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 with vacuum, add 5 ml sterile phosphate buffered saline (PBS), pH 7.4 to the culture dish, and gently shake the dish to wash the culture surface, remove the PBS by vacuum.</li>
	<li>Add 2 ml trypsin-EDTA (0.05% Trypsin and 0.53 mM EDTA) to the culture dish, incubate it in the CO2 incubator at 37 &#176;C for about 5 min. Check cells on an inverted microscope to make sure that all cells are detached from the bottom of the culture dish.</li>
	<li>Quench the trypsin by adding 8 ml serum-containing medium, transfer cells to a 15-ml conical tube and centrifuge the cells at approximately 450 x g for 3 min at RT.</li>
	<li>Remove the supernatant by vacuum and resuspend cells in 10 ml PBS. Centrifuge cells at approximately 450 x g for 3 min. Remove the supernatant by vacuum, and resuspend cells in 10 ml PBS.</li>
	<li>Pipette 10 &#956;l of the cell suspension onto a hemocytometer under the cover glass. Count the cells to determine the cell concentration. Resuspend cells in PBS at a density of 2 x 105 cells/ml HBSS. Transfer cells to a 1.5 ml sterile microfuge tube and keep cells on ice until use.</li>
	<li>Use female BALB/c mice of 6 - 8 week for tumor cell injection.
	<ol>
		<li>Shave hair near surrounding nipple # 3 using an electronic hair trimmer. Injection of 4T1 cell into the mammary gland 3 fat pad reproducibly generates lung metastasis. Clean the shaved area by using the cotton swab dipped into 70% ethanol. Resuspend cells by inverting the microcentrifuge tube 2 - 3 times. Gently aspirate 50 &#181;l of cell suspension into a &#189; CC 27 G 1/2 tuberculin syringe.</li>
		<li>Inject the tumor cells into the mammary fat pad under #3 mammary gland of a BALB/c mouse. Place the mouse in a clean cage and return the cage to animal housing facility. Wait approximately 21 - 30 days for tumor development.&#160;Tumor growth should be monitored on a regular basis as per institution&#8217;s policies for tumor studies.</li>
	</ol>
	</li>
</ol>
2. Live Mouse Bioluminescence Imaging of Tumor Growth
<ol>
	<li>Image mice every 3 days after tumor injection Transfer mice to the imaging facility. Inject 100 &#956;l luciferin (30 mg/ml in PBS) intraperitoneally (i.p.) to mice prior to imaging. After approximately 10 min, anesthetize mice in an induction chamber with 2% isoflurane/O2 flow. Use the forceps to touch the leg of the mouse. No response to the touch confirms that the mouse is fully unconscious.
	<ol>
		<li>Place the mice in the imaging chamber with continuous 2% isoflurane and O2&#160;at a flow rate of 2 L/min.&#160;&#160;Place the mouse such that the area of interest (i.e., the mammary pad of the initial tumor injection) faces the camera of the imaging system. Ensure the nose and mouth of the mouse are firmly set within the anesthetic tubing.&#160;Animals should be anesthetized per veterinary standards at the institution.</li>
	</ol>
	</li>
	<li>Acquire bioluminescence imaging with an array of various exposure time. For instance, set exposure to 30, field of view (FOV) to 25, object height to 1.5 cm, and obtain luminescent photographic images with low power X-ray. Set the units of measurement to &#34;radiance.&#34;
	<ol>
		<li>Use an automatic linear color range with a maximum of approximately 2.4 x 107 radiance units. After acquiring initial images, adjust settings and obtain an optimized-image (Figures 3 &#38; 4).</li>
	</ol>
	</li>
	<li>Return mice to clean cages and ensure mice regain consciousness with ambulation before returning the animals to the housing facility.</li>
	<li>Analyze data with the living imaging software associated with the imaging instrument. Using an imaging program, draw an enclosed line around the region of interest (ROI). Use the imaging software to determine the radiance intensity of the ROI. Record the relative intensities of each ROI for each mouse. 
	NOTE: Higher radiance intensities indicate greater levels of luminescent cells, and thus greater tumor growth.</li>
	<li>Visually examine the mouse every 3 days for adverse effects. 
	NOTE: Mice will be weighed once a week. A weight loss of 15% body weight will be considered adverse effect and the tumor-bearing mice will be euthanized as described in step 5. Mice with ulcerated tumors will also be euthanized.</li>
</ol>
3. Surgical Removal of Primary Tumors
NOTE: Autoclave scissors, forceps and the wound clips and wound clip applier (Figure 1).
<ol>
	<li>Perform the surgery in a sterile hood to maintain a sterile condition to reduce risk of infection. One day before surgery, shave the area surrounding the tumors of the tumor-bearing mice with an electric hair trimmer.</li>
	<li>Approximately 2 - 4 hr before surgery, feed the mice chewable carprofen tablets formulated for mice at a dose of 5 mg/kg body weight.</li>
	<li>Put the mice in a chamber containing 2% isoflurane in oxygen for approximately 30 sec. Continuously anesthetize the mouse using an anesthesia apparatus during the entire surgical period.</li>
	<li>Use a sterile cotton stick to apply vet ointment to the eyes of the mouse to prevent dryness while under anesthesia. Use the forceps to touch the leg of the mouse. No response to the touch confirms that the mouse is fully unconscious.</li>
	<li>Disinfect the skin area around the tumor site with three alternating wipes of a soap disinfectant scrub. Wipe the surgical area with a 70% alcohol wipe.</li>
	<li>Use a sterilized scalpel (Figure 1) to cut a small incision near the tumor.</li>
	<li>Insert the tip of a pair of sterilized scissors to the incision to cut the skin around the tumor to expose the tumor nodule (Figure 2A). Hold the tumor with sterile forceps and use the scissors to separate the tumor from the skin (Figure 2B). Save the tumor sample by freezing in a -80 &#176;C freezer or by fixing in 10% formalin solution for future use.</li>
	<li>Close the surgical site with 9 mm wound clips using the auto-clip applier (Figure 1). Return the mouse to a clean cage with autoclaved bedding.&#160;The animals should be monitored per institutional guidelines. 
	NOTE: The mouse usually regains sufficient consciousness and starts to move in the cage approximately 10 - 30 min after the surgery.</li>
	<li>Administer carprofen to mice again 24 hr after surgery. Carprofen is for post-surgery analgesia and prescribing of post-surgery analgesia should be verified by direct consultation with the institution&#8217;s veterinary staff.&#160;&#160;Remove wound clips after the wound heals (usually within 5 - 7 days after surgery). Remove wound clips using the auto-clip remover.</li>
</ol>
4. Live Mouse Bioluminescence Imaging of Tumor Metastasis
<ol>
	<li>Image mice every 3 days after surgery as described in step 2.</li>
</ol>
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.
<ol>
	<li>Keep mice in their existing home cage for euthanasia. When housed with other animals, relocate those mice not being euthanized to a different cage.</li>
	<li>Remove the cage filter top and replace with the CO2 filter top. Turn on the compressed CO2 gas cylinder (100%) that is connected to the CO2 filter top. Set the flow rate at 2 L/min and keep CO2 on for at least 10 min. If the mice are still breathing at the end of 10 min, leave the CO2 on until at least 1 min after mice stop breathing. Turn off CO2 cylinder control and the flow meter. Wait for 3 more minutes and remove the filter top.</li>
	<li>Place the sacrificed-mouse on its back on a polystyrene board. Pin the legs to ensure unobstructed access to the trachea. Spray the mice with 70% ethanol.</li>
	<li>Using a pair of scissors, cut along the midline of the mid-abdomen of the mouse through the ribcage and up toward the salivary glands. Observe the trachea. Use forceps to remove tissues surrounding the trachea to expose the trachea.</li>
	<li>Thread a pipette tip underneath the trachea. Holding the tip with one hand, gently lift the trachea up and away from the body.</li>
	<li>Rotate the platform holding the mouse 180&#176;. Use a &#189; CC 27 G 1/2 tuberculin syringe to inject India Ink (10% India ink and 0.1% Ammonium Hydroxide) into the lungs via the trachea. Completely inflate the lungs with ink until a strong resistance is felt.</li>
	<li>Use a pair of scissors to cut the trachea. Use forceps to hold the mouse and insert another set of forceps under the lungs and pull the lung lobes out of the mouse. Rinse the lungs briefly in a beaker containing water.</li>
	<li>In a chemical fume hood, transfer the lungs to a glass scintillation vial containing 3 ml of Fekete&#39;s solution (50% Ethanol, 6% Formaldehyde, and 3% glacial acetic acid). Cap the vial to prevent the solution from evaporating. 
	NOTE: The tissues can be stored in Fekete&#39;s solution indefinitely.</li>
	<li>After a few minutes, observe tumor nodules as white dots on the black lungs (Figure 5). The white tumor nodule is visible by eyes. Transfer the lungs to a dish in a fume hood and count the number of white spots. Each white spot represents a single metastatic tumor nodule.</li>
</ol>

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Mice were purchased from the Jackson Laboratory and housed in the Georgia Regents University animal facility. Experiments and care/welfare were in agreement with federal regulations and an approved protocol by the Georgia Regents University IACUC committee. The authors have nothing to disclose.

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.

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

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 ...

an-orthotopic-mouse-model-of-spontaneous-breast-cancer-metastasis

Research

JoVE Journal

Medicine

30.7K Views.  Augusta University.  Een orthotope borstkanker primaire tumor model en chirurgische verwijdering van de primaire tumor de muis het leven uit te breiden tot spontane metastase genereren beschreven. De tumorgroei en progressie worden gecontroleerd en gekwantificeerd luciferase fluorescentiebeeldvorming. 

Video: An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Cancer Research

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

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&#39; 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.

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, ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

Orthotopic Injection: Implanting Tissue Specific Cancer Cells into an Adult Mouse

Source: Katsuta, E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57849/generating-murine-orthotopic-metastatic-breast-cancer-model">Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy.</a>&#160;J. Vis. Exp.&#160;(2018).
The video demonstrates the breast cancer orthotopic inoculation technique in a mouse model. This technique produces a high tumorigenesis rate with less variability in tumor size and shape compared to subcutaneous or non-surgical injection.

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

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