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

All procedures follow guidelines and approved protocols by Georgia Regents University Animal Use and Care Committee.
1. Establishment of Orthotopic Breast Cancer Tumor
<ol>
	<li>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 &#176;C and 5% CO2.</li>
	<li>On the day of tumor cell injection, remove culture medium from the culture dish...

<ol>
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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...

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 There is currently no effective therapy for metastatic breast cancer. Therefore, development of chemotherapeutic and immunotherapeutic agents to suppress metastatic breast cancer is of great significance.
Various breast cancer spontaneous metastasis mouse models have been developed to study the molecular mechanisms underlying breast tumor cell progression and metastasis and to be used as models for the development of therapeutic agents.1,3-5 However, most of these mouse models are genetic tumor models that, while excellent models for mechanistic studies, are not suitable for testing therapeutic agents since the metastasis takes months to develop in these genetic models, thereby requiring costly long-term administration of the anti-cancer agents.6,7 Monitoring tumor progression in live mice is also technically challenging. In contrast, a transplanted breast cancer metastasis model has the advantages of short term tumor progression and easy tracking of tumor progression in live mice. The 4T1 orthotopic breast cancer spontaneous metastasis mouse model is such a transplanted tumor model.8 In this model, the breast tumor cells are transplanted into the mammary fat pad to establish primary tumor nodules. The primary tumor can then be surgically removed as in human breast cancer patients. 4T1 tumor cells are highly invasive.8,9,3,10 Almost all tumor-bearing mice develop metastasis in 30 days after tumor transplant into the mammary fat pad. However, 4T1 tumors grow aggressively in the primary sites and the tumor sizes often exceed the limits that are allowed in most animal protocols. At this stage, the metastases are often micrometastases. Therefore, it is essential to remove primary tumors to allow metastasis progress for testing therapeutic agents. Here, we report the establishment of a simple and yet sensitive procedure of primary tumor transplant, surgical removal of primary tumor and bioluminescence imaging-based quantification of the 4T1 tumor growth and progression in vivo.

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an orthotopic mouse model of spontaneous breast cancer metastasis

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.

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...

Watch this Scientific Journal Video about An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis at JoVE.com

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

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 ...

The overall goal of this procedure is to establish a mouse spontaneous metastasis breast cancer model that allows the quantification of primary tumor breast cancer growth as well as the visualization and measurement of the breast cancer metastasis kinetics.This method can help answer key questions in the field of spontaneous cancer metastasis, such as how can altering tumor-associated genes affect cancer growth and metastasis?The main advantage of this technique is that this model mimics human breast cancer development, making it ideal for testing the efficacy of therapeutic agents.On the day of the experiment, harvest an overnight culture of 4T1 luciferase tumor cells, and pellet the cells by centrifugation.Remove the supernatant by vacuum, then wash the cells in 10ml of PBS, and re-suspend the pellet in 10ml of fresh PBS for counting.Next, dilute the cells to 2x10^5 cells/ml concentration in HBSS, and output the suspension into 1.5ml sterile microcentrifuge tubes on ice.Then, restraining the mouse by hand, label the mammary glands of a 6-8 week-old female BALB/c mouse with a black marker, and use an electronic hair trimmer to shave the hairs surrounding the number 3 nipple.Proceed to re-suspend the cells by 2-3 inversions of the microfuge tube, then carefully aspirate 50

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29.8K Views.  Augusta University. The overall goal of this procedure is to establish a mouse spontaneous metastasis breast cancer model that allows the quantification of primary tumor breast cancer growth as well as the visualization and measurement of the breast cancer metastasis kinetics.This method can help answer key questions in the field of spontaneous cancer metastasis, such as how can altering tumor-associated genes affect cancer growth and metastasis?The main advantage of this technique is that this model mimics huma...