The authors would like to acknowledge the Netherlands Organization for Scientific Research (NWO, grant 17.106.329)

Animal experiments were approved by the animal welfare committee of the Leiden University Medical Center(LUMC).
1. Preparation of Cells, Instruments and Mice
<ol>
	<li>A day before operation, shave the NOD/SCID gamma (NSG) mice from the fourth nipple to the midline and weigh the mice to verify that all mice have roughly comparable weights.</li>
	<li>Autoclave a scissor, two tweezers and two straight mosquito forceps (Figure 1A) .</li>
	<li>On the day of operation, wash the h...

<ol>
	<li>Aaronson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+and+cancer.">Growth factors and cancer.</a> Science. 254 (5035), 1146-1153 (1991).</li><li>Bao, L., Matsumura, Y., Baban, D., Sun, Y., Tarin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+inoculation+site+and+Matrigel+on+growth+and+metastasis+of+human+breast+cancer+cells.">Effects of inoculation site and Matrigel on growth and metastasis of human breast cancer cells.</a> Br. J. Cancer. 70 (2), 228-232 (1994).</li><li>Brouxhon, S. 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Orthotopic injection of breast cancer cells is a powerful model to study all aspects of cancer growth. Implantation of these cells in the mammary fat pad of the mice should be carefully performed in order to prevent variation in tumor growth. Most importantly, injecting the same amount of cells to each mouse is crucial. To do so, one should trypsinize the cells rigorously without affecting viability of the cells. Non-viable cells should be disregarded during the cell counting and reagents (i.e. trypan blue) that...

Cancer is a very complex disease that has been subject to studies for over centuries. Breast cancer is the most common cancer type; it occurs predominantly in females but may sporadically also occur in males27. The disease is mainly caused by the loss of control mechanism governing cell division which in turn leads to an infinite growth of cells in the body. These malfunctions can be caused by several mechanisms: first, healthy cells need growth signals from the surrounding cells in order to proliferate whereas cancer cells make their own growth factors and increase the expression of growth factor receptors thus obtaining a higher proliferative rate1; second, cancer cells are less susceptible to anti-proliferative signals8; third, to balance the cell number in the body cell death is also required; however, cancer cells escape from programmed cell death, termed apoptosis14; fourth, cells adhere to extracellular matrices in order to survive but tumor cells can grow without the need of attachment and show resistance to anoikis19; fifth, activation of telomerase circumvents the telomere shortening and prevents the replicative senescence21; last but not least, skipping of DNA quality control following mitosis results in altered genetic content15,16. In order to identify oncogenes or tumor suppressors that play a role in this deregulated proliferation, tumor growth experiments in mice are crucial.
Primary tumor growth is generally not the main reason of death. Migration of cancer cells from the primary site to a secondary site, termed metastasis, is the leading cause of death in most cancer patients22. Metastasis entails tumor cell invasion, intravasation, travelling through the circulation, avoiding immune attack, extravasation and growth at the secondary site. Epithelial to mesenchymal transition (EMT) is a key process in metastasis and involves a switch in gene expression profiles yielding cells with higher motility and invasiveness, which are pre-requisites for the metastasizing cell12. As the cancerous process is the resultant of a combination of various actions, including reciprocal interactions between cancer cells, stromal cells and pro- and anti-inflammatory cells, an in vitro approach to cancer often does not provide full insight into the cancerous process. Similarly, anticancer treatments impacting the tumor vasculature can often not be studied in vitro, thus the use of in vivo approaches is inevitable.
To study breast cancer progression, different experimental methods have been developed. The most widely used model is the subcutaneous injection of breast cancer cells into mice5. In this experimental setup, the investigator may introduce a wide range of alterations to a cell line of choice in vitro (i.e. upregulation, downregulation of proteins) and inject the cells under the skin. Although this method is straightforward and the injection process is simple without any need to perform surgery on the mice, the site at which the tumor is injected does not represent the local mammary tumor environment and the absence of this environment may result in breast cancer development that differs from that observed in human pathology. Secondly, genetically engineered mice are used frequently as an in vivo tool to study breast cancer progression. In this model, oncogene (i.e. PyMT, Neu) expression is driven by a mammary tissue specific promoter leading to the formation of spontaneous breast tumor s. This experimental setup is useful to study the treatment aspect of the disease by injecting drugs or antibodies while checking tumor size in time3. However, breeding these mice with other mouse strains deficient or mutated in a gene of interest might also give insights into the role of different proteins in breast tumor growth24. The downside of this model is that it is prone to variation in tumor size and number. Moreover, the level of transgene expression depends on the integration site in the genome and can change from one mouse strain to another4. In this model, the expression of the transgene can be achieved by all the cells with epithelial origin whereas in human disease, only a subpopulation of cells express the oncogene or downregulate the tumor suppressor levels26. To study metastasis, breast cancer cells may also be injected intravenously (a model termed experimental metastasis)25. However, this approach only recapitulates the metastatic process partially; it circumvents the requirement for tumor cells to invade and intravasate, and starts from the point at which tumor cells are readily present in the circulation.
In our work, we use an orthotopic injection model to study the involvement of genes of interest in breast cancer progression13. We overexpress the protein in human breast cancer cells and inject them into the mammary fat pad of NOD/SCID gamma (NSG) mice. This method is advantageous in many ways: it allows very rapid and diverse genetic changes in the injected cell line, it covers the entire process of breast cancer progression from primary tumor growth to metastasis at pathologically relevant sites, and it also provides a good experimental model for studying the impact of therapeutic treatments at early or late stages of the disease. In addition, using this model one can investigate the role of stromal versus cancer cell-derived proteins in disease progression by using genetically modified mice or cells. Although subcutaneous injections are easier to perform, orthotopic models give rise to a more tumorigenic and more metastatic cancer cell population. Thus, results obtained by means of the subcutaneous injections might be either false-negative or false-positive6,17 encouraging the use of orthotopic models to study the tumor growth.

