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 human breast adenocarcinoma (MDA-MB-231) cells that will be injected once with phosphate buffered saline (PBS), pH 7.4 and trypsinize the cells. Quench the trypsin by adding 10 ml serum-containing DMEM media on top of the cells. Centrifuge the cells at 1,200 rpm for 7 min at RT to remove serum by resuspending the cells either in PBS or in media without serum. Centrifuge them again to remove traces of serum completely. Resuspend the cells in PBS or media.</li>
	<li>Count the cells with a hemocytometer and calculate the amount of cells. Use 500,000 cells/mouse in not more than 150 &#956;l. Optional to PBS or media, resuspend the cells in matrigel. 
	NOTE: Determine the amount of cells depending on the cell type used.</li>
	<li>Place cells in a sterile microcentrifuge tubes and keep them on ice.</li>
	<li>Fill one 50 ml conical centrifuge tube with 35 ml of PBS pH 7.4 and another 50ml conical centrifuge tube with 70% ethanol. Soak cotton swabs into each tube.</li>
</ol>
2. Orthotopic Injection
<ol>
	<li>Perform the surgery in a sterile hood to maintain a sterile atmosphere. Anesthetize the mouse by subcutaneously injecting Xylazin/Ketamine mix at a dose of 10mg/kg, 100 mg/kg body weight respectively. Fix the mouse on a heating pad. Confirm the success of anesthesia by the lack of reaction to toe pinch. 
	NOTE: If one is not very familiar with surgical procedures, surgery may take longer. Arrange the anaesthesia dose accordingly.</li>
	<li>Apply ophthalmic ointment on the eyes of mice, to prevent the eyes from drying out.</li>
	<li>Clean the shaved area by using the cotton swab dipped into ethanol prepared in step 1.6. Alternating scrubs using betadine and alcohol three times in a circular fashion rotating away from the planned incision site should be performed. Alternatively, use ionophore as a disinfectant.</li>
	<li>Make a small incision between the fourth nipple and the midline with a scissor and make a pocket by inserting the cotton swab moistened with PBS pH 7.4.</li>
	<li>Use a tweezer to expose the mammary fat pad. Observe the fat pad by its white colour.</li>
	<li>Squeeze the fat pad with the other tweezer from its base; by doing this, fully expose the fat pad (Figure 1B) to perform injections easily.</li>
	<li>Homogenize the cell mixture by pipetting up and down. Gently aspirate 50 &#181;l of cell suspension into an insulin syringe and inject into the mammary fat pad by holding the needle horizontally. Confirm successful injection by checking for swelling of the fat pad.</li>
	<li>Release the fat pad gently.</li>
	<li>Suture the incision by using a needle driver. Make three throws by turning the suture around the needle driver in clockwise, anti-clockwise and clockwise direction. Two knots per suture should be used. 
	NOTE: Make sure the knots are tight otherwise mice can open up the sutures.</li>
	<li>After surgery, inject an analgesic, such as temgesic at 0.05-0.1 mg/kg bodyweight, subcutaneously in order to relieve the pain.</li>
	<li>Do not leave the animals unattended until they gain consciousness and maintain sternal recumbence. Place all the animals into different cages until they are fully recovered.</li>
	<li>To maintain sterility, use autoclaved cages and water. Change the cages every three days to keep the atmosphere clean.</li>
</ol>
3. Harvesting Organs for Analysis
<ol>
	<li>At the day of harvest, 8 weeks after the implantation of the cells, fill 15 ml conical centrifuge tubes with 3 ml Bouin&#8217;s solution for each mouse. In addition, use two 15 ml tubes filled with 5 ml formalin solution per mouse.</li>
	<li>Anesthetize the animals by injecting Xylazin/Ketamine, at a dose of 10 mg/kg 100 mg/kg body weight subcutaneously. Wait until the animal loses toe pinch withdrawal reflex.</li>
	<li>Fix the animal with needles on a base. Make a long, vertical midline incision with scissors. Make two horizontal incisions right below the front leg and above the rear leg. Expose the tumor by pinning the skin to the base. Measure the tumor volume by using a caliper.</li>
	<li>Dissociate the tumor from the skin using scissors. Snap freeze a part of the tumor in liquid nitrogen for RNA isolation. Place the other part, into the conical centrifuge tube filled with formalin to perform immunohistochemistry following paraffin embedding.</li>
	<li>Gently take out the lungs. Place the left lung into Bouin&#8217;s solution. Keep the lung in solution for 3 days. Observe superficial metastatic foci clearly to naked eye. Optionally, place the right lung into formalin to verify micrometastasis using haematoxylin and eosin (H&#38;E) staining. 
	NOTE: Although, lung metastasis observed frequently in breast cancer, one might also want to collect bone, liver, brain and spleen to analyse metastasis. The cells at the metastatic area are denser and morphologically different and therefore can be distinguished easily from lung tissue.</li>
	<li>A day after harvest, aspirate the formalin solution from 15 ml tubes and replace with 70% ethanol. Embed the tissues in paraffin and perform immunohistochemistry studies.</li>
	<li>For blood analysis, open up the chest cavity with scissors. Withdraw 450 &#181;l blood via cardiac puncture. Place blood sample in a microcentrifuge tube containing 50 &#181;l of 3.2% sodium citrate. After blood is collected, spin it at 2,000 x g for 10 min. Store the plasma samples at -20 &#176;C until further use. 
	NOTE: There are several other blood collection techniques commonly used in practice20 and the technique that needs to be used depends on experimental setup and scientist&#8217;s choice. If blood sample is not required, euthanize the animal according to the animal protocol or as per the institution guidelines.</li>
</ol>

