We thank Dr. Gary Chamness for his helpful comments on this manuscript. This work was supported by the Department of Defense (DOD) CDMRP BC191649 (YL) and BC191646 (YL) as well as the National Institutes of Health (NIH) CA271498 (YL). The authors would like to thank the Breast Center Pathology Core Facility supported by SPORE P50CA186784, and the Cytometry and Cell Sorting Core supported by CPRIT-RP180672, NIH CA125123, and RR024574 with the assistance of Joel M. Sederstrom.

All procedures using mice were performed in compliance with the Institutional Animal Care and Use Committee-approved animal protocol. For the present study, 9-12-week-old FVB/N or MMTV-tva female mice were used. The mice were obtained commercially or self-made (see Table of Materials). The Lenti-EGFP (FUCGW) and RCAS-Erbb2 (Neu) viruses were used. Virus preparation and titer determination were performed following the previously published reports10,12.
1. Syringe preparation
<ol>
	<li>Cut the 33 G metal hub needles (see Table of Materials) to approximately 1 cm in length. Store the needles and the 50 &#181;L syringe in 70% alcohol after autoclaving.</li>
	<li>Take out syringes and needles from the alcohol. Propel the remaining alcohol out of the syringe and needle. Disassemble the syringe for air drying on an autoclaved absorbent bench pad.</li>
	<li>Assemble the syringe after drying.</li>
</ol>
2. Virus preparation
<ol>
	<li>Take out one or several viral stock tubes as needed from the -80 &#730;C freezer and thaw the virus on ice. 
	NOTE: Depending on the number of cells to be infected, the virus may need to be diluted into intended titers using 1x PBS at this time.</li>
	<li>Add bromophenol blue by dipping the pipette tip to a depth of 1 cm into the bromophenol blue powder (see Table of Materials). Then, bring the attached trace amount of bromophenol blue into the virus suspension. 
	NOTE: For the present study, the viral stock is usually 200 &#181;L per tube, so the final dye concentration is approximately 0.2% (0.2 &#181;g/100 &#181;L). Bromophenol blue should be sterilized with microwave radiation for 45 seconds at high power of 1250 W&#160;(see&#160;Table of Materials). Ensure to carry whole procedure in a sterile environment.</li>
	<li>Mix the bromophenol blue with the virus solution by pipetting up and down 10 times.</li>
	<li>Place the virus on ice and bring the ice bucket to the vivarium.</li>
</ol>
3. Animal preparation
<ol>
	<li>Anesthetize a female mouse by intraperitoneal injection of 2 &#181;L/g of anesthetic (37.6 mg/mL ketamine, 1.92 mg/mL xylazine, and 0.38 mg/mL acepromazine, see Table of Materials). Check the depth of anesthesia with a toe pinch. Apply ointment on the eyes to prevent dryness while under anesthesia. 
	NOTE: The mouse must be non-responsive to toe&#160;pinch under good anesthesia. Isoflurane may also be used to anesthetize the mice.</li>
	<li>Put the mouse in a supine position on a warm pad and attach all four limbs to the bench using adhesive tape.</li>
	<li>Identify one nipple (or more) to be injected (number 4 is preferred for the present study). Expose it by trimming the surrounding hair using a pair of scissors.</li>
	<li>Apply&#160;70% alcohol and iodophor swabs to the nipple area in three rounds to&#160;clean and expose the nipple.</li>
</ol>
4. Intraductal injection of virus
NOTE: A magnifying lamp may be used to help visualize the nipple opening.
<ol>
	<li>Transect the distal tip of a nipple using a pair of sterile&#160;microdissection spring scissors, until a small central ductal opening can be seen under a magnifier lamp (see Table of Materials).</li>
	<li>Load 10 &#181;L of virus/bromophenol blue mixture into the syringe. 
	NOTE: Lenti-EGFP (FUCGW) and RCAS-Erbb2 (Neu) are used in this demonstration.</li>
	<li>Carefully insert the needle into the nipple opening with the help of the magnifier lamp. The orientation of the needle is adjusted slightly from medial to lateral to align with the main duct.</li>
	<li>Inject the entire 10 &#181;L of the virus into the duct tree. 
	NOTE: The ducts under the skin should turn blue, if the injection is successful. Any resistance or the appearance of a localized color change indicates the failure of the injection.</li>
	<li>Put the mouse on a slide warmer set at 45 &#176;C until the mouse fully wakes up(~30-60 min).</li>
</ol>
5. Detection of infected cells by a fluorescent stereomicroscope
<ol>
	<li>Three to five days after injection of the virus carrying GFP or other fluorescent genes, euthanize the mouse by over-dosing it with 4 uL/g of anesthetic (37.6 mg/mL ketamine, 1.92 mg/mL xylazine, and 0.38 mg/mL acepromazine).</li>
	<li>Open the thoracic cavity. Cut the skin along the ventