Name: orthotopic injection of breast cancer cells into the mice mammary fat pad
Uploaded: 2019-01-20T13:00:00
Description: Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad at JoVE.com

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).&#160;We thank Prof. Chang-Zhen Liu, from Experimental Research Center, China&#160;Academy of Chinese Medical Sciences, for bioluminescent images.

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
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	<li>One day before the operation, shave the hair around the fourth nipples to expose the operating area.
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		<li>Use one ...

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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...

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 cancer2,3,4. Xenograft models are widely used for cancer study to understand the pathological characters and to screen drugs for safety and efficacy5,6,7. Genetically engineered mice (GEM) are generated to mimic human breast cancer by targeting certain oncogenes or tumor suppressor genes8. GEM have a relatively simple and uniformed background to understand the role of genes in cancerous progress; however, the artificial environment and background are limited to investigate the metastasis pathology and related therapies9. Human cancer cells, although with human pathological features, can only be implanted in immune-deficient mice, and the insufficiency of the tumor-host immune interaction may lead to biased results10.
Where the solid tumor initiates has a direct influence on the biological and pathological characters of the disease11,12,13. Since cancerous progress is the complicated outcome of interactions among tumor cells, stromal cells, immunology cells, inflammatory cells, growth factors, and proteases, primary tumors implanted in situ will provide better insight and mimic the cancerous process more accurately than tumors induced by chemical agents or a subcutaneous injection of tumor cells. Chemical agents used to induce tumors may be harmful to researchers and environment and are even forbidden in some countries. Because of the absence of a mammary fat pad environment, the pathological progress of a subcutaneous injection may differ with that in real breast cancer patients, whose cancer originates and irritates from the mammary fat pad. The disadvantages of subcutaneous injections encourage the use of orthotopic models to study tumor growth. In earlier research, highly metastatic MDA-MB-231 tumors, developed after seven orthotopic transplantations, indicated the importance of the injection location14. Recently, the orthotopic implantation of breast cancer cells into the mammary fat pad with surgery was reported15,16. With the mammary pad environment, the tumor growth and the migration into distant organs cover the entire process of breast cancer at pathologically relevant sites, which makes this model a miniature of the progress of human diseases. However, after the surgery, the skin automatically attempts to heal itself, which may bring the potential risk of interfering with the normal breast cancer origination and bias the results.
We have compared some breast cancer models and established a minimally invasive orthotopic model to investigate the potential effect of drugs on breast cancer progression17,18. In this study, a video protocol on how to orthotopically inject breast cancer cells into the mammary fat pad in a simple, less invasive way is presented. This orthotopic injection method without surgery is advantageous in many ways. First, the operation is simple and rapid, about 1 minute per mouse. Second, with primary tumor foci starting at the right pathological sites, it covers the whole tumorigenic process of breast cancer progression from the tumor growth to other organs metastasis, which provides a good experimental animal model for studying the interaction of tumor cells and tumor microenvironment. Besides, it can be a valuable model to estimate the treatment effects at all stages of breast cancer. The goal of this method is to provide an animal model to maximumly mimic human breast cancer progression.

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			<td height="48" style="height:49px;width:149px;">anesthesia machine</td>
			<td style="width:235px;">Midmark Corporation, Dayton, OH, USA</td>
			<td style="width:96px;">Matrx VMS</td>
			<td style="width:172px;">anesthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">In-Vivo Imaging System</td>
			<td style="width:235px;">PerkinElmer</td>
			<td style="width:96px;">IVIS Spectrum</td>
			<td style="width:172px;">used for bioluminescence detecion</td>
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			<td height="42" style="height:43px;width:149px;">&#160;isoflurane</td>
			<td style="width:235px;">Hebei yipin chemical reagents&#160; company</td>
			<td style="width:96px;">O21400</td>
			<td style="width:172px;">anesthesia</td>
		</tr>
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			<td height="40" style="height:40px;width:149px;">1 ml syringe</td>
			<td style="width:235px;">Becton,Dickinson and&#160; Company</td>
			<td style="width:96px;">A257</td>
			<td style="width:172px;">cell injection</td>
		</tr>
		<tr height="48">
			<td height="48" style="height:49px;width:149px;">digital caliper</td>
			<td style="width:235px;">Shang Hai Shen Han Measuring Tools Co., Ltd.</td>
			<td style="width:96px;">S-H</td>
			<td style="width:172px;">volume measurement</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">tramadol</td>
			<td style="width:235px;">Mundipharma company</td>
			<td style="width:96px;">-</td>
			<td style="width:172px;">pain killer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">D-luciferin</td>
			<td style="width:235px;">Gold Biotechnology Inc.</td>
			<td style="width:96px;">LUCK-1G</td>
			<td style="width:172px;">used for bioluminescence detecion</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:52px;width:149px;">The primary antibody against cluster of differentiation (CD) 31</td>
			<td style="width:235px;">Abcam</td>
			<td style="width:96px;">&#160;ab28364</td>
			<td style="width:172px;">used for MVD detection</td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;width:149px;">hematoxylin and eosin staining kit</td>
			<td style="width:235px;">&#160;Beijing Zhong Shan Jinqiao Biotechnology Co., Ltd.</td>
			<td style="width:96px;">ZLI-9615</td>
			<td style="width:172px;">histology&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:149px;">hair removal cream</td>
			<td style="width:235px;">Veet</td>
			<td style="width:96px;">-</td>
			<td style="width:172px;">hair removal cream</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:149px;">Carbomer Eye Gel</td>
			<td style="width:235px;">Dr.GerhardMannChem-Pharm.FabrikGmbH</td>
			<td style="width:96px;">-</td>
			<td style="width:172px;">ophthalmic ointment</td>
		</tr>
		<tr height="59">
			<td height="59" style="height:59px;">sewing needle</td>
			<td style="width:235px;">Shanghai Pudong Jinhuan Medical Products Co., Ltd.&#160;</td>
			<td>17U0302J</td>
			<td style="width:172px;">suture</td>
		</tr>
	</tbody>
</table>

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.

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...

Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad at JoVE.com

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

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, ...

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