JoVE Logo
Faculty Resource Center

Sign In





Representative Results






An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Published: August 14th, 2016



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

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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


Log in or to access full content. Learn more about your institution’s access to JoVE content here

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
Trypsin-EDTA Mediatech Inc 25-040-CI

  1. Fantozzi, A., Christofori, G. Mouse models of breast cancer metastasis. Breast Cancer Res. 8, 212 (2006).
  2. Weigelt, B., Peterse, J. L., van 't Veer, L. J. Breast cancer metastasis: markers and models. Nat Rev Cancer. 5, 591-602 (2005).
  3. Kocaturk, B., Versteeg, H. H. Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth. J Vis Exp. , (2015).
  4. Stewart, T. J., Abrams, S. I. How tumours escape mass destruction. Oncogene. 27, 5894-5903 (2008).
  5. Lopez, J. I., et al. CD44 attenuates metastatic invasion during breast cancer progression. Cancer Res. 65, 6755-6763 (2005).
  6. Khanna, C., Hunter, K. Modeling metastasis in vivo. Carcinogenesis. 26, 513-523 (2005).
  7. Cuevas, B. D., Winter-Vann, A. M., Johnson, N. L., Johnson, G. L. MEKK1 controls matrix degradation and tumor cell dissemination during metastasis of polyoma middle-T driven mammary cancer. Oncogene. 25, 4998-5010 (2006).
  8. Danna, E. A., et al. Surgical removal of primary tumor reverses tumor-induced immunosuppression despite the presence of metastatic disease. Cancer Res. 64, 2205-2211 (2004).
  9. Tao, K., Fang, M., Alroy, J., Sahagian, G. G. Imagable 4T1 model for the study of late stage breast cancer. BMC Cancer. 8, 228 (2008).
  10. Hu, X., et al. Deregulation of apoptotic factors Bcl-xL and Bax confers apoptotic resistance to myeloid-derived suppressor cells and contributes to their persistence in cancer. J Biol Chem. 288, 19103-19115 (2013).
  11. Kwan, H., et al. Transgenes expressing the Wnt-1 and int-2 proto-oncogenes cooperate during mammary carcinogenesis in doubly transgenic mice. Mol Cell Biol. 12, 147-154 (1992).
  12. Guy, C. T., Cardiff, R. D., Muller, W. J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol. 12, 954-961 (1992).
  13. Almholt, K., et al. Reduced metastasis of transgenic mammary cancer in urokinase-deficient mice. Int J Cancer. 113, 525-532 (2005).
  14. Gallego, M. I., Bierie, B., Hennighausen, L. Targeted expression of HGF/SF in mouse mammary epithelium leads to metastatic adenosquamous carcinomas through the activation of multiple signal transduction pathways. Oncogene. 22, 8498-8508 (2003).
  15. Lin, S. C., et al. Somatic mutation of p53 leads to estrogen receptor alpha-positive and -negative mouse mammary tumors with high frequency of metastasis. Cancer Res. 64, 3525-3532 (2004).
  16. Ridgeway, A. G., McMenamin, J., Leder, P. P53 levels determine outcome during beta-catenin tumor initiation and metastasis in the mammary gland and male germ cells. Oncogene. 25, 3518-3527 (2006).
  17. Zimmerman, M., Hu, X., Liu, K. Experimental metastasis and CTL adoptive transfer immunotherapy mouse model. J Vis Exp. , (2010).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved