin 0.05%, EDTA 0.02% | Life Technologies Corporation, Tullamarine, VIC, Australia | 25300054 | For pancreatic stellate cells |
| Trypsin 0.25%, EDTA 0.02% | Life Technologies Corporation, Tullamarine, VIC, Australia | 25200056 | For ASPC-1 cells |
| U-100 insulin syringes, 0.5 mL with 29 G (0.33 mm) × 13 mm needle | Terumo Medical Corporation, Elkton, MD, USA | ||
| Equipment for Resection Procedure | |||
| Alm self-retaining retractor | Generic stainless steel microsurgical instrument set | ||
| Autoclip wound clips 9 mm | Becton Dickson Pty Ltd, North Ryde, NSW | 500346 | |
| Basic Dressing Pack | Multigate Medical Products Pty Ltd, Villawood, NSW, Australia | 08-559NP | |
| Disposable stainless-steel scalpel blade with handle, size 15 | Livingstone International, Mascot, NSW, | SCP15 | |
| Gilles fine tooth forceps 12 cm | Generic stainless steel microsurgical instrument set | ||
| Hand-held high temperature fine tip cautery | Bovie Medical Corporation, Melville, NY, USA | AA01 | |
| Heated mats to maintain body temperature during surgery and postoperative recovery | Generic | ||
| IVIS Lumina II Bioluminescent Imaging Device | Caliper Life Sciences, Hopkinton, MA, USA | ||
| Jewellers forceps 11.5 cm | Generic stainless steel microsurgical instrument set | ||
| Micro needle holder (round handle) 15 cm straight | Generic stainless steel microsurgical instrument set | ||
| Micro scissors (round handle) 15 cm straight | Generic stainless steel microsurgical instrument set | ||
| Polyglycolic acid suture, size USP 5/0 on 13mm half-circle round-bodied needle | Braun Australia Pty Ltd, Bella Vista, NSW, Australia | C1049407 | |
| Portable weighing scale | Precision balances, Bradford, MA, USA | ||
| Reflex wound clip applier and clip remover | World Precision Instruments, Sarasota, FL, USA | 500345 | |
| Round bodied vessel dilator 15 cm, 0.1 mm tip | Generic stainless steel microsurgical instrument set | ||
| Titanium “Weck style” Ligaclip, small | HZMIM, Hangzhou, China | ||
| Titanium Ligaclip applier for open surgery, small | HZMIM, Hangzhou, China | ||
| Volatile anaesthetic machine, including vapouriser and induction chamber | Generic | Generic vapouriser and induction chamber | |
| Drugs for Procedures | |||
| 70% w/w ethanol solution | Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia | Applied topically as surgical skin preparation | |
| Buprenorphine 0.3 mg/mL | Troy Laboratories Pty Ltd, Glendenning, NSW, Australia | Dose: 0.05 mg/kg s.c. | |
| D-Luciferin (1 U/g) | PerkinElmer, Inc., Waltham, MA, USA | 122799 | diluted in PBS to 15 mg/mL. Dose: 150 mg/kg i.p |
| Enrofloxacin 50 mg/mL | Troy Laboratories Pty Ltd, Glendenning, NSW, Australia | Dose: 5 mg/kg s.c. | |
| Flunixin 50 mg/mL | Norbrook Laboratories Australia, Tullamarine, VIC, Australia | Dose: 2.5 mg/kg s.c. | |
| Isoflurane | Zoetis Australia Pty Ltd., Rhodes, NSW, Australia | Dose (vapourised with oxygen): 4% induction, 3% maintenance | |
| Ketamine 100 mg/mL | Maylab, Slacks Creek, QLD, Australia | Dose: 80 mg/kg i.p. | |
| Povidone-Iodine 10% w/v solution | Perrigo Australia, Balcatta, WA, Australia | RIO00802F | Applied topically to the anterior abdomen as surgical skin preparation |
| Refresh eye ointment (liquid paraffin 42.5% w/w, soft white paraffin 57.3% w/w) | Allergan Australia Pty Ltd, Gordon, NSW, Australia | Applied to both eyes | |
| Sodium chloride 0.9% w/v | Braun Australia Pty Ltd, Bella Vista, NSW, Australia | 9481P | Dose: 900 μL s.c. |
| Water for injections BP | Pfizer Australia, Sydney, NSW, Australia | For dilution of drugs | |
| Xylazine 20 mg/mL | Troy Laboratories Pty Ltd, Glendenning, NSW, Australia | Dose: 10 mg/kg i.p. |
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