A Preclinical Murine Model of Hepatic Metastases

Podsumowanie

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Streszczenie

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

Wprowadzenie

Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer related deaths in the United States¹. The five year survival for patients with metastatic PDA is 2-12%¹. Although adjuvant and neoadjuvant chemotherapy for pancreatic cancer is more effective today than ever, there still remains a desperate need for novel therapies.

The development of novel therapeutics necessitates reliable preclinical animal models. Although subcutaneous tumor models are straightforward to use, drawbacks include the inability of these tumors to metastasize and mimic the tumor progression and microenvironments seen in human cancers. Genetically engineered models, such as the genetically engineered mouse containing Kras and p53 knock-in oncogenic mutations (the KPC model)² and the KPC model with a YFP lineage tracer (the PKCY model)³ allow for the study of naturally occurring tumors in immunocompetent mice. Additionally, the tumor microenvironment is unaltered and more closely resembles that seen in human tumor progression. Unfortunately, the timing of tumor development is variable within these models, which makes it difficult to study experimental therapies. Additionally, backcrossing of these mice requires a significant amount of time and financial commitment, which is not always feasible. Finally, implanted xenograft PDA mouse models necessitate immunocompromised mice, which have altered or absent tumor microenvironments. As a result, the efficacy of experimental therapies demonstrated in these preclinical models are often not reproducible in human clinical trials⁴.

This hemisplenectomy mouse model utilizes Panc02 tumor cells, which are a highly tumorigenic, methylcholanthrene induced pancreatic tumor cell line derived from C57BL/6 mice^5,6. Additionally, other murine PDA cell lines derived from the KPC mice can be used in this model. Schulick et al. have previously developed a hemisplenectomy technique and created a syngeneic mouse model of colon cancer metastases⁷. This hemisplenectomy model (Figure 1) offers consistent and equal tumor inoculation in immunocompetent mice lending itself to the investigation of novel therapeutic agents^7-10.

Protokół

All animal experiments conformed to the guidelines of the Animal Care and Use Committee of the Johns Hopkins University, and animals were maintained in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Care (AAALAC). The surgical procedure is performed under sterile conditions in an operating room that undergoes a minimum of fifteen air exchanges per hour. All instruments are sterilized by autoclaving prior to the procedure and again with 70% ethanol in between each mouse during the procedure. All mice not expected to survive the surgical procedure are euthanized under CO₂ for 3 min followed by cervical dislocation while still under anesthesia.

1. Cell Preparation

1. Resuspend cultured Panc02 cells in phosphate buffered saline at 2 x10⁷ cells/ml, and keep on ice.

2. Mouse Preparation

1. Administer Ketamine (100 mg/kg) mixed with Xylazine (10 mg/kg) intraperitoneally to 8-12 week old C57BL/6 female mice in divided doses.
2. Check to see that the toe pinch withdrawal reflex is abolished to ensure that the mouse is fully anesthetized.
3. Administer additional doses of Ketamine (100 mg/kg) mixed with Xylazine (10 mg/kg) as needed to fully anesthetize the mice.
4. Apply Puralube Vet Ointment to the eyes of the mice to prevent drying while the mice are under anesthesia.
5. Shave the surgical area of the mice to avoid contamination of the wound.
6. Prep the left subcostal area of the incision with 70% ethanol and then iodine.
7. Place a surgical drape around the abdomen to maintain sterility.

3. Laparotomy

1. Make a left subcostal incision in line with the left ear through the skin and through the peritoneum using a scalpel.
2. Express the spleen through the incision by applying simultaneous digital pressure along the cranial and caudal aspects of the incision.
3. Locate the splenic blood vessels at the inferior end of the spleen.
4. Divide the spleen by placing two Horizon medium size ligating clips in the center of the spleen being careful to avoid damaging the splenic blood vessels at the splenic poles.
5. Place the upper pole of the spleen back into the peritoneum to avoid contamination.