<table border="0" cellpadding="0" cellspacing="0" width="1093">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:185px;">Name of material/equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:95px;">Catalog number</td>
			<td style="width:641px;">Comments/Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bouin&#39;s solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT10132</td>
			<td>Used for investigating the metastasis on lungs</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>Used to fix the tissues</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Matrigel, growth factor reduced</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Cells can be resuspended in matrigel for injection</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mosquito forceps</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td>Used for stiching</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Angled forceps</td>
			<td>Electron microscopy sciences</td>
			<td>72991-4c</td>
			<td>These make the exposure of mammary fat pad easier</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissors</td>
			<td>B Braun Medicals</td>
			<td>BC056R</td>
			<td>Used to cut open the mice</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Straight forceps</td>
			<td>B Braun Medicals</td>
			<td>BD025R</td>
			<td>This is used to open up the skin to expose mammary fat pad</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NOD scid gamma mice</td>
			<td>Charles River</td>
			<td>005557</td>
			<td>Experimental animal used for experiment</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MDA-MB-231</td>
			<td>Sigma-Aldrich</td>
			<td>92020424</td>
			<td>Experimental cells used for injections</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oculentum simplex</td>
			<td>Teva Pharmachemie</td>
			<td></td>
			<td>Opthalmic ointment used to prevent drying out of eyes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Betadine</td>
			<td>Fischer Scientific</td>
			<td>19-898-859</td>
			<td>Ionophore, used to disinfect the surgical area</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazin/Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>X1251, K2753</td>
			<td>Use injected anesthesia as 10mg/kg and 100mg/kg body weight respectively</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Temgesic</td>
			<td>Schering-Plough</td>
			<td></td>
			<td>Use the painkiller as 0,05-0,1mg/kg body weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Life sciences</td>
			<td>11995</td>
			<td>For trypsin neutralization,use media with serum(FBS:media 1:10 volume); for injection, use media with no serum</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Buffered sodium citrate</td>
			<td>Aniara</td>
			<td>A12-8480-10</td>
			<td>Use the volume ratio as citrate:blood; 1:9</td>
		</tr>
	</tbody>
</table>

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.

Successful application of the &#8220;orthotopic breast cancer model&#8221; is based on proper injection of cells into the mammary fat pad. Experimental errors such as imprecise inoculation of cells or leakage might lead to variations in tumor size or even the absence of a tumor which leads to the formation of a structure looking similar to a mammary fat pad injected with a control buffer (Figure 2A). The growth rate of the tumor is dependent on the nature of the injected cell line and in general, can be ...

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

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

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 ...

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78.0K Views.  Leiden University Medical Center. 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.