En orthotopic Resectional Mouse Model af kræft i bugspytkirtlen
Summary
I den kliniske sammenhæng, patienter med lokaliseret kræft i bugspytkirtlen vil gennemgå pancreatectomy efterfulgt af adjuvans behandling. Denne protokol rapporteret her har til formål at etablere en sikker og effektiv metode til modellering dette kliniske scenario i nøgne mus, gennem orthotopic implantation af kræft i bugspytkirtlen efterfulgt af distal bugspytkirtelektomi og splenectomy.
Abstract
Der er mangel på tilfredsstillende dyremodeller til at studere adjuvans og / eller neoadjuvans behandling hos patienter, der overvejes til operation af kræft i bugspytkirtlen (PC). For at løse denne mangel, beskriver vi en mus model, der involverer orthotopic implantation af PC efterfulgt af distal pancreatectomy og splenectomy. Modellen har vist sig at være sikker og passende fleksibel til undersøgelse af forskellige terapeutiske tilgange i adjuvans og neo adjuvans indstillinger.
I denne model, en bugspytkirtel tumor er først genereret ved at implantere en blanding af humane kræftceller i bugspytkirtlen celler (luciferase-tagged AsPC-1) og human kræft forbundet bugspytkirtel stjernernes celler i distale bugspytkirtel af Balb / c athymisk nøgen mus. Efter tre uger, kræften er resected af re-laparotomi, distal pancreatectomy og splenectomy. I denne model, bioluminescens imaging kan bruges til at følge udviklingen i kræft udvikling og virkningerne af resektion / behandlinger. Efter resektion kan adjuvansbehandling gives. Alternativt kan neoadjuvansbehandling gives før resektion.
Repræsentative data fra 45 mus præsenteres. Alle mus gennemgik vellykket distal pancreatectomy / splenectomy uden problemer med hemostase. En makroskopisk proksimal pancreas margin større end 5 mm blev opnået i 43 (96%) Mus. Den tekniske succesrate for pancreas resektion var 100%, med 0% tidlig dødelighed og sygelighed. Ingen af dyrene døde i løbet af ugen efter resektion.
Sammenfattende beskriver vi en robust og reproducerbar teknik til en kirurgisk resektionsmodel af kræft i bugspytkirtlen hos mus, der efterligner det kliniske scenarie. Modellen kan være nyttig til testning af både adjuvans- og neoadjuvansbehandlinger.
Introduction
Bugspytkirtelkanalen adenocarcinoma (kræft i bugspytkirtlen [PC]) er forbundet med en dårlig prognose1. Kirurgisk resektion er fortsat den eneste potentielt helbredende behandling for PC og bør overvejes for patienter, der præsenterer med tidlig sygdom. Desværre, selv med R0 resektion (dvs. resektion margener fri for tumor), gentagelseshastigheden (lokal eller fra uopdaget metastatisk sygdom) er høj2,3. Derfor er systemisk adjuvansbehandling angivet hos næsten alle patienter, der gennemgår resektion4. Desuden, mens neoadjuvans terapi nu kun anbefales til borderline-resectable kræftformer, dens indikationer er ved at udvide sådan, at dens rutinemæssige brug er i fokus for meget klinisk forskning5,6,7,8. For at udvikle nye terapeutiske tilgange til pc,der involverer resektion, skal disse tilgange først vurderes i prækliniske modeller, der nøjagtigt opsummerer kliniske indstillinger.
Orthotopic mus modeller af PC er blevet ofte brugt i fortiden til at teste medicinske behandlinger9,10. Mange af disse blev produceret ved injektion af kræftceller alene i musen bugspytkirtel, hvilket resulterer i tumorer, der manglede den fremtrædende stroma, der er karakteristisk for PC. For nylig, co-injektion orthotopic modeller, som den, vi først udviklet ved at injicere en blanding af human pc og menneskelige bugspytkirtel stellate celler (PSC’ er, de primære producenter af kollagen stroma i PC), er kommet i regelmæssig brug11,12. Tumorerne produceret af en sådan co-injektion af kræft og stromale celler udviser (i) både kræftelementer og den karakteristiske stromale (desmoplastiske) komponent af PC, og (ii) forbedret kræftcellespredning og metastase11. Således ligner denne model meget menneskelig pc. Mens en række resectional modeller af orthotopic PC er blevet beskrevet13,14,15,16, ingen har afspejlet de kliniske realiteter i bugspytkirtlen resektion hos mennesker så nøjagtige som denne model, og derfor har været suboptimal for test adjuvans eller neoadjuvans behandlinger.
Formålet med den fremlagte musemodel var at demonstrere, hvordan man: (i) med succes implanterer orthotopic kræft i bugspytkirtlen og samtidig minimere utilsigtet peritoneal formidling og (ii) efterfølgende helt resect kræften. Papiret fremhæve tips og potentielle faldgruber i denne teknik.
Protocol
Representative Results
Discussion
En resectional orthotopic mus model af kræft i bugspytkirtlen er vigtig, fordi det giver mulighed for test af adjuvans og neoadjuvan behandlinger. Dette er især vigtigt i kræft i bugspytkirtlen, hvor kirurgi er fortsat den mest effektive behandling, men er forbundet med høj risiko for tilbagefald. Dette papir beskriver en metode, som pålideligt vil producere en kræft i bugspytkirtlen, som potentielt kan helbredes med resektion, replikere det kliniske scenario, hvor neoadjuvans / adjuvans behandling er påkrævet.</…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfattere har modtaget støtte fra Avner Kræft i bugspytkirtlen 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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