4. Tumor Injection

1. Draw up 150 μl of phosphate buffered saline into a 26 G x 5/8” syringe.
2. Draw up 100 μl of Panc02 (2 x10⁶) cells into the same syringe maintaining the syringe in an upright position at all times during the injection.
3. Dip the needle in a 70% ethanol solution. The needle is dipped in 70% ethanol to ensure sterility.
4. Inject the cells slowly into the exposed hemispleen being sure the syringe is kept upright at all times. The bevel of the syringe should be observed at all times through the splenic capsule to avoid injecting the cells under the spleen. A whitening of the spleen and blood vessels should be observed upon injection. 
5. Elevate the spleen and apply one medium Horizon clip under the spleen to occlude the splenic blood vessels.
6. Apply one small Horizon clip to the most distal aspect of the pancreas and splenic vessels.
7. Ligate the pancreas and splenic vessels from the hemispleen by cutting above the Horizon clips.
8. Place the mouse on its’ side, and irrigate the incision with distilled water.

5. Abdominal Closure and Recovery from Anesthesia

1. Close the peritoneum with a 4-0 running stitch.
2. Apply two to three skin clips to close the skin incision.
3. Administer 0.1 mg/kg buprenorphine or 5-10 mg/kg Carprofen subcutaneously to alleviate post surgical pain.
4. Place the mouse on a heating pad for recovery until mobile and the mouse demonstrates regular breathing patterns. Animals are only returned to the company of other animals after fully recovering from the surgical procedure.

6. Necropsy Examination and Harvesting of the Liver

1. Between 30 to 60 days following the surgery, mice will begin to show clinical symptoms of metastasis and will require euthanasia. Signs of metastatic disease requiring humane euthanasia include enlarged abdomens and the development of ascites. Euthanize mice by inhalation of CO₂.
2. Following inhalation, perform cervical dislocation.
3. After the animal has been determined to be non responsive, make an incision through the peritoneum to expose the abdomen using scissors.
4. Using forceps and scissors, gently cut the liver out of the abdomen.
5. Place the liver in OCT compound, and flash freeze using liquid nitrogen. Store the liver at -80°C. Alternatively, the liver can be fixed in 16% neutral buffered formalin at RT for 24 hr and paraffin embedded.

Wyniki

Panc02 tumor cells injected into the hemispleen (Figure 1) form liver metastases in 100% of the mice, while lung and peritoneal metastases are not observed if the technique is performed correctly. This technique has been performed on over one hundred mice using Panc02 cells in multiple, independent experiments, and reproducibly, all untreated mice die with liver metastases between 30 and 60 days following the hemisplenectomy procedure (Figure 2). To obtain statistical significance betwee...

Dyskusje

This preclinical murine model of pancreatic cancer metastasis to the liver via a hemispleen injection technique is the first model described that mirrors many of the clinical and immunological conditions of patients with metastatic disease to the liver. This model offers several advantages over other murine models. First, unlike transgenic models of pancreatic cancer in which only 30% of the mice develop metastases to the liver and the timing of metastases formation is quite variable, this model offers the advantage of c...

Materiały

Name	Company	Catalog Number	Comments
Cell culture media and components			will vary depending on cell line
Phosphate Buffered Saline	Gibco by Life Technologies	10010-023
15 ml centrifuge tubes	Corning	430052
Curved Operating Scissor	Roboz Surgical Store	RS-6835
Micro Dissecting Graefe Forceps	Roboz Surgical Store	RS-5130
Needle holder (regular)	World Precision Instruments, Inc
3-0 or 4-0 suture			courtesy of dept of surgery, Johns Hopkins
Weck Horizon Open Ligating Clip Applier, small, angled	Teleflex	137085
Weck Horizon Open Ligating Clip Applier, medium, angled	Teleflex	237115
Weck Horizon medium ligating clips	Teleflex	002200
Weck Horizon small ligating clips	Teleflex	001200
Syringe, 1 cc, tb syringe, 3/8" slip tip	Becton Dickinson	309625
Syringe, 1 cc, tb syringe, 5/8" slip tip	Becton Dickinson	309597
9 mm Autoclip Applier	Mikron	427630
9 mm Autoclip	Becton Dickinson	427631
Heating pad	Sunbeam	CAT93
70% Ethanol			sold by numerous vendors
Ketamine Hydrochloride	Hospira	22395DD
Xylazine hydrochloride	Sigma	X1251