Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Summary

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Abstract

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Introduction

Pancreatic cancer is the fourth leading cause of cancer-related death, with a 5-year survival rate of 4-6%.^1,2 Only 15% of patients are diagnosed early enough in the disease to be eligible for surgery, and tumors recur in >80% of those patients.^3,4 Gemcitabine is used for treatment of pancreatic adenocarcinomas, however, chemoresistance is common and often the drug has little impact on overall survival.⁵ New pharmacological strategies to treat pancreatic cancer are critically needed. Their development depends on significantly improved understanding of the key steps of disease progression that may be sensitive to therapeutic intervention.

Orthotopic models of pancreatic cancer emulate key aspects of the human disease, making them ideal tools for studying the biology of pancreatic cancer.^6-9 In contrast to in vitro cell-based assays of pancreatic cancer cell behavior and subcutaneous in vivo models of pancreatic cancer, orthotopic models allow investigation of tumor cell interactions with the pancreatic microenvironment. The kinetics of disease progression are highly reproducible in orthotopic models and occur over a short time frame (weeks), which makes them well suited to pre-clinical testing of novel therapeutics. This is in contrast to transgenic models where disease onset occurs over a longer and more variable time frame (months to 1 year).¹⁰ When used with more aggressive cell lines, orthotopic models of pancreatic cancer have patterns of spontaneous metastasis similar to those seen in patients.⁸ Expression of bioluminescent reporter genes such as firefly luciferase facilitates longitudinal monitoring of tumor growth, metastatic dissemination, recurrence and response to therapeutics.^6,11

Here we describe an orthotopic model of pancreatic cancer that utilizes Matrigel for localized cell delivery and in vivo bioluminescence imaging for non-invasive monitoring of tumor progression. This orthotopic model of pancreatic cancer allows non-invasive analyses of disease progression and response to therapeutic interventions in syngenic or xenograft models.

Protocol

The protocol being demonstrated is performed under the guidance and approval of the author's institution's animal care and use committee. All experiments are executed in compliance with all relevant guidelines, regulations and regulatory agencies.

1. Transducing Pancreatic Cancer Cell Lines

1. Transduce pancreatic cancer cells to express luciferase as previously described.^12,13 Panc-1 and Capan-1 pancreatic cancer cell lines transduced with firefly luciferase are used here.

Note: Renilla luciferase or bacterial luciferase may also be used.

2. Pancreatic Cancer Cell Preparation

1. Culture transduced pancreatic cancer cells until 70% confluent.
2. Lift the pancreatic cells and ensure viability is greater than 90%.
3. Resuspend at 2 x 10⁷ cells/ml in a 3:2 mixture of chilled Matrigel:Phosphate buffered saline (PBS).
4. Keep the Matrigel-cell suspension on ice prior to injection into the pancreas.

Notes: To ensure rapid solidification of Matrigel, reduce the PBS volume to account for the volume of the cell pellet. Handle Matrigel using ice-cold instruments and syringes at all times to prevent solidification prior to injection. The suggested cell number is a guide and should be determined empirically for each cell line.

3. Mouse Preparation

1. Anesthetize the mouse using inhaled 2-3% isoflurane. Determine depth of anesthesia by lack of pedal reflex to a gentle toe pinch.
2. Apply lubricant to the eyes to prevent desiccation.
3. Position the mouse on its back on a 37 °C heating pad and gently turn the mouse to raise the left side of the abdomen.
4. Prepare the abdomen with a 10% povidone iodine solution.

Notes: Injectable anesthesia may be used instead of inhaled anesthesia. Pre-operative fasting is not necessary.

4. Laparotomy

1. Using sterile surgical instruments make a 1.5 cm incision in the skin approximately 1 cm left lateral from the midline.
2. Make a 1.5 cm incision in the underlying abdominal muscle.
3. Locate the spleen using the forceps and gently remove the spleen from the abdominal cavity. Secure the spleen along a sterile cotton bud to expose the underlying pancreas.
4. Locate the tail of the pancreas adjacent to the spleen.
5. Using a 29 G 0.3 ml insulin syringe, inject 20 μl of the Matrigel-cell suspension into the pancreas.
6. Following injection, hold the syringe in the pancreas for 30-60 sec until the Matrigel has solidified. This important step minimizes cell leakage.
7. Inspect the site of injection to ensure no leakage occurred.
8. Return the spleen and pancreas to the abdominal cavity.

Note: Take care to avoid puncturing the dorsal side of the pancreas which may be thin.

5. Abdominal Wall Closure

1. Close the abdominal musculature of the mouse with an absorbable braided 4-0 suture with a round needle using a continuous stitch.
2. Close the external skin with a non-absorbable monofilament 6-0 suture with a cutting needle using a continuous stitch.
3. Remove the mouse from the inhaled anesthetic and injected 0.05-0.1 mg/kg buprenorphine subcutaneously.
4. Allow the mouse to recover in its cage placed on a 37 °C heating pad with free access to food and water. If mice demonstrate signs of pain such as hunching or reduced mobility, buprenorphine may be given every 12 hr over a 36 hr period.
5. After wound healing (7-10 days), anesthetize the mouse and remove the external sutures.

6. Bioluminescent Tracking of Pancreatic Cancer Progression

1. Anesthetize the mouse using inhaled isoflurane.
2. Inject 150 mg/kg D-luciferin via tail vein.
3. Place the mouse in the bioluminescent imaging system and capture white-light and bioluminescence images as previously described.^14,15
4. Remove the mouse from the inhaled anesthetic and allow it to recover in its home cage.

Note: Bioluminescent imaging is non-invasive and can be conducted periodically to investigate tumor growth kinetics. To image tumor in the pancreatic tail it is important to put the mouse on its left side, so the tumor points towards the camera. We imaged once per week with the frequency increased up to three times per week prior to the experimental endpoint using a Lumina II imaging system (Perkin-Elmer, formerly Caliper Life Sciences) running Living Imaging 4.3.1 software with binning 4, FOV 12.5, F-stop 1, exposure 1 - 60 sec (determined by the highest exposure without pixel saturation).

Results

This method describes an orthotopic model of pancreatic cancer using surgical procedures, including induction of anesthesia, laparotomy, injection of cancer cells in Matrigel and abdominal closure (Figure 1A). The injected cells form a bubble in the surface of the pancreas (Figure 1B). Pancreatic cancer progression may be non-invasively monitored using in vivo bioluminescence imaging to track cancer cell proliferation and dissemination (Figure 2). Liver me...

Discussion

Here we describe an orthotopic model for longitudinal assessment of pancreatic tumor development and progression. Primary tumor growth kinetics are reproducible (Figure 3) and may be non-invasively monitored using bioluminescence imaging of luciferase-tagged cells, e.g. for analyses of tumor response to novel anti-pancreatic cancer therapeutics. Consistent with the human disease, the model shows local pancreatic invasion (Figure 4A) which allows investigation of tumor cel...

Materials

Name	Company	Catalog Number	Comments
Clean Bench coat
Heating pad			Set to 37 °C
Ivis Lumina ll Bioluminescent imager	Caliper		Alternative bioluminescent imaging systems include In vivo F PRO (Carestream) and Photon Imager (Biospace Lab)
Dissecting scissors
Iris forceps (serrated)
Needle holder
27 G 0.3 ml insulin syringe	Terumo	T35525M2913