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 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.
All procedures follow guidelines and approved protocols by Georgia Regents University Animal Use and Care Committee.
1. Establishment of Orthotopic Breast Cancer Tumor
2. Live Mouse Bioluminescence Imaging of Tumor Growth
3. Surgical Removal of Primary Tumors
NOTE: Autoclave scissors, forceps and the wound clips and wound clip applier (Figure 1).
4. Live Mouse Bioluminescence Imaging of Tumor Metastasis
5. Validation of Lung Metastases Using India Ink Inflation of Tumor-bearing Lungs
NOTE: To validate the luciferase live tumor imaging results, perform India ink inflation of tumor-bearing mouse lungs to quantify tumor nodules. 4T1 cells also metastasize to other organs and tissues (Figure 5) and therefore, histological examination of these organs to validate tumor metastasis should be performed if needed. This protocol use lung metastasis as a example.
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 imaging (Figure 3). Tumors are detectable approximately 3 days after tumor injection using a bioluminescence imaging system, and the luminescence intensity increases as tumor size increases (Figure 3). Therefore, this model is an ideal orthotopic breast cancer model for testing efficacy of chemotherapeutic and immunotherapeutic agents against all stages of mammary carcinoma.
Spontaneous Metastasis
In human breast cancer patients, the primary tumors are surgically removed after diagnosis. However, a large number of patients already have LN or distant metastasis at the time of the diagnosis. In the procedure, most of the mice have developed LN and distant metastasis after 30 days of tumor transplant. The described surgical procedure completely removes the primary tumor (Figure 2B). No residual primary tumors are detected at the primary tumor sites 10 – 30 days after surgery (Figure 2B). Surgical removal of the primary tumor extends the life span of the tumor-bearing mice but does not prevent tumor metastasis to various parts of the mice. These metastases can be detected by the live imaging method (Figure 4). This model thus is a spontaneous metastasis breast cancer model that mimics human breast cancer patients. This model is useful for: 1) studying spontaneous breast cancer metastasis (for example, 4T1 cells can be transfected with a gene of interest to test the functions of these genes in breast cancer spontaneous metastasis); and 2) testing the efficacy of chemotherapeutic and immunotherapeutic agents against spontaneous breast metastasis in an immune-competent host. The sites of and the degrees of metastasis can be detected by live imaging (Figure 4) and validate with a second approach (Figure 5). Luminescence imaging can quantify tumor burden of the entire lung. Ink inflation of tumor-bearing lungs allows accurate counting of the actual tumor nodules of each lung (Figure 5), thereby, representing a complimentary quantification method of tumor metastasis.
Figure 1. Surgery Tools. 1.Stainless scalpel, 2. Forceps. 3. Stainless scissors. 4. Autoclip wound clip applier. 5. Autoclip wound clip remover. Please click here to view a larger version of this figure.
Figure 2. The Orthotopic 4T1 Tumor Model. A. 4T1 tumor cells (1 x 104 cells in 100 μl PBS) cells were injected into the mammary fat pad. Thirty days after tumor injection, the mouse was sacrificed and examined for tumor growth. Shown is the tumor-bearing mice with a single tumor nodule at the site of injection (yellow arrow). B. The tumor as shown in A was surgically removed. Shown is the 4T1 tumor-bearing mice 10 days after surgical removal of the primary tumor. Please click here to view a larger version of this figure.
Figure 3. Live Tumor Imaging by Luciferase-based Live Tumor Imaging. 4T1 tumor cells (1 x 104 cells in 100 μl PBS) cells were injected into the mammary fat pad. The mouse was imaged at days 7, 14 and 28. Shown is the luminescence intensity of the primary tumor. Please click here to view a larger version of this figure.
Figure 4. Visualization of Tumor Progression by Luciferase-based Live Tumor Imaging. 4T1 tumor cells (1 x 104 cells in 100 μl PBS) cells were injected into the mammary fat pad. The primary tumor was surgically removed as shown in Figure 3. The mouse was then imaged 17 days after surgical removal of the primary tumor. Shown is image of metastasis. Please click here to view a larger version of this figure.
Figure 5. Visualization of Lung Tumor Nodules by India Ink Inflation. The lung of the tumor-bearing mouse was inflated with India ink and fixed in Fekete's solution. The white dots are lung metastases. Please click here to view a larger version of this figure.
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 models of breast cancer metastasis are excellent systems for studying the genetic and molecular mechanisms underlying breast cancer metastasis. However, the low metastasis incidence and long metastasis latency limit the usefulness of these models in the test of anti-cancer agents.
The orthotopic implantation of 4T1 cells in the mammary fat pad, with the formation of primary tumors and subsequent metastatic growth, resembles multiple stages from malignant breast cancer, including primary tumor formation, lymph node metastasis and distant organ metastasis. Moreover, syngenic transplantation avoids immunologic host-versus-graft reaction allowing the study of the contribution of tumor microenvironment, including immune system, to malignant tumor progression. This model is also useful for studying host anti-tumor immune response. Therefore, injection of 4T1 cells in the mammary fat pad represents a fast and quantitative method to study breast cancer metastasis.
The orthotopic 4T1 transplant tumor model has a 100% tumor incidence in less than 30 days and 100% metastasis incidence in less than 60 days (Figure 2-4). Furthermore, unlike most of the transgenic mouse models of breast cancer metastasis, 4T1 tumor cells also metastasize to the bone (Figure 4), thereby, resembling human breast cancer metastasis.
Stable expression of luciferase-coding cDNA in 4T1 tumor cells allows monitoring of tumor growth and progression in live mice over time (Figure 3). The tumor burden and metastasis kinetics can be quantified based on the luminescence intensity. Furthermore, the metastasis sites can also be identified by the luciferase imaging (Figure 4) and validated with a complementary approach (Figure 5). Therefore, this breast cancer spontaneous metastasis model is an excellent model for determining the efficacy of anti-metastasis agents in vivo.
Most of the surgery involves closure of cuts with sutures. Here, we observed that stainless wound clips work well (Figure 1). The wound clip can be easily sterilized and easily applied with the autoclip applier (Figure 1). The wound clips can be easily removed with the clip remover (Figure 1) after the wound has been healed. We have observed no infections with this surgical technique in over 20 surgeries.
One limitation of this model is the fast tumor growth rate. Majority of the metastases-bearing mice dies in 1 – 2 months after surgery due to extensive metastasis burden. Another limitation is that 4T1 is a mouse cell line and its response to experimental drugs could differ to the response from human cells. This should be considered in study design for experimental drug testing.
In summary, we have optimized a simple and yet sensitive procedure of orthotopic breast cancer spontaneous metastasis model. This model mimics human breast cancer metastasis. The tumor progression kinetics can be monitored and quantified over time. This model is an ideal one not only for studying breast cancer growth and metastasis, but also for testing the efficacy of chemotherapeutic and immunotherapeutic agents17 in suppression of spontaneous breast cancer metastasis.
The authors have nothing to disclose.
Supported by grants from the National Institute of Health grants CA133085, CA182518 and CA185909 (to KL) and VA Merit Review Award BX001962 (to KL).
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 |