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Cancer Research

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Published: January 20th, 2019



1Oncology Department, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs' metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Breast cancer is the leading cause of female mortality worldwide. With its progressively increasing incidence, breast cancer has become a serious challenge to public health1. Murine cancer models are good bridges between preclinical and clinical studies, and a good mimic murine disease model will increase the accuracy of research on disease and medicine.

Primary tumor growth starts the progress of malignant disease, while metastasis and complications are the main causes of death and poor life qualities in most cancer patients. Several murine models are used to mimic the pathology of human breast cancer.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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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 .......

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After a successful injection, one white transparent flat sphere with the nipple as the out-surface round center can be observed (Figure 1). Primary tumor growth can be measured by tumor volume and living tumor cell bioluminescence (Figure 2). Both the tumor volume and the total flux increased during the experiment before resection. At an early stage, metastasis cannot be found because no secondary tumor has happened or the strong.......

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The cells used in this study are 4T1-luc2, murine triple-negative breast cancer cells with luciferase labeling, which are a useful tool for investing antitumor and antimetastatic effects of drugs due to their highly invasive nature2,19. Luciferases, stable to the next cell generation, are used to indicate the living tumor cells, both at the mammary gland and at other distant organs20. In some cases, hypoxia and nutrient deficiency within t.......

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The authors would like to thank the National Natural Science Foundation of China (grant no. 81873111, 81673924, 81774039, 81503517), the Beijing Natural Science Foundation (grant no. 7172095, 7162084, 7162083), and the Xu Xia Foundation of the Beijing Hospital of Traditional Chinese Medicine (grant no, xx201701). We thank Prof. Chang-Zhen Liu, from Experimental Research Center, China Academy of Chinese Medical Sciences, for bioluminescent images.


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Name Company Catalog Number Comments
anesthesia machine Midmark Corporation, Dayton, OH, USA Matrx VMS anesthesia
In-Vivo Imaging System PerkinElmer IVIS Spectrum used for bioluminescence detecion
 isoflurane Hebei yipin chemical reagents  company O21400 anesthesia
1 ml syringe Becton,Dickinson and  Company A257 cell injection
digital caliper Shang Hai Shen Han Measuring Tools Co., Ltd. S-H volume measurement
tramadol Mundipharma company - pain killer
D-luciferin Gold Biotechnology Inc. LUCK-1G used for bioluminescence detecion
The primary antibody against cluster of differentiation (CD) 31 Abcam  ab28364 used for MVD detection
hematoxylin and eosin staining kit  Beijing Zhong Shan Jinqiao Biotechnology Co., Ltd. ZLI-9615 histology 
hair removal cream Veet - hair removal cream
Carbomer Eye Gel Dr.GerhardMannChem-Pharm.FabrikGmbH - ophthalmic ointment
sewing needle Shanghai Pudong Jinhuan Medical Products Co., Ltd.  17U0302J suture

  1. Torre, L. A., et al. Global cancer statistics. CA: A Cancer Journal for Clinicians. 65 (2), 87-108 (2012).
  2. Rashid, O. M., Takabe, K. Animal models for exploring the pharmacokinetics of breast cancer therapies. Expert Opinion on Drug Metabolism & Toxicology. 11 (2), 221-230 (2015).
  3. Wagner, K. U. Models of breast cancer: quo vadis, animal modeling. Breast Cancer Research. 6 (1), 31-38 (2004).
  4. Horas, K., Zheng, Y., Zhou, H., Seibel, M. J. Animal models for breast cancer metastasis to bone: opportunities and limitations. Cancer Investigation. 33 (9), 459-468 (2015).
  5. Kawaguchi, T., Foster, B. A., Young, J., Takabe, K. Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research. Journal of Mammary Gland Biology and Neoplasia. 22 (2), 131-139 (2017).
  6. Wright, L. E., et al. Murine models of breast cancer bone metastasis. BoneKEy Reports. 5, 804 (2016).
  7. Cassidy, J. W., Batra, A. S., Greenwood, W., Bruna, A. Patient-derived tumour xenografts for breast cancer drug discovery. Endocrine-Related Cancer. 23 (12), T259-T270 (2016).
  8. Stiedl, P., Grabner, B., Zboray, K., Bogner, E., Casanova, E. Modeling cancer using genetically engineered mice. Methods in Molecular Biology. 1267, 3-18 (2015).
  9. Menezes, M. E., et al. Genetically engineered mice as experimental tools to dissect the critical events in breast cancer. Advances in Cancer Research. 121, 331-382 (2014).
  10. Talmadge, J. E., Singh, R. K., Fidler, I. J., Raz, A. Murine models to evaluate novel and conventional therapeutic strategies for cancer. The American Journal of Pathology. 170 (3), 793-804 (2007).
  11. Spaw, M., Anant, S., Thomas, S. M. Stromal contributions to the carcinogenic process. Molecular Carcinogenesis. 56 (4), 1199-1213 (2017).
  12. Joyce, J. A., Pollard, J. W. Microenvironmental regulation of metastasis. Nature Reviews Cancer. 9 (4), 239-252 (2009).
  13. Hanahan, D., Weinberg, R. A. Hallmarks of cancer: the next generation. Cell. 144 (5), 646-674 (2011).
  14. Yano, S., et al. Tumor-targeting adenovirus OBP-401 inhibits primary and metastatic tumor growth of triple-negative breast cancer in orthotopic nude-mouse models. Oncotarget. 7 (51), 85273-85282 (2016).
  15. Kocaturk, B., Versteeg, H. H. Orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth. Journal of Visualized Experiments. (96), e51967 (2015).
  16. Tavera-Mendoza, L. E., Brown, M. A less invasive method for orthotopic injection of breast cancer cells into the mouse mammary gland. Lab Animal. 51 (1), 85-88 (2017).
  17. Zhang, Y., et al. Establishment of a murine breast tumor model by subcutaneous or orthotopic implantation. Oncology Letters. 15 (5), 6233-6240 (2018).
  18. Zhang, Y., et al. Gubenyiliu II Inhibits Breast Tumor Growth and Metastasis Associated with Decreased Heparanase Expression and Phosphorylation of ERK and AKT Pathways. Molecules. 22 (5), (2017).
  19. Kwon, Y. S., Lee, K. S., Chun, S. Y., Jang, T. J., Nam, K. S. Suppressive effects of a proton beam on tumor growth and lung metastasis through the inhibition of metastatic gene expression in 4T1 orthotopic breast cancer model. International Journal of Oncology. 49 (1), 336-342 (2016).
  20. Kalra, J., et al. Validating the use of a luciferase labeled breast cancer cell line, MDA435LCC6, as a means to monitor tumor progression and to assess the therapeutic activity of an established anticancer drug, docetaxel (Dt) alone or in combination with the ILK inhibitor, QLT0267. Cancer Biology & Therapy. 11 (9), 826-838 (2011).
  21. Hoffman, R. M. Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Investigational New Drugs. 17 (4), 343-359 (1999).
  22. Hoffman, R. M. Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts. Nature Reviews Cancer. 15, 451 (2015).

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