Development of Orthotopic Pancreatic Tumor Mouse Models

Abstract

Genetically engineered mouse models of pancreatic cancer that recapitulate human pancreatic tumorigenesis have been established. However, the cost associated with generating and housing these mice can be prohibitive. Tumor latency and progression to invasive diseases in these models are also highly variable. Xenograft mouse models of human pancreatic cancer including heterotopic and orthotopic have been widely used in preclinical studies for their comparatively low cost and rapid, predictable tumor growth. Of the two, orthotopic tumor mouse models are preferred because they offer tissue site-specific pathology, allow studies of metastasis, and are generally deemed more clinically relevant. Here we describe the procedures of implanting cancer cell lines to generate orthotopic mouse models for pancreatic cancer.

1. Introduction

Pancreatic cancer is the fourth leading cause of cancer death in the developed countries including the United States. The disease has the worst prognosis in the gastrointestinal malignancies with an overall 5-year survival rate of less than 5%. Unfortunately, this poor survival rate has not improved in the past decades, although dramatic progress has been achieved in understanding the molecular genetics of human pancreatic carcinogenesis (1). Therefore, search for biomarkers of early detection and novel target genes and development of novel therapeutic strategies for this disease are still in urgent demand. Mouse models of pancreatic cancer including genetically engineered mouse models and tumor xenograft mouse models have proved to be very useful in advancing these fronts of cancer research (2–5).

By the locations of implanted tumor or tumor cells, human xenograft mouse models are majorly classified into two types: heterotopic and orthotopic, which both are commonly used in the cancer research field. Subcutaneous heterotopic mouse modeling is rapid, is of lower cost, requires easily manageable techniques, and is best suited for expanding human tumor specimens, a means to convert human tumor specimens into cell lines, angiogenesis studies, etc. However, promising results of therapeutic regiments demonstrated in the subcutaneous heterotopic mouse models often have little effects on human patients; preclinical drug testing now inclines toward employing genetically engineered or orthotopic mouse models. In orthotopic xenograft models, tumors or tumor cells are either implanted or injected into the equivalent organ from which the cancer originated. The orthotopic xenograft models have similar tumor microenvironment as the original tumor and are therefore deemed to more closely resemble the natural tumorigenesis in human. In addition, orthotopic mouse models are better suited for metastasis studies because subcutaneous xenograft mouse model rarely develops metastasis (6). However, the generation of orthotopic pancreatic cancer mouse models is labor intensive, technically challenging, and needs more complex imaging methods to monitor the growth of the implanted tumors. Orthotopic implantation of tumor cells or mass into the pancreas by surgery can also inflict significant physical trauma to the recipient animals and the animals may require lengthy postoperative recovery. Thus, an ultrasound-guided method of injection of tumor cells into pancreas for the development of orthotopic pancreatic cancer mouse model has recently been established in order to minimize the surgical wound to the recipient animals (7).

Immunodeficient mouse models of orthotopic implantation do not completely replicate the process of natural tumorigenesis in human due to the lack of comparable tumor microenvironment. It is commonly accepted that the host immune cells in the tumor microenvironment play critical roles in the progression and metastasis of pancreatic tumor. However, xenografting human cancer cells directly into an immunocompetent murine host would only result in graft rejection. Recently several genetically engineered mouse models have been validated to recapitulate the full spectrum of pancreatic tumorigenesis in human (2, 3, 8). Invasive pancreatic tumors from these mouse models can be a good source for the generation of syngeneic orthotopic pancreatic mouse models in an immunocompetent mouse host (9). Syngeneic orthotopic pancreatic mouse models encompass both the advantages of orthotopic mouse models and genetically engineered mouse models and are a lower cost alternative to genetically engineered mouse models. Here, we focus on the procedures for surgical generation of orthotopic pancreatic cancer mouse models using cancer cell lines.

2. Materials

2.1. Reagents

1. RPMI-1640 or DMEM medium for tumor cell culture.

2. Phosphate-buffered saline (PBS).

3. 70% (vol/vol) ethanol.

4. Betadine solution (10% providone-iodine).

5. Trypsin–EDTA, 0.05% (Gibco, cat. no. 15400).

6. Ketamine HCl (100 mg/ml).

7. Xylazine hydrochloride (Sigma, cat. no. X1251-5G).

8. Matrigel (BD Biosciences, cat. no. 35620).

9. Antibiotic Enrofloxacin (Enzo life science, lot: L22571).

10. Immunodeficient mice (athymic nude mice or NOD/SCID mice, female, 4–8 weeks) (see Notes 1 and 2).

2.2. Equipment

1. Scale.

2. Luminar flow hood.

3. 1.5 ml sterile Eppendorf tubes.

4. 50 ml centrifuge tubes (Corning, cat. no. 430828).

5. Sterile petri dishes, 10 cm dishes.

6. 26G needles.

7. 100 μl Hamilton Syringe (Hamilton Syringe Co., Nevada, USA).

8. Electric clipper (if mouse hosts have hair).

9. Microscissor.

10. Curved forceps.

11. Splinter forceps.

12. Homostatic forceps.

13. Gauze pads, 10 × 10 cm.

14. Sterile Cotton tipped applicator (Puritan medical, Maine, USA).

15. Surgical tape (Fisher).

16. 4-0 polysorb sutures.

3. Procedures

1. Purchase sufficient number of female athymic nude or other type of immunodeficient mice such as SCID for the experiments (see Note 2).

2. Administer antibiotic Enrofloxacin (5 μg/mg) daily by subcutaneous injection for 2 days before surgery.

3. All surgical instruments should be autoclaved freshly and allowed to cool to room temperature before use.

4. Prepare ketamine/xylazine solution containing 10 mg/ml ketamine and 2 mg/ml xylazine by a combination of 1 ml ketamine stock (100 mg/ml), 0.2 ml xylazine stock (100 mg/ml), and 8.8 ml distilled water. This freshly made solution should be filter-sterilized and could be stored at room temperature for continuous use up to 2 months.

5. Prepare cell suspension (from human pancreatic cancer cell lines) to a concentration of 500,000–1,000,000 cells in 50 μl PBS solution, and leave on ice with occasional agitation (see Note 3).

6. Weigh the recipient nude mouse for orthotopic surgery, and anesthetize the animal by intraperitoneal administration of the prepared ketamine/xylazine solution at a dose of 10 μl/mg. Before starting the operation, make sure that the mouse completely lose consciousness by stimulating the abdominal skin with a pair of splinter forceps.

7. Sterilize the left side of abdominal skin by painting the area with betadine solution from the neck level to the tail, and then completely remove the stain of the bedatine with 70% ethanol.

8. Cover the animal with a gauze sponge previously cut with an ~2 cm diameter hole at the center to expose the left abdominal flank, and fix the animal to the platform with surgical tapes (Fig 1a).

9. The site of incision is chosen at left abdominal flank, which is slightly medial to the splenic silhouette. Lift the abdominal skin with forceps and make a 1-cm incision with sterile microscissors (Fig 1b).

10. Grasp and lift the underlying muscle with forceps and incise to enter the abdominal cavity without injury to the underlying organs. Extend the muscle incision to 1 cm. At this point, you should be able to note the spleen organ.

11. Gently pull out and expose the entire pancreatic body together with spleen to the outside of the peritoneal cavity by using a pair of blunt-nose forceps (Fig 1c).

12. Make a knot surrounding the pancreas tail at ~2 mm from the end of the tail using a string of 4-0 polysorb suture, but do not tight the knot (Fig 1d).

13. Remove the syringe containing the cell suspension from the ice and agitate to disrupt the cell clumps or clusters.

14. While gently retracting the pancreas laterally, insert the needle through the knot into the tail of the pancreas and pass into the pancreas head (Fig 1e). Slowly inject 50 μl of cell suspension while withdrawing the needle. A fluid-filled region should be formed within the pancreatic parenchyma.

15. Remove the needle from the pancreas and immediately tight the knot (see Note 4) (Fig 1f).

16. Leave the pancreas externalized and untouched for 1 min to inspect for any hemorrhage and any leakage.

17. The pancreas and spleen are carefully returned back to the peritoneal cavity with blunt forceps, and the abdominal muscle layer and the skin layer are sequentially closed with interrupted 4-0 polysorb sutures (Fig. 1g, h).

18. After tumor implantation, all mice should be monitored at least twice weekly for the tumor growth and general signs of morbidity such as ruffled fur, hunched posture, and immobility. Eight weeks after surgery, mice will be sacrificed to examine the growth of pancreatic tumors. The tumor mass will be removed from the normal pancreatic tissues. The weight and size of tumor should be documented (measurement of tumor volume = 1/2 length × breath × width) (see Note 5).

Fig. 1.

Orthotopic implantation of pancreatic cancer cells. (a) Let the mouse sleep on its right side. (b) Make about 1-cm incision with sterile microscissors beside the splenic silhouette. (c) Expose the entire pancreas and spleen by using a pair of blunt-nose forceps. (d) Make a loose knot at the tail of pancreas using a 4-0 polysorb suture. (e) Insert the needle into the tail of the pancreas via the knot, and pass into the pancreatic head area. (f) Immediately tighten the knot after the administration of cancer cell solution. (g) Suture the abdominal muscle layer first as putting back the pancreas and spleen into the abdominal cavity. (h) Close the skin with interrupted 4-0 polysorb sutures.

Fig. 2.

An example of dynamic and quantitative monitoring of tumor burdens by measuring bioluminescence emission using IVIS (Xenogen Corp). (a) Tumor burden displayed as ROI 2 = 3.151e + 09, 3 weeks after pancreatic orthotopic implantation with half million human pancreatic cancer cells. (b) and (c) The growth of pancreatic tumor can be precisely measured by bioluminescence emission using IVIS.