This work was supported by NIH grant R01CA160688 and Susan G. Komen Foundation Investigator Initiated Research grant (IIR12222224) to K.T. Mice bioluminescence images were acquired by shared resource Translational Imaging Shared Resource at Roswell Park Comprehensive Cancer Center, which was supported by the Cancer Center Support Grant (P30CA01656) and Shared Instrumentation grant (S10OD016450).

Approval from the Roswell Park Comprehensive Cancer Center Institutional Animal Care and Use Committee was obtained for all experiments.
NOTE: Nine&#160;to twelve weeks-old female BALB/c mice are obtained. 4T1-luc2 cells, a mouse mammary adenocarcinoma cell line derived from BALB/c mice that has been engineered to express luciferase, are used. These cells are cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum (FBS).
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<ol>
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The authors have nothing to disclose.

For the last decade, we have been establishing multiple murine cancer models, including breast cancer models3,7,13,16,20,21. Previously, we demonstrated that breast cancer cell orthotopic inoculation into the mammary gland tissue under direct vision produced a larger tumor with less size variability compared to injecting cell...

Animal models play a key role in cancer research. When a hypothesis is proven in vitro, it should be tested in vivo to evaluate its clinical relevance. Cancer progression and metastasis are often better captured by animal models as compared to in vitro models, and it is essential to test a new drug in an animal model as a preclinical study for drug development1,2. However, the technical details of animal experiments are often not well described in published articles, making it challenging to reproduce the model successfully. Indeed, the authors who established these orthotopic inoculation and mastectomy models went through long and rigorous processes of trial and error. The success rate of tumorigenesis after cancer cell inoculation is one of the key factors to determine the success and efficiency of an animal study3. The cell line and the number of cells to inoculate, the inoculation site, and the strain of the mice are all important factors. It is well known that there are huge variations in the results of animal experiments due to individual differences, compared to in vitro techniques. Therefore, using a well-established model with a standard technique is important to obtain stable results, to improve the efficiency of animal experiments, and to avoid misleading results.
This paper provides well-established techniques4 to generate breast cancer orthotopic and mastectomy mouse models. The aims of these methods are 1) to mimic human breast cancer progression and treatment courses, and 2) to conduct in vivo experiments with greater efficiency and higher success rates compared to other breast cancer inoculation or mastectomy techniques. In orthotopic cancer cell inoculation, to mimic human breast cancer progression, we choose the #2 mammary fat pad as an inoculation site, which is located in the chest. In most of the studies, breast cancer cells are inoculated subcutaneously5. This technique does not require surgery and, thus, it is simple and straightforward. However, the subcutaneous microenvironment is quite different from the mammary gland microenvironment, which results in different cancer progression and even molecular profiles6,7. Some studies use the #4 mammary gland, which is located in the abdomen, as an inoculation site6. However, since #4 mammary glands are located in the abdomen, the most common metastatic pattern is peritoneal carcinomatosis7, which occurs with less than 10% of metastatic breast cancer8. Breast cancer generated by the technique presented here, in the #2 mammary gland, metastasizes to the lung, which is one of the most common breast cancer metastatic sites9.
With this technique, the goal is also to achieve a higher tumorigenesis rate with minimal tumor size variability compared to other breast cancer inoculation techniques. To do so, cancer cells suspended in a gelatinous protein mixture are inoculated under direct vision through a median anterior chest wall incision. This technique produces a high tumorigenesis rate with less variability in tumor size and shape compared to subcutaneous or non-surgical injection, as previously reported3,7.
We also introduce a mouse radical mastectomy technique in which the orthotopic breast tumor is resected with the surrounding tissues and axillary lymph nodes. In the clinical setting, the standard of care for breast cancer patients without distant metastasis disease is mastectomy10,11. Before a mastectomy, axillary lymph node metastasis is surveyed by imaging and sentinel lymph node biopsy. If there is no evidence of axillary lymph node metastasis, the patient is then treated with a total or partial mastectomy, in which the axillary lymph node resection is omitted. Total mastectomy is a technique to resect breast cancer with the whole breast tissue en bloc, whereas partial mastectomy is to resect breast cancer with a margin of surrounding normal breast tissue only, thus conserving the remaining normal breast tissue in the patient. However, patients who preserve remaining normal breast tissue after a partial mastectomy require postoperative radiotherapy to avoid local recurrence10. Patients who have axillary lymph node metastasis undertake radical mastectomy which removes the breast cancer with all normal breast tissue and axillary lymph nodes and invaded tissues en bloc10,11. In the mouse model, surveillance for axillary lymph node metastasis and/or post-operative radiation is not reasonable or feasible. Thus, we utilize the radical mastectomy technique to avoid local or axillary lymph node metastasis.
Cancer cell inoculation via the tail vein is the most common lung metastasis mouse model12, the so-called &#34;experimental metastasis&#34;. This model is easy to generate and does not require surgery; however, it does not mimic human breast cancer progression which may result in different metastatic disease behavior. In order to mimic the human breast cancer treatment course where metastasis often occurs after mastectomy, the primary tumor is removed after orthotopic cancer cell inoculation. This technique produces less local recurrence compared to simple tumor resection, as previously reported13, and is useful for novel therapeutics, preclinical studies, and for metastatic breast cancer research studies. The techniques described here are applicable for most breast cancer orthotopic model experiments. However, it is important to consider that the gelatinous protein mixture can affect the microenvironment and surgery can affect the stress/immune response14. Therefore, investigators studying the microenvironment and/or the stress/immune response should be aware of potential confounding factors.

<table>
	<tbody>
		<tr>
			<td>Micro Dissection Scissors</td>
			<td>Roboz</td>
			<td>RS-5983</td>
			<td>For cancer cell inoculation and masstectomy</td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>Roboz</td>
			<td>RS-5233</td>
			<td>For cancer cell inoculation and masstectomy</td>
		</tr>
		<tr>
			<td>Needle Holder</td>
			<td>Roboz</td>
			<td>RS-7830</td>
			<td>For cancer cell inoculation and masstectomy</td>
		</tr>
		<tr>
			<td>Mayo</td>
			<td>Roboz</td>
			<td>RS-6873</td>
			<td>For ex vivo</td>
		</tr>
		<tr>
			<td>5-0 silk sutures</td>
			<td>Look</td>
			<td>774B</td>
			<td>For cancer cell inoculation and masstectomy</td>
		</tr>
		<tr>
			<td>Dry sterilant (Germinator 500)</td>
			<td>Braintree Scientific</td>
			<td>GER 5287-120V</td>
			<td>For cancer cell inoculation and masstectomy</td>
		</tr>
		<tr>
			<td>Clipper</td>
			<td>Wahl</td>
			<td>9908-717</td>
			<td>For cancer cell inoculation and masstectomy</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>354234</td>
			<td>For cancer cell inoculation</td>
		</tr>
		<tr>
			<td>D-Luciferin, potassium salt</td>
			<td>GOLD-Bio</td>
			<td>LUCK-1K</td>
			<td>For bioluminescence quantification</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Insitute 1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serub</td>
			<td>Gibco</td>
			<td>10437028</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco</td>
			<td>25200056</td>
			<td>For cell culture</td>
		</tr>
	</tbody>
</table>

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.

The purpose of the orthotopic model is to mimic human cancer progression (i.e., the growth of the primary tumor followed by lymph node metastasis and then distant lung metastasis)15. After cancer cell inoculation, the bioluminescence is quantified regularly (two to three times/week) (Figure 1A). The bioluminescence in the lungs is deeper and smaller than the primary lesion. The bioluminescence mainly reflects the primary tumor...

Watch this Scientific Journal Video about Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy at JoVE.com

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

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 ...

This method can help answer key questions in the breast cancer field about how to mimic human breast cancer progression and treatment in a mouse model. The main advantage of this mouse breast cancer model is that it has a high tumorigenesis rate after orthotopic implantation and a low local recurrence rate after mastectomy. Begin by confirming a lack of response to toe pinch in an anesthetized female mouse and taping the animal's limbs to a surgical board.Using sterile cotton swabs sterilize the surgical area with chlorhexidine, iodine and 75%ethanol. And make a five-millimeter skin incision in the middle of the anterior chest wall. Next lift the right side of the skin adjacent to the incision and use sterile microdissection scissors to detach the skin from the chest wall.Invert the skin to expose the right number two mammary fat pad and use a one-milliliter insulin syringe equipped with a 28.5-gauge needle to carefully inject 20 microliters of cancer cell suspension through the wound into the fat pad. Hold the needle within the fat pad for five seconds to allow the gelatinous cancer cell protein mixture to solidify before removing the needle, and closing the incision with sterile 5-0 nonabsorbable sutures. Then place the animal in a clean cage with monitoring until full recumbency.To perform the mastectomy eight days after cancer inoculation prepare the mouse for surgery as just demonstrated, and make a five-millimeter skin incision two millimeters to the left of the surgical scar from the cancer cell inoculation. Extend the incision toward the root of the forelimb to remove the tumor, and the skin, including the surgical scar, and the lesion in contact with the tumor, and the axillary lymph node basin in which typically no visible lymph nodes are present at the time of the mastectomy. Then close the skin defects with sterile 5-0 nonabsorbable sutures in the shape of a Y.And return the animal to its cage with monitoring until full incumbency.For lung metastasis quantification 21 days after inoculation inject 150 milligrams per kilogram of D-Luciferin IP 15 minutes before euthanasia. And use curved Mayo scissors to open the skin and peritoneum at the mid-abdomen. Press down the liver until the diaphragm can be visualized, and cut the diaphragm and the bilateral ribs from caudad to cephalad.Flip the anterior thorax wall to expose the lungs and lift the lungs to identify the thoracic esophagus, which looks like a cord connecting the lungs to the spine. Cut the esophagus with microdissection scissors and use forceps to pull the bilateral lungs and heart toward the caudad. Then cut the trachea and major vessels to the lung apex at the cephalad and place the lungs into a 10-centimeter Petri dish for bioluminescent imaging.Use the scissors to remove the heart from the lung tissue. Beginning at about eight days after mammary fat pad cancer cell inoculation the bioluminescence in the lungs is deeper and smaller than in the primary lesion reflecting the primary tumor burden in live mice. Measurement of the tumor size with calipers allows estimation of the tumor volume in situ.This mastectomy model also allows serial metastatic tumor burden monitoring without having to euthanize the animals. The tumor burden of the lung metastases can also be quantified by ex vivo imaging, but the limitation is that the mice must be euthanized. Mouse survival after surgical treatment can also be monitored as a translatable clinical endpoint.After its development this technique paved the way for researchers in the field of breast cancer to explore not only new drug development, but also breast cancer research in general.

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10.3K Views.  Roswell Park Comprehensive Cancer Center. This method can help answer key questions in the breast cancer field about how to mimic human breast cancer progression and treatment in a mouse model. The main advantage of this mouse breast cancer model is that it has a high tumorigenesis rate after orthotopic implantation and a low local recurrence rate after mastectomy. Begin by confirming a lack of response to toe pinch in an anesthetized female mouse and taping the animal's limbs to a surgical board.Using sterile cotton swabs ste...