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

<ol>
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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>
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			<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>
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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>
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			<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>
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			<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>
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			<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, ...

This method can help answer key questions in the cancer field about how the microenvironment supports tumor growth or whether a specific drug can effectively inhibit the breast cancer progression. The main advantages of the tumor induction technique are that no surgery is needed and it is a less invasive manner in which to establish breast cancer in situ. Using this technique, the tumor cells grow within the mammary fat pad, in the appropriate tissue environment, mimicking human breast cancer progression more closely than subcutaneously injection or xenograft to mice cells.Ke-Xin Cao, our technician, and I, will assist Gan-Lin Zhang in demonstrating this procedure. On the day before the operation, manually restrain the recipient mouse and turn the animal belly side up to the fur to be shaved around the fourth nipples. Wipe the fur with a thin layer of depilatory cream.Use distilled water to remove the cream after 30 to 60 seconds and dry the mouse with soft paper towels. On the day of the operation, dilute the murine breast cancer cell suspension to a two times ten to the fifth cells per milliliter of PBS concentration, in a sterile microcentrifuge tube on ice. Before each injection, load one, one milliliter syringe equipped with a 45 by 16 needle, with 50 microliters of cells per mouse.And confirm a lack of response to toe pinch in each sedated animal. Apply ointment to the animal's eyes and use tweezers to tent the skin around the fourth nipple of the first mouse, to create a tunnel for the syringe to follow. Hold the syringe with the bevel facing upwards and subcutaneously enter the skin at five to 10 millimeters from the nipple, following the tunnel until the needle tip almost reaches the nipple.Using tweezers to tent the skin around the first nipple creates a tunnel for the syringe to follow. The needle should be entered between the tweezer tips and should raise up instead of depressing into the tissue. Carefully move the needle tip up into the mammary fat pad until the bevel is under the nipple, and release the skin tent to allow a slow injection of the cancer cells.Upon insertion, carefully move the needle tip up into the mammary fat pad until the bevel is under the nipple. And you will feel resistance to ensure delivery of the cells to the correct location. When all of the cells have been injected, turn the needle slightly to the side and retract the syringe slowly.Use a cotton swab to apply pressure to the needle wound for 10 to 20 seconds, to make there is no fluid seepage. The injected nipple should appear as a transparent, white, flat sphere, with the nipple in the center. To establish the tumor volume, restrain the mouse belly side up and use a caliper to measure the tumor length and width.To capture the tumor bioluminescence, 10 minutes before imaging inject 150 milligrams per kilogram of D-luciferin into the back of the mouse's neck. And capture bioluminescence images of the primary tumor and metastases under the appropriate bioluminescence imaging parameters. At the appropriate experimental time point, use scissors to harvest the tumor from the skin.And apply the analgesic solutions to the surgical site to relieve post-operative pain. Then close the skin with surgical sutures according to standard protocols. Allow the animal to recover under a warming lamp with monitoring until full recovery.Use a scalpel to cut the primary tumor into two equal pieces. Store one-half of the tissue in 4%paraformaldehyde for subsequent hematoxylin and eosin staining and immunohistochemistry. Freeze the other half in liquid nitrogen for western blotting or RNAi isolation.At the appropriate experimental endpoint harvest the lungs according to standard dissection protocols and wash the organ in PBS. Then fix the lungs in 4%paraformaldehyde to verify metastasis by hematoxylin and eosin staining. Primary tumor growth can be measured by tumor volume and living tumor cell bioluminescence, as demonstrated.Metastases are not observed during the early stages of the inoculation as either no secondary tumor has been established, or the strong bioluminescent signals of the primary tumor obstruct the detection of small metastatic foci with weak signals. After resection, the bioluminescence signal from the metastatic foci of distant organs can be detected and analyzed. Analysis of the morphology of both primary breast tumor and lung sections by hematoxylin and eosin staining reveals increased angiogenesis in the tumor cell inoculated animals as evidenced by anti-CD31 microvessel marker staining.When attempting this procedure, it's important to remember to make sure the needle tip is in the correct location before delivering the tumor cells. Following this procedure, atomizers, like animal ultrasound, can be performed to answer additional questions about bladder supplementation of the primary tumor in vivo. After its development, this technique paved the way for researchers in the field of tumorigenesis and the drug development in exploring the key factors and pathways involved in tumorigenesis and the mechanisms of anti-cancer drug in murine animal models.Don't forget that working with isoflurane or other gas anesthesia can be extremely hard to dose and that precautions such as using an activated carbon mask, should always be taken while performing this procedure.

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Cancer Research

48.4K visualizações.  Capital Medical University. Este método pode ajudar a responder a perguntas-chave no campo do câncer sobre como o microambiente apoia o crescimento do tumor ou se uma droga específica pode efetivamente inibir a progressão do câncer de mama. As principais vantagens da técnica de indução do tumor são que não é necessária cirurgia e é uma forma menos invasiva para estabelecer o câncer de mama in situ. Usando essa técnica, as células tumorais crescem dentro da almofada de gordura mamária, no ambiente de tec...