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Abstract

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Medicine

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Published: August 14th, 2016

DOI:

10.3791/54040

1Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, 2Georgia Cancer Center, Augusta University, 3Charlie Norwood VA Medical Center

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

According to the American Cancer Society, breast cancer is the most frequently diagnosed form of cancer in women in the United States. Early detection in combination with recently developed targeted therapies has significantly reduced the mortality of breast cancer in the last two decades. However, breast cancer is still the second leading cause of cancer-related death in women in the United States1. The majority of deaths of breast cancer patients are due to tumor cell metastasis. Unfortunately, most breast cancer is invasive and frequently metastasizes to the lymph node and subsequently to distant organs, including bone, lung, liver and brain.1,2

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All procedures follow guidelines and approved protocols by Georgia Regents University Animal Use and Care Committee.

1. Establishment of Orthotopic Breast Cancer Tumor

  1. One day before the experiment, culture approximately 2 x 106 4T1-Luc tumor cells in a 10-cm culture dish in 10 ml RPMI medium containing 10% FBS. Incubate the culture dish in a CO2 incubator at 37 °C and 5% CO2.
  2. On the day of tumor cell injection, remove culture medium from the culture dish.......

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Establishment of Orthotopic Breast Cancer Mouse Model
4T1 is an aggressive mammary carcinoma cell line. Injection of as little as 1 x 104 cells into the mammary fat pad can lead to establishment of a single tumor nodule in the site of injection (Figure 2A). Therefore, the tumor mimics human primary mammary carcinoma. Almost 100% mice develop the orthotopic tumor. The tumor size can be quantified using a digital caliper or by live tumor im.......

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Many types of transgenic mouse models of breast cancer metastasis have been developed.1 These transgenic mice have high tumor incidence ranging from 60 to 100%. However, the metastasis incidence of these transgenic mice is much lower than the tumor incidence (14 - 100%). Tumor cells metastasize to LN and lungs in the majority of these transgenic mouse models of breast cancer metastasis. The metastasis latency varies from model to model and ranges from 2 to 8 months.6,11-16 These transgenic mouse mod.......

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Supported by grants from the National Institute of Health grants CA133085, CA182518 and CA185909 (to KL) and VA Merit Review Award BX001962 (to KL).

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Name Company Catalog Number Comments
Ami-X Imaging System  Spectral Instruments Imaging Inc. Tucson, AZ
IsoTec SurgiVet Anesthesia Service & Equipment , Inc., Atlanta, GA
AutoClip Physicians Kit Becton Dickinson Primary Care Diagnostics. Sparks, MD 427638
9 MM AutoClip Applier  Becton Dickinson Primary Care Diagnostics. Sparks, MD 427630
9 MM AutoClip Remover Becton Dickinson Primary Care Diagnostics. Sparks, MD 427637
9 MM AutoClip Wound Clip Becton Dickinson Primary Care Diagnostics. Sparks, MD 427631
Sharp-Pointed Dissecting Scissors Fisher 8940
Dissecting Fine-Pointed Forceps Fisher 8875
1/2 CC 27G1/2 tuberculin syringe  Becton Dickinson and Co. NJ  305620
RPMI 1640 medium Mediatech Inc 10-040-CV
PBS Mediatech Inc 21-040-CV
70% Ethanol Ultrapure-usa.com
Trypsin-EDTA Mediatech Inc 25-040-CI

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