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 borderline-resectable cancers, its indications are expanding such that its routine use is the focus of much clinical research^5,6,7,8. In order to develop novel therapeutic approaches for PC involving resection, these approaches need to be first assessed in pre-clinical models that accurately recapitulate clinical settings.

Orthotopic mouse models of PC have been frequently used in the past to test drug treatments^9,10. Many of these were produced by injection of cancer cells alone into mouse pancreas, resulting in tumors that lacked the prominent stroma that is characteristic of PC. More recently, co-injection orthotopic models, such as the one we first developed by injecting a mixture of human PC and human pancreatic stellate cells (PSCs, the primary producers of the collagenous stroma in PC), have come into regular use^11,12. The tumors produced by such co-injection of cancer and stromal cells exhibit (i) both the cancer elements and the characteristic stromal (desmoplastic) component of PC, and (ii) enhanced cancer cell proliferation and metastasis¹¹. Thus, this model closely resembles human PC. While a number of resectional models of orthotopic PC have been described^13,14,15,16, none have reflected the clinical realities of pancreatic resection in humans as accurate as this model, and therefore have been suboptimal for testing adjuvant or neoadjuvant treatments.

The aims of the mouse model presented were to demonstrate how to: (i) successfully implant orthotopic pancreatic cancer while minimizing inadvertent peritoneal dissemination and (ii) subsequently completely resect the cancer. The paper highlight tips and potential pitfalls of this technique.

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 animal).
   1. Maintain these cells in a humidified temperature-controlled CO₂ incubator and perform routine mycoplasma testing. Culture medium used for AsPC-1 and CAhPSCs are RPMI 1640 (with 300 mg/L L-glutamine, 20% v/v foetal bovine serum, 1% v/v penicillin/streptomycin) and IMDM (with 4 mM L-glutamine, 10% v/v foetal bovine serum, 1% v/v penicillin/streptomycin).
   2. Use standard cell culture techniques to trypsinize the cells into a cell suspension. Neutralize the trypsin using the respective complete culture medium at a volume twice that of the trypsin solution used.
   3. Wash these cells twice with phosphate buffered saline (PBS) and resuspend into a mixture containing 1 x 10⁶ AsPC-1 cells and 1 x 10⁶ CAhPSCs in a 50 μL cell suspension.
   4. Keep this suspension on ice until use.
2. Prepare a class II biosafety cabinet for the procedure. Use a heating mat overlaid by a sterile plastic drape. For magnification during the procedure, use a pair of 2.5x to 3.5x magnification surgical loupes.
3. Prepare purse-string swabs by cutting a hole, 1 cm in diameter, into a gauze swab. Secure this hole with a purse-string suture. Any fine braided suture can be used for this (e.g., 5/0 polyglycolic acid suture). Braided suture material is recommended as it allows the loose knot to stay in place after tightening. This is illustrated in Figure 1a.
4. Anesthetize the mouse with 80 mg/kg of ketamine and 10 mg/kg of xylazine by intraperitoneal injection.
5. Administer 5 mg/kg enrofloxacin antibiotic prophylaxis, 2.5 mg/kg flunixin analgesia and 1 mL of 0.9% saline subcutaneously.
6. Once anesthetized, place the mouse on the sterile field in a supine position and apply povidone-iodine followed by 70% ethanol for skin preparation.
7. Make a longitudinal incision in the skin of the left cranial quadrant of the abdomen, and then enter the abdomen by incising the muscular layer between forceps.
8. Load a 29 G insulin syringe with 50 μL of cell suspension–this equates to 1 x 10⁶ CAhPSCs and 1 x 10⁶ luciferase-tagged AsPC-1 cells per injectate. Mount it on the injection device. The design and function of this injection device is explained in detail in Figure 1b and its legend.
9. Place the purse-string swab over the laparotomy incision and then exteriorize the spleen and pancreatic tail through the opening of this swab. Tighten the purse-string to gently encircle the body of the pancreas, exposing the pancreatic tail for injection. It is important to be tight enough that the gauze contacts the pancreas circumferentially while at the same time not constricting it.
10. Using a pair of forceps, grasp the tail of the pancreas and gently place lateral tension on it. Puncture the ventral peritoneal surface with the needle at a shallow angle and then inject the cell suspension into the pancreas in a slow and controlled fashion (over 10−15 s) with the injection device.
11. During the injection process, carefully observe for leakage—both around the injection site (from reflux) and on the other side of the pancreatic lobule (in case of through-and-through penetration). If visible leakage occurs, stop the injection and note the volume of leakage by checking the volume of remaining injectate in the syringe. If the leakage is of small volume (<10 μL), and then absorb any leakage with gauze and reposition the needle into a different pancreatic lobule to complete the injection.
12. Replace the spleen and pancreas and close the abdominal wall with 5/0 polyglycolic acid suture in a continuous fashion. Close the skin with clips.
13. Monitor the mouse in a warmed cage until recovered from the anaesthetic. Once awake and alert, move the mouse back to its cage.