<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. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibody+against+the+ectodomain+of+E-cadherin+(DECMA-1)+suppresses+breast+carcinogenesis:+involvement+of+the+HER/PI3K/Akt/mTOR+and+IAP+pathways.">Monoclonal antibody against the ectodomain of E-cadherin (DECMA-1) suppresses breast carcinogenesis: involvement of the HER/PI3K/Akt/mTOR and IAP pathways.</a> Clin. Cancer Res. 19 (12), 3234-3246 (2013).</li><li>Dobie, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variegated+transgene+expression+in+mouse+mammary+gland+is+determined+by+the+transgene+integration+locus.">Variegated transgene expression in mouse mammary gland is determined by the transgene integration locus.</a> Proc. Natl. Acad. Sci. U S A. 93 (13), 6659-6664 (1996).</li><li>Ewens, A., Mihich, E., Ehrke, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distant+metastasis+from+subcutaneously+grown+E0771+medullary+breast+adenocarcinoma.">Distant metastasis from subcutaneously grown E0771 medullary breast adenocarcinoma.</a> Anticancer Res. 25 (6B), 3905-3915 (2005).</li><li>Fidler, I. J., Naito, S., Pathak, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+implantation+is+essential+for+the+selection,+growth+and+metastasis+of+human+renal+cell+cancer+in+nude+mice+[corrected.">Orthotopic implantation is essential for the selection, growth and metastasis of human renal cell cancer in nude mice [corrected.</a> Cancer Metastasis Rev. 9 (2), 149-165 (1990).</li><li>Fridman, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+tumor+growth+of+both+primary+and+established+human+and+murine+tumor+cells+in+athymic+mice+after+coinjection+with+Matrigel.">Enhanced tumor growth of both primary and established human and murine tumor cells in athymic mice after coinjection with Matrigel.</a> J. Natl. Cancer Inst. 83 (11), 769-774 (1991).</li><li>Fynan, T. M., Reiss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+inhibition+of+cell+growth+by+transforming+growth+factor-beta+and+its+role+in+oncogenesis.">Resistance to inhibition of cell growth by transforming growth factor-beta and its role in oncogenesis.</a> Crit Rev. Oncog. 4 (5), 493-540 (1993).</li><li>Huang, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypsin-induced+proteome+alteration+during+cell+subculture+in+mammalian+cells.">Trypsin-induced proteome alteration during cell subculture in mammalian cells.</a> J. Biomed. Sci. 17, 36 (2010).</li><li>Iorns, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mouse+model+for+the+study+of+human+breast+cancer+metastasis.">A new mouse model for the study of human breast cancer metastasis.</a> PLoS. One. 7 (10), e47995 (2012).</li><li>Jessani, N., Niessen, S., Mueller, B. M., Cravatt, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cell+lines+grown+in+vivo:+what+goes+in+isn't+always+the+same+as+what+comes+out.">Breast cancer cell lines grown in vivo: what goes in isn't always the same as what comes out.</a> Cell Cycle. 4 (2), 253-255 (2005).</li><li>Kalluri, R., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> J. Clin. Invest. 119 (6), 1420-1428 (2009).</li><li>Kocaturk, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternatively+spliced+tissue+factor+promotes+breast+cancer+growth+in+a+beta1+integrin-dependent+manner.">Alternatively spliced tissue factor promotes breast cancer growth in a beta1 integrin-dependent manner.</a> Proc. Natl. Acad. Sci. U S A. 110, 11517-11522 (2013).</li><li>Lowe, S. W., Lin, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+cancer.">Apoptosis in cancer.</a> Carcinogenesis. 21 (3), 485-495 (2000).</li><li>Meek, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p53+response+to+DNA+damage.">The p53 response to DNA damage.</a> DNA Repair (Amst). 3 (8-9), 1049-1056 (2004).</li><li>Meek, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppression+by+p53:+a+role+for+the+DNA+damage+response).">Tumor suppression by p53: a role for the DNA damage response).</a> Nat. Rev. Cancer. 9 (10), 714-723 (2009).</li><li>Miller, F. R., Medina, D., Heppner, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+growth+of+mammary+tumors+in+intact+mammary+fatpads.">Preferential growth of mammary tumors in intact mammary fatpads.</a> Cancer Res. 41 (10), 3863-3867 (1981).</li><li>Mueller, B. M., Ruf, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+binding+of+catalytically+active+factor+VIIa+in+tissue+factor-dependent+experimental+metastasis.">Requirement for binding of catalytically active factor VIIa in tissue factor-dependent experimental metastasis.</a> J. Clin. Invest. 101 (7), 1372-1378 (1998).</li><li>Paoli, P., Giannoni, E., Chiarugi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anoikis+molecular+pathways+and+its+role+in+cancer+progression.">Anoikis molecular pathways and its role in cancer progression.</a> Biochim. Biophys. Acta. 1833 (12), 3481-3498 (2013).</li><li>Parasuraman, S., Raveendran, R., Kesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+sample+collection+in+small+laboratory+animals.">Blood sample collection in small laboratory animals.</a> J. Pharmacol. Pharmacother. 1 (2), 87-93 (2010).</li><li>Shay, J. W., Zou, Y., Hiyama, E., Qright, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+and+cancer.">Telomerase and cancer.</a> Hum. Mol. Genet.. 10 (7), 677-685 (2001).</li><li>Sporn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+war+on+cancer.">The war on cancer.</a> Lancet. 347 (9012), 1377-1381 (1996).</li><li>Tao, K., Fang, M., Alroy, J., Sahagian, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imagable+4T1+model+for+the+study+of+late+stage+breast+cancer.">Imagable 4T1 model for the study of late stage breast cancer.</a> BMC. Cancer. 8, 228 (2008).</li><li>Versteeg, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-activated+receptor+(PAR)+2,+but+not+PAR1,+signaling+promotes+the+development+of+mammary+adenocarcinoma+in+polyoma+middle+T+mice.">Protease-activated receptor (PAR) 2, but not PAR1, signaling promotes the development of mammary adenocarcinoma in polyoma middle T mice.</a> Cancer Res. 68 (17), 7219-7227 (2008).</li><li>Versteeg, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+tissue+factor+signaling+suppresses+tumor+growth.">Inhibition of tissue factor signaling suppresses tumor growth.</a> Blood. 111 (1), 190-199 (2008).</li><li>Wagner, K. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+breast+cancer:+quo+vadis,+animal+modeling?.">Models of breast cancer: quo vadis, animal modeling?.</a> Breast Cancer Res. 6 (1), 31-38 (2004).</li><li>Weigelt, B., Peterse, J. L., van't Veer, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+metastasis:+markers+and+models.">Breast cancer metastasis: markers and models.</a> Nat. Rev. Cancer. 5 (8), 591-602 (2005).</li></ol>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>Bouin&#039;s solution</td><td>Sigma-Aldrich</td><td>HT10132</td><td>Used for investigating the metastasis on lungs</td></tr><tr><td>Formalin solution</td><td>Sigma-Aldrich</td><td>HT501128</td><td>Used to fix the tissues</td></tr><tr><td>Matrigel, growth factor reduced</td><td>Corning</td><td>356230</td><td>Cells can be resuspended in matrigel for injection</td></tr><tr><td>Mosquito forceps</td><td>Fine Science Tools</td><td>13008-12</td><td>Used for stiching</td></tr><tr><td>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><td>Scissors</td><td>B Braun Medicals</td><td>BC056R</td><td>Used to cut open the mice</td></tr><tr><td>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><td>NOD scid gamma mice</td><td>Charles River</td><td>005557</td><td>Experimental animal used for experiment</td></tr><tr><td>MDA-MB-231</td><td>Sigma-Aldrich</td><td>92020424</td><td>Experimental cells used for injections</td></tr><tr><td>Oculentum simplex</td><td>Teva Pharmachemie</td><td></td><td>Opthalmic ointment used to prevent drying out of eyes</td></tr><tr><td>Betadine</td><td>Fischer Scientific</td><td>19-898-859</td><td>Ionophore, used to disinfect the surgical area</td></tr><tr><td>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><td>Temgesic</td><td>Schering-Plough</td><td></td><td>Use the painkiller as 0.05-0.1 mg/kg body weight</td></tr><tr><td>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><td>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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78.7K 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.

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

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, ...

Cancer Research

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

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. ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

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 ...

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

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.

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 ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

Orthotopic Injection into the Mammary Fat Pad:  Establishing Breast Cancer in Mice

Source: Zhang, G. L., et al. <a href="https://www.jove.com/video/58604/orthotopic-injection-breast-cancer-cells-into-mice-mammary-fat?access=v2wuhgcg">Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad.</a>&#160;J. Vis. Exp. (2019).
In this article we discuss how to establish a breast cancer model in female mice by injecting breast cancer cells into the mammary fat pad. The example protocol demonstrates the orthotopic injection.

Source: Zhang, G. L., et al. Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad.&#160;J. Vis. Exp. (2019).
In this article we ...

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

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Chapters in this video

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

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

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

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

Orthotopic Injection into the Mammary Fat Pad: Establishing Breast Cancer in Mice