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Encyclopedia of Experiments

Orthotopic Injection into the Mammary Fat Pad: Establishing Breast Cancer in Mice


In this article we discuss how to establish a breast cancer model in female mice by injecting breast cancer cells into the mammary fat pad. The example protocol demonstrates the orthotopic injection.


Animal experiments were conducted in accordance with the Provision and General Recommendation of Chinese Experimental Animal Administration Legislation and were approved by the Institution of Animal Care and Use Committee of Capital Medical University (Ref no. AEEI-2014-052). Female Balb/c mice aged 6 to 8 weeks were used.

1. Preparation of the Cells and Animals

  1. One day before the operation, shave the hair around the fourth nipples to expose the operating area.
    1. Use one hand to hold the mouse tightly at the back to make sure the mouse will not move freely, turn up the mouse to expose its belly to the operator, and then use another hand to shave the fur around the fourth nipples with an electronic shaver.
    2. Put a thin layer of hair removal cream around the nipples for 30 - 60 s, clean away the cream with distilled water, and then dry the mouse with soft paper.
  2. On the day of the operation, collect murine breast cancer cells (4T1-luc2), count the cell number, and adjust the cell density to 2 x 105 cells/mL in phosphate-buffered saline (PBS) in sterile microcentrifuge tubes and keep them on ice. For each mouse, 1 x 104 cells/50 µL in a 1-mL syringe is prepared.

2. Orthotopic Injection

  1. Anesthesia of mice
    1. Induce anesthesia. Put the mice in a closed transparent container and turn on 2% isoflurane gas to anesthetize the mice for about 5 min, till they all lie down and fall asleep.
    2. Maintain anesthesia. Transfer the mice from the container to individual anesthesia masks, put some vet ointment on their eyes to avoid dryness, keep the mice supinely with their nipples exposed to the operator, and let them continue to inhale isoflurane gas for a few minutes until the injection has been performed.
    3. Confirm the success of anesthesia by the lack of a toe pinch withdrawal reflex.
    4. Apply ophthalmic ointment to the eyes of the mice to prevent eye dryness.
      NOTE According to the container size and the number of individual anesthesia masks available, a group of mice can be anesthetized together and injected one by one under individual anesthesia.
  2. Performance of the injection
    1. Use one hand to tent up the skin around the fourth nipple with tweezers, to set up an uplifted “tunnel” for the syringe to follow. Use the other hand to hold the syringe with the needle turned upwards to enter the skin subcutaneously at 5 - 10 mm from nipple; then, follow the “tunnel” until the needle tip comes close to the nipple, carefully move the needle tip up into the mammary fat pad till the needle eye comes under the nipple tip, discard the tweezers, and inject the cells slowly.
    2. Turn around the needle a little and retract the syringe slowly; use a cotton swab to press the needle wound for 10 - 20 s to make sure there is no fluid seepage.
    3. Observe the fourth nipple to confirm a successful injection: one white transparent flat sphere with the nipple as the center can be observed (Figure 1).
  3. Keep the mice anesthetized for another minute to avoid letting the mice recover and move too quickly, which will cause the injection wound to open and the tumor cells to leak out.

Figure 1
Figure 1: Pictures before and after the orthotopic injection at the mammary fat pad of female Balb/c mice. The operation was performed, and the pictures were taken while the mice were under isoflurane anesthesia. A, before injection, B, after injection

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Name Company Catalog Number Comments
anesthesia machine Midmark Corporation, Dayton, OH, USA Matrx VMS anesthesia
 isoflurane Hebei yipin chemical reagents  company O21400 anesthesia
1 mL syringe Becton, Dickinson and  Company A257 cell injection
hair removal cream Veet - hair removal cream
Carbomer Eye Gel Dr.GerhardMannChem-Pharm.FabrikGmbH - ophthalmic ointment


Orthotopic Injection into the Mammary Fat Pad:  Establishing Breast Cancer in Mice
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Source: Zhang, G. L., et al. Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad. J. Vis. Exp. (2019).

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