2. Cancer resection surgery: Distal pancreatectomy and splenectomy

1. The timing of resection in relation to implantation can vary depending on the experimental protocol. In general, allow the tumors to grow at least for 3 weeks prior to resection, but optimize this empirically for the particular implanted cancer cell line.
2. On the day prior to the resection surgery, perform bioluminescence imaging on the animals to confirm the presence of a localized primary tumor. Note that this imaging study is simply used to exclude mice with obvious extra-pancreatic disease from resection. Neither size nor radiant flux should be used as thresholds for determining eligibility for resection.
   1. Weigh mice and inject with D-luciferin intraperitoneally (150 mg/kg).
   2. Determine the timing of the imaging step in relation to luciferin injection for each experiment by the performance of a luciferin kinetic curve. The period of time where the radiant flux is above 90% of its maximum represents the optimal time for bioluminescence imaging (in this experiment, 18 to 26 minutes post-injection)
   3. Induce anaesthesia and maintain using isoflurane (4% and 3% with oxygen, respectively) and perform imaging using a bioluminescent imaging device (e.g., IVIS Lumina II). Use automatic exposure and binning settings (this can, however, be optimized for the expected radiant flux).
3. Prepare the class II biosafety cabinet for procedure. Use a heating mat overlaid by a sterile plastic drape. For magnification during dissection, use a pair of 2.5x to 3.5x magnification surgical loupes.
4. Anesthetize the mouse with 80 mg/kg of ketamine and 10 mg/kg of xylazine by intraperitoneal injection.
5. Administer 5 mg/kg enrofloxacin antibiotic prophylaxis, 2.5 mg/kg flunixin analgesia and 1 mL of 0.9% saline subcutaneously.
6. Place the mouse on the sterile field in a supine position and apply povidone-iodine followed by 70% ethanol for skin preparation.
7. Make a longitudinal incision in the skin of the left cranial quadrant of the abdomen, preferably through the previous incision site.
8. Bluntly dissect the skin off the underlying muscular abdominal wall, and then place an Alm self-retaining retractor to hold the skin wound open.
9. Incise the muscular layer between forceps just to one side of the suture line of the previous operation, and then extend the incision to excise the entire previous suture line.
10. Exteriorize the spleen and distal pancreas and retract it cranially. At the caudal aspect of the pancreas, the colon may be found attached by filmy adhesions. If this is found, bluntly dissect the colon off.
11. Carefully pass a pair of forceps dorsal to the body of the pancreas and splenic vessels and open this space. This frees up a segment of pancreas for subsequent ligation.
12. Ligate the body of the pancreas proximal to the tumor with a titanium ligation clip, and then transect the pancreas distal to this with cautery. An alternative way to control the pancreatic stump is to ligate it in continuity with 5/0 polyglycolic acid suture before transection.
13. Retract the pancreas caudally and cauterize the gastrosplenic vessels between the cranial pole of the spleen and the stomach.
14. Remove the specimen and confirm haemostasis.
15. Close the abdominal wall with 5/0 polyglycolic acid suture in a continuous fashion. Close the skin with clips.

3. Postoperative management

1. In the immediate post anaesthetic period (for both of the above procedures), monitor the mouse in a warmed cage until recovered from the anaesthetic. Once awake and alert, move the mouse back to its cage. In the postoperative period, monitor the animals for pain and signs of distress. Administer 0.05 mg/kg buprenorphine by subcutaneous injection and closely observe the animals for 12 hours. 
2. Subsequently, monitor mice daily for weight, food intake and activity. Examine incision sites and palpate for tumor size. Remove skin clips on the seventh postoperative day.
3. Euthanize the mouse if humane endpoints are reached. These humane endpoints include: loss of body weight >20%, features of untreatable distress (including hunched posture, lack of movement or grooming) and tumor size greater than 1 cm³ as estimated by external palpation.

Results

Fifty-nine consecutive mice underwent implantation surgery. Gross leakage occurred in eight (14%) mice. The degree of leakage at the time of injection is estimated as described above in the protocol section. After three weeks to allow these implanted tumors to grow, pre-resection bioluminescence imaging was performed to exclude mice with gross metastatic disease prior to resection. Forty-five (76%) mice underwent surgical resection.

All 45 (100%) mice underwent successful distal pancreatectomy...

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

List of materials used in this article

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.