An Orthotopic Resectional Mouse Model of Pancreatic Cancer

Summary

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.

Abstract

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.

Introduction

Pancreatic ductal adenocarcinoma (pancreatic cancer [PC]) is associated with a poor prognosis¹. Surgical resection remains the only potentially curative treatment for PC and should be considered for patients presenting with early stage disease. Unfortunately, even with R0 resection (i.e., resection margins free of tumor), the recurrence rate (local or from undetected metastatic disease) is high^2,3. Therefore, systemic adjuvant therapy is indicated in almost all patients who undergo resection⁴. Furthermore, while neoadjuvant therapy is now recommended only for bor....

Protocol

All procedures were approved by the Animal Care and Ethics Committee of the University of New South Wales (17/109A). Female athymic Balb/c nude mice, aged 8-10 weeks weighing 16-19 g, were used for this protocol. Mice were housed in micro-isolator cages and fed commercially available pelleted food and water ad libitum.

1. Orthotopic pancreatic cancer implantation

1. Prepare the cells for implantation. First, calculate the number of cells required for the procedure (1 x 10⁶ luciferase-tagged AsPC-1 cells and 1 x 10⁶ cancer-associated human pancreatic stellate cells [CAhPSCs] are required for each anima....

Discussion

A resectional orthotopic mouse model of pancreatic cancer is important because it allows for the testing of adjuvant and neoadjuvant treatments. This is particularly important in pancreatic cancer where surgery remains the most effective treatment but is associated with high risk of recurrence. This paper describes a method which will reliably produce a pancreatic cancer which is potentially curable with resection, replicating the clinical scenario where neoadjuvant/ adjuvant therapy is required.

Materials

Name	Company	Catalog Number	Comments
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.