Mouse 4T1 Breast Tumor Model

Beth A. Pulaski1, Suzanne Ostrand‐Rosenberg1

1 University of Maryland Baltimore County, Baltimore, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 20.2
DOI:  10.1002/0471142735.im2002s39
Online Posting Date:  May, 2001
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The 4T1 mammary carcinoma is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone The 4T1 tumor has several characteristics that make it a suitable experimental animal model for human mammary cancer. First, tumor cells are easily transplanted into the mammary gland so that the primary tumor grows in the anatomically correct site, as described in this unit. Second, as in human breast cancer, 4T1 metastatic disease develops spontaneously from the primary tumor. Also, the progressive spread of 4T1 metastases to the draining lymph nodes and other organs is very similar to that of human mammary cancer. In this unit, a protocol describes surgical removal of the primary tumor, so that metastatic disease can be studied in an animal setting comparable to the clinical situation where the primary tumor is surgically removed, and metastatic foci remain intact. Another advantage of 4T1 is its resistance to 6‐thioguanine. This property enables precise quantitation of metastatic cells, even when they are disseminated and at sub‐microscopic levels in distant organs, as described here.

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Table of Contents

  • Basic Protocol 1: Establishing the Primary 4T1 Tumor
  • Basic Protocol 2: Quantification of Distant‐Site Metastases
  • Support Protocol 1: Surgical Removal of the Primary 4T1 Tumor
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Establishing the Primary 4T1 Tumor

  • 4T1 tumor cells (ATCC; also available from Fred Miller, University of Michigan)
  • IMDM, DMEM, or RPMI with and without 10% (w/v) FBS ( appendix 2A)
  • 100× antibiotic‐antimycotic (Life Technologies)
  • 0.25% trypsin/1 mM EDTA (Life Technologies)
  • 8‐week‐old female BALB/c mice (The Jackson Laboratory)
  • 10‐cm tissue culture plates or T25 or T75 culture flasks
  • 37°C tissue culture incubator, 5% CO 2
  • 15‐ml conical tubes
  • Benchtop centrifuge
  • 1‐ml tuberculin syringe and 18‐ and 27‐G hypodermic needles
  • Vernier calipers
  • Additional equipment and reagents for culturing cells, determining cell concentration with a hemacytometer ( appendix 3A), handling and restraining mice (unit 1.3) and animal identification (unit 1.5)

Basic Protocol 2: Quantification of Distant‐Site Metastases

  • 1× Hank's balanced salt solution (HBSS; appendix 2A)
  • recipeHeparin solution (see recipe)
  • 8‐week‐old female BALB/c mice infected with appropriate sized primary 4T1 tumors (see protocol 1)
  • Anesthetic (e.g., methoxyflurane; Mallinckrodt Veterinary)
  • Culture medium: IMDM supplemented with 10% FBS ( appendix 2A), 1× antibiotic‐antimycotic (Life Technologies), and recipe60 µM 6‐thioguanine (see recipe)
  • recipeCollagenase type IV/elastase cocktail (see recipe)
  • recipeCollagenase type I cocktail (see recipe)
  • recipeHyaluronidase cocktail (see recipe)
  • Methanol
  • recipe0.03% (w/v) methylene blue (see recipe)
  • 15‐ and 50‐ml conical tubes
  • 6‐well tissue culture plates
  • 1‐ml tuberculin syringe and 27‐G needle
  • Dissecting equipment: scissors, curved scissors, and forceps
  • Benchtop centrifuge
  • 6‐ and 10‐cm tissue culture plates
  • 37°C tissue culture incubator, 5% CO 2
  • Frosted slides or cell strainer
  • Rotating wheel or platform rocker
  • 70‐µm nylon cell strainers
  • Additional reagents and equipment for mouse anesthesia (unit 1.4), blood collection (unit 1.7), and euthanasia using CO 2 asphyxiation (unit 1.8)
NOTE: All solutions and equipment must be sterile, and aseptic technique should be used accordingly.

Support Protocol 1: Surgical Removal of the Primary 4T1 Tumor

  • 70% ethanol
  • Nolvasan surgical scrub (Henry Schein)
  • 70% isopropanol
  • Saline, sterile
  • 8‐week‐old female BALB/c mice infected with appropriate sized primary 4T1 tumors and established metastatic disease (see protocol 1 and protocol 22)
  • Tribromoethanol or other injectable anesthetic (unit 1.4)
  • Food cubes
  • Pyrex dish filled with dH 2O
  • 27‐G needles
  • Spill pads
  • Sterile surgical drapes
  • 10‐cm tissue culture dish
  • Specimen cups
  • 1‐ and 5‐ml syringes
  • Cages
  • Heat lamp
  • Oster Henry Schein finisher trimmers
  • Gauze pads, sterile
  • Surgical mask and sterile surgical gloves
  • Surgical instruments: sharp‐blunt and sharp‐sharp type scissors, needle‐nose forceps, and hemostats
  • 4‐0 silk, nonabsorbable surgical sutures (optional)
  • Nexaband liquid (Henry Schein), 9‐mm wound clamps preloaded in a wound clamp applier, or sutures
  • Wound clamp remover (optional)
  • Additional reagents and equipment for mouse anesthesia (unit 1.4), blood collection (unit 1.7),and euthanasia using CO 2 asphyxiation (unit 1.8)
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Literature Cited

Literature Cited
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