The authors would like to thank Michael Goldberg for preparing stabilized siRNA solution and Heather Tobin for her assistance with the animals. This study was funded by DOD Innovator Award W81XWH-08-1-0659 to DEI. The postdoctoral training of SK was supported by the Susan G Komen foundation (KG101329). The authors would like to thank Kristin Johnson for preparation of the schematic.

1. Preparation of Injection Solution
<ol>
	<li>Prepare 0.2% Evans blue dye in phosphate buffered saline (PBS) or other aqueous solution of interest. This vital dye is useful for assessing the success of intraductal injections and is recommended as a visualization aid when developing expertise in the technique.</li>
</ol>
The volume required will depend on the number and location of the glands to be injected. All 10 mammary glands of female mice can be injected but due to the smaller gland size, gland pairs 1 and 5 are typically injected with 10 &mu;l of solution. All other mammary pairs are injected with 20 &mu;l. These volumes are sufficient to fill the entire ductal tree.
2. Preoperative Preparation
<ol>
	<li>Record the body weight of each mouse. As with all preclinical studies, animal weights should be monitored regularly (twice a week or greater) to assess potential toxicity.</li>
	<li>Anesthetize the mouse using an isoflurane chamber and apply eye lubricant. During the procedure mice will continue to be anesthetized using inhaled 2-4% concentration of isoflurane in oxygen via a nose cone. Carefully monitor the mouse for changes in respiratory rate, adjusting the level of isoflurane accordingly.</li>
	<li>Inject meloxicam (5-10 mg/kg) subcutaneously prior to the procedure as analgesia.</li>
	<li>Apply an over-the-counter hair removal cream to the nipple area. Wait 5 min and gently remove loose hair with a cotton-tip applicator using a circular motion. Remove the cream using damp paper towels wetted with warm water. Shaving is not recommended due to the risk of damaging the nipples.</li>
	<li>Secure the mouse under the stereoscope by gently taping down the extremities.</li>
	<li>Clean injection sites with alcohol swabs.</li>
</ol>
3. Intraductal injection
<ol>
	<li>Locate appropriate nipples to be injected under the stereoscope. Use fine micro-dissecting tweezers to remove any dead skin that covers the nipple opening.</li>
	<li>Load 10-20 &mu;l of injection solution into a 50 &mu;l syringe with a 33 G metal hub needle affixed. Sometimes, after injection, small amounts (1 &mu;l or less) can leak out of the nipple upon pulling out the needle. Therefore, it is recommended to inject 11 or 21 &mu;l respectively to account for the potential loss.</li>
	<li>Hold the nipple gently with the fine tweezers and lift it slightly to position it for injection. It is not necessary to cut the nipple.</li>
	<li>Inject the solution slowly to minimize potential damage caused by rapidly moving fluid within the ductal lumens. The injection rate should be maintained at approximately 40 &mu;l/min.</li>
</ol>
4. Postoperative Care
<ol>
	<li>Observe the injection site. There should be no signs of trauma to the nipple region or surrounding tissue. Swelling in the area surrounding the nipple likely indicates a mammary fat pad injection rather than a successful intraductal injection.</li>
	<li>Remove the animal from the nose cone and move to a separate cage for recovery. Place the cage under a heat lamp to prevent hypothermia and assist in recovery. Mice are housed singly and will be monitored closely until they regain consciousness and mobility.</li>
</ol>
5. Analysis of Mammary Gland Tissue
<ol>
	<li>Upon completion of the study, mice are euthanized by cervical dislocation following CO2 compressed gas in an isolation chamber. Mammary glands are excised and may be used for whole mount preparation 11, histology, or RNA and protein isolation.</li>
</ol>

<ol>
	<li>Dooley, W. C., 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=Ductal+lavage+for+detection+of+cellular+atypia+in+women+at+high+risk+for+breast+cancer.">Ductal lavage for detection of cellular atypia in women at high risk for breast cancer.</a> J Natl Cancer Inst. 93, 1624-1632 (2001).</li><li>Jacobs, L., Sukumar, S., Stearns, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraductal+therapy+for+the+prevention+of+breast+cancer.">Intraductal therapy for the prevention of breast cancer.</a> Curr Opin Investig Drugs. 11, 646-652 (2011).</li><li>King, B. L., Love, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraductal+approach+to+the+breast:+raison+d'etre.">The intraductal approach to the breast: raison d'etre.</a> Breast Cancer Res. 8, 206 (2006).</li><li>Murata, S., 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=Ductal+access+for+prevention+and+therapy+of+mammary+tumors.">Ductal access for prevention and therapy of mammary tumors.</a> Cancer Res. 66, 638-645 (2006).</li><li>Stearns, V., 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=Preclinical+and+clinical+evaluation+of+intraductally+administered+agents+in+early+breast+cancer.">Preclinical and clinical evaluation of intraductally administered agents in early breast cancer.</a> Sci Transl Med. 3, 106ra108 (2011).</li><li>Flanagan, M., Love, S., Hwang, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+of+Intraductal+Therapy+for+Ductal+Carcinoma+in+Situ.">Status of Intraductal Therapy for Ductal Carcinoma in Situ.</a> Curr Breast Cancer Rep. 2, 75-82 (2010).</li><li>Nguyen, D. -. A., Beeman, N., Lewis, M., Schaack, J., Neville, M. C., Ip, M. M., Asch, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in mammary gland biology and breast cancer research. , 259-270 (2000).</li><li>Behbod, F., 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=An+intraductal+human-in-mouse+transplantation+model+mimics+the+subtypes+of+ductal+carcinoma+in+situ.">An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.</a> Breast Cancer Res. 11, R66 (2009).</li><li>Bu, W., Xin, L., Toneff, M., Li, L., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+vectors+for+stably+introducing+genes+into+mammary+epithelial+cells+in+vivo.">Lentivirus vectors for stably introducing genes into mammary epithelial cells in vivo.</a> J Mammary Gland Biol Neoplasia. 14, 401-404 (2009).</li><li>Reddy, J. P., Li, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RCAS-TVA+system+for+introduction+of+oncogenes+into+selected+somatic+mammary+epithelial+cells+in+vivo.">The RCAS-TVA system for introduction of oncogenes into selected somatic mammary epithelial cells in vivo.</a> J Mammary Gland Biol Neoplasia. 14, 405-409 (2009).</li><li>Plante, I., Stewart, M. K., Laird, 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=Evaluation+of+mammary+gland+development+and+function+in+mouse+models.">Evaluation of mammary gland development and function in mouse models.</a> J. Vis. Exp. , e2828 (2011).</li></ol>

The authors have no financial disclosures.

Transgenic and knockout mice are invaluable tools for studying the in vivo role of individual genes in breast cancer. However, it is costly and time-consuming to generate these animals and the gene knockdown is usually ubiquitous which sometimes precludes us from recognizing the specific effects of that gene on the mammary gland. Therefore, a targeted knockdown would alleviate issues of non-specific side-effects and toxicities in other organs.
The intraductal injection of siRNA presented here enables the localized gene knockdown in the mouse mammary gland. It is a rapid method for a targeted and localized drug delivery to the mammary gland not limited to siRNA delivery. Such a mammary gland-specific drug delivery avoids unspecific side-effects and toxicity to other organs and allows for the study of gene alterations at specific time points during mammary gland development and breast tumor formation. There is no evidence of siRNA particles in non-injected glands, the liver or other organs providing further evidence that this method presents a preferred way for localized gene knockdown in the mammary gland. Furthermore, the intraductal injections can be repeated weekly or biweekly over several months to allow for long-term monitoring of therapeutic agents.
This technique opens up the possibility for assaying the intraductal delivery of various reagents to the mouse mammary ducts. Advances in localized delivery in well-characterized mouse models should accelerate the application of non-invasive, targeted therapeutic strategies in humans.

