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Abstract

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Protocol

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Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Published: September 20th, 2016

DOI:

10.3791/54337

1College of Pharmacy, Western University of Health Sciences, 2UVP, LLC, 3Department of Surgery, School of Medicine, University of California San Diego, 4Anticancer Inc.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

Pancreatic cancer is diagnosed with increased frequency compared to other cancers and is the 4th leading cause of cancer-related deaths in the United States. From the time of diagnosis, over 90% of patients die within five years 1,2. Currently, surgical tumor removal is the only cure for pancreatic cancer, but less than 20% of patients are eligible to undergo surgery mainly because at the time of diagnosis the disease is at an advanced stage and has metastasized 3,4. The lack of specific symptoms makes pancreatic cancer a silent disease; some of the symptoms include abdominal pain, back pain, loss of appetite, jaundice and nausea; whic....

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The protocol described below is executed under guidance and approval of Western University's Animal Care and Use Committee. All experiments are performed in compliance with all relevant guidelines, regulation and regulatory agencies.

1. Cell Culture

  1. Preparation of Complete Medium
    1. Using a Class II biological safety cabinet, prepare complete medium by aseptically adding fetal bovine serum (FBS) and penicillin streptomycin (P/S) to a 500 ml bottle of R.......

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This method describes a surgical orthotopic implantation of fluorescent human pancreatic cancer cells, focusing on the preparation of the cell suspension for injection, proper anesthesia for rodents, delivery of cell suspension via laparotomy, and the use of fluorescent in vivo small animal imaging. The detection of a green fluorescence signal (GFP signal) between two and three weeks post-implantation, provides researchers a visual cue to confirm the presence of a developing panc.......

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We describe an orthotopic murine model of pancreatic cancer which expresses GFP, thus allowing non-invasive monitoring of tumor growth using whole body in vivo fluorescent imaging (Figure 1). This technique allows us to monitor the tumor development in real time (Figure 3); it can be an important tool for researchers to study the therapeutic efficacy of novel agents against pancreatic cancer. Another important aspect of this model is that GFP fluorescence provides a visual cue i.......

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We thank the Western University of Health Sciences for the Intramural Grant.

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Name Company Catalog Number Comments
RPMI media 1640  Caisson Labs  RPL03-500ML
Fetal Bovine Serum  Gibco 10437-077
Penicillin Streptomycin   Thermo Ficher Sci 15140-122
Matrigel HC  Corning  354248
SutureVet PGA 6-0 PGA Henry Schein 39010
Alcare or Foamed Antiseptic Handrub Steris 639680
DPBS (Dubelcco's Phosphate-Buffered saline)  Thermo Ficher Sci 21300025
TB Syringe 27G1/2 Becton Dickinson 305620
Isoflurane  Blutler Schein 50562
Ketoprofen  Fort Dodge Animal Health 
Surgical Scissors, 5.5"straight mayo  Henry Schein 22-1600
PANC-1 GFP cell line  Anticancer, Inc
Small Animal Imaging System:
iBOx Scientia, UVP : UVP, LLC  Upland, CA.  Small Animal Imaging System to observe the fluorescent tumor in live animals

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