The mouse mammary gland is composed of a complex ductal-alveolar tree that converges into a central duct at the nipple. In mice as well as humans, the majority of breast tumors originate in the epithelial cells lining the milk ducts. Current available treatments include intravenous chemotherapy, radiation, and surgery. Localized intraductal treatments have been explored 1-3 and offer significant advantages in terms of reduced toxicity and side effects. However, several challenges remain before these can be applied more widely. The ability to perform comparable procedures in the mouse mammary gland facilitates the characterization of intraductal interventions. Here we present a non-invasive method for the delivery of reagents directly into the mouse mammary duct.
Intraductal delivery of chemotherapeutic agents in both breast cancer patients and mouse models has previously been shown to be highly effective with no evidence of systemic toxicity or long-term histopathological changes 3-6. Furthermore, tumor cell injection and lentiviral vector delivery of oncogenes have been shown previously to be feasible via intraductal injection 7-10.
In addition to drug delivery, this procedure is useful for local silencing of mammary gland genes through intraductal injection of siRNA. Injection of small volumes of siRNA solution is effective without altering the anatomy and physiology of the mammary gland. In our laboratory we have recently shown successful silencing of key mediator genes in transgenic mice that spontaneously develop mammary tumors resulting in prevention of tumor progression (Brock et al., unpublished). We have been able to repeat biweekly injections into the same nipples up to 7 times in 2-week intervals with no visible tissue damage or toxicity.
Intraductal injections can be timed in an adult mouse to address developmentally-specific events relevant to mammary gland development during pregnancy, lactation, and involution or to target later stages during tumor development and progression. Virgin females as young as 8 weeks of age have been injected non-invasively in our laboratory.

<table><tbody><tr><td>isoflurane</td><td></td><td></td><td>2-4%</td></tr><tr><td>meloxicam</td><td></td><td></td><td>5-10 mg/ml</td></tr><tr><td>Evans blue dye</td><td>Sigma-Aldrich</td><td>E2129-10G</td><td>0.2% in PBS</td></tr><tr><td>hair removing cream</td><td>Veet</td><td></td><td>Choose sensitive option</td></tr><tr><td>stereoscope</td><td>Zeiss</td><td>Stemi DV4</td><td></td></tr><tr><td>micro-dissecting tweezers</td><td>Roboz</td><td>rs-4976</td><td>With a 0.10 x 0.06 mm tip made of dumostar</td></tr><tr><td>Hamilton syringe</td><td>Hamilton</td><td>7637-01</td><td></td></tr><tr><td>33-gauge needle</td><td>Hamilton</td><td>7803-05</td><td>point style 4, 12 degree bevel (standard)</td></tr><tr><td>siGLO cyclophilin B control siRNA</td><td>Dharmacon</td><td>D-001610-01-05</td><td></td></tr></tbody></table>

intraductal injection for localized drug delivery to the mouse mammary gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

The nipples can be easily localized using a stereomicroscope once hair has been removed in the area surrounding it (Figure 1A). To master the injection technique, it is recommended to inject Evans blue dye and monitor the integrity of the gland (Figures 1B and C). This approach also allows for the determination of appropriate volumes to be injected into each gland as one can visually assess whether the dye reaches the entire ductal system (Figures 1C and 2A). An image of the injected Evans blue image can then be compared to the whole-mounted mammary gland, in which the entire ductal tree is stained with Carmine alum dye (Figure 2B).
Please note that robustness of this injection method is highly dependent on the operator. For example, perforating the duct will result in a mammary fat pad injection and injecting the solution too fast may damage the ductal epithelial cells and provoke an inflammatory response. Successful intraductal injection enables the localized drug delivery to the mammary gland and reduces non-specific side-effects often observed otherwise.
An example image of a gland injected with fluorescently tagged siRNA targeting cyclophilin (a non-essential gene) is shown in Figure 3.
<img alt="Figure 1" src="/files/ftp_upload/50692/50692fig1.jpg" /> 
Figure 1. An inguinal mammary gland injected through the nipple with Evans blue dye. A) Appearance of the nipple before injection and B) after injection with 20 &mu;l Evans blue dye. No swelling or tissue damage is observed. C) Upon opening the skin, the dye permits visualization of the entire mammary ductal tree.
<img alt="Figure 2" src="/files/ftp_upload/50692/50692fig2.jpg" /> 
Figure 2. Whole-mount analysis of Evans blue-injected gland. A) Representative image of a dissected gland excised from the animal and spread onto a glass slide immediately after injection. B) Whole mount staining of the same gland with carmine dye after fat had been cleared. Comparison with A) confirms that the injected solution fills the mammary ductal tree and the gland is anatomically intact.
<img alt="Figure 3" src="/files/ftp_upload/50692/50692fig3.jpg" /> 
Figure 3. Intraductal delivery of fluorescent siRNA. Representative image of fresh tissue sample, excised 48 hr postinjection.

Watch this Scientific Journal Video about Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland at JoVE.com

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

intraductal-injection-for-localized-drug-delivery-to-mouse-mammary

Research

JoVE Journal

Biology

34.9K Views.  Boston Children's Hospital and Harvard Medical School.  Een protocol voor de niet-invasieve intraductale afgifte van waterige reagentia voor de muis borstklier wordt beschreven. De methode maakt gebruik van gelokaliseerde injectie in de tepels van melkklieren targeting mamma-kanalen specifiek. Deze techniek kan worden aangepast voor verschillende verbindingen waaronder siRNA, chemotherapeutische middelen en kleine moleculen. 

Video: Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, as visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse1.
Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-&kappa;B luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-&kappa;B signaling within the LPS-injected gland4.
These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse.

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

Medicine

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 ...

Intraductal Delivery and X-ray Visualization of Ethanol-Based Ablative Solution for Prevention and Local Treatment of Breast Cancer in Mouse Models

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic mastectomy is the most effective primary prevention strategy. Prophylactic mastectomy is an aggressive surgical procedure that completely removes the mammary epithelial cells from which breast cancer arises along with the surrounding tissue. We seek to develop a minimally invasive intraductal procedure as an alternative to prophylactic mastectomy to locally ablate the mammary epithelial cells before they can become malignant. We and others have developed an intraductal delivery procedure to reach and treat these epithelial cells in rodent models of breast cancer. While the mouse mammary gland with a single non-anastomosed ductal tree opening at the nipple has a much less complex and tortuous architecture than the human breast, chemically induced and genetically engineered mouse models of breast cancer are valuable to produce proof-of-concept studies of new preventative strategies. Here, we describe a procedure for intraductal delivery of an ethanol-based ablative solution containing micro-CT/X-ray tantalum-based contrast agent within the mouse mammary ductal tree for the therapeutic purpose of primary prevention of breast cancer. Intraductal delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) has been previously described in mouse models. Thus, we focus our protocol description on methodological modifications and unique experimental considerations for optimizing delivery of ethanol, for minimizing local and systemic side effects of ethanol administration, and for in vivo visualization of ductal tree filling via micro-CT/fluoroscopy imaging. Visualization of the ductal tree immediately after injection of a contrast-containing solution allows for confirmation of complete filling or unsuccessful outcomes such as underfilling or overfilling. This procedure can be applied for delivery and imaging of other ablative compounds aimed at either preventing tumor formation or locally treating early-stage tumors accessible via the ductal tree.

A method of intraductal injection of reagents for an ethanol-based ablative solution to the mouse mammary ductal tree for in vivo imaging and breast cancer prevention is described. Injection directly into the nipple opening allows for targeting mammary epithelial cells with minimal collateral tissue damage.

Breast cancer is the most prevalent cancer and the second-leading cause of cancer-related death for women in the USA. For high-risk women, prophylactic ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

X-Ray Visualization of Intraductal Ethanol-Based Ablative Treatment for Prevention of Breast Cancer in Rat Models

There are still a limited number of primary interventions for prevention of breast cancer. For women at a high risk of developing breast cancer, the most effective intervention is prophylactic mastectomy. This is a drastic surgical procedure in which the mammary epithelial cells that can give rise to breast cancer are completely removed along with the surrounding tissue. The goal of this protocol is to demonstrate the feasibility of a minimally invasive intraductal procedure that could become a new primary intervention for breast cancer prevention. This local procedure would preferentially ablate mammary epithelial cells before they can become malignant. Intraductal methods to deliver solutions directly to these epithelial cells in rodent models of breast cancer have been developed at Michigan State University and elsewhere. The rat mammary gland consists of a single ductal tree that has a simpler and more linear architecture compared to the human breast. However, chemically induced rat models of breast cancer offer valuable tools for proof-of-concept studies of new preventive interventions and scalability from mouse models to humans. Here, a procedure for intraductal delivery of an ethanol-based ablative solution containing tantalum oxide nanoparticles as X-ray contrast agent and ethyl cellulose as gelling agent into the rat mammary ductal tree is described. Delivery of aqueous reagents (e.g., cytotoxic compounds, siRNAs, AdCre) by intraductal injection has been described previously in mouse and rat models. This protocol description emphasizes methodological changes and steps that pertain uniquely to delivering an ablative solution, formulation consideration to minimize local and systemic side effects of the ablative solution, and X-ray imaging for in vivo assessment of ductal tree filling. Fluoroscopy and micro-CT techniques enable to determine the success of ablative solution delivery and the extent of ductal tree filling thanks to compatibility with the tantalum-containing contrast agent.

A procedure for the delivery of a chemical ablative solution to the rat mammary ductal tree for image-guided preventive treatment of breast cancer is described. Mammary epithelial cells can be targeted with minimal collateral tissue damage through cannulation directly into the nipple opening and intraductal infusion of a 70% ethanol-based ablative solution.

There are still a limited number of primary interventions for prevention of breast cancer. For women at a high risk of developing breast cancer, the most ...

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play a major role in mammary gland function and are the origin of most mammary tumors. Introducing genes of interest into mouse mammary epithelial cells is a critical step in evaluating gene function in epithelial cells and generating mouse mammary tumor models. This goal can be accomplished through the intraductal injection of a viral vector carrying the genes of interest into the mouse mammary ductal tree. The injected virus subsequently infects mammary epithelial cells, bringing in the genes of interest. The viral vector can be lentiviral, retroviral, adenoviral, or adenovirus-associated viral (AAV). This study demonstrates how a gene of interest is delivered into mammary epithelial cells through mouse mammary intraductal injection of a viral vector. A lentivirus carrying GFP is used to show stable expression of a delivered gene, and a retrovirus carrying Erbb2 (HER2/Neu) is used to demonstrate oncogene-induced atypical hyperplastic lesions and mammary tumors.

In Vivo Gene Delivery into Mouse Mammary Epithelial Cells Through Mammary Intraductal Injection

The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

Mouse mammary glands comprise ductal trees, which are lined by epithelial cells and have one opening at the tip of each nipple. The epithelial cells play ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Bioengineering

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 ...

JoVE Encyclopedia of Experiments

Breast Cancer

Procedures and techniques

Intraductal Injection: Delivering Injection Mix into the Ducts of the Mouse Mammary Gland

Source: Xiang, D., et al. <a href="https://www.jove.com/v/59502/modeling-breast-cancer-via-an-intraductal-injection-cre-expressing">Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland.</a> J. Vis. Exp (2019).
This video describes an intraductal injection technique where we deliver injection mix into the ducts of mouse mammary glands.

Source: Xiang, D., et al. Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland. J. Vis. Exp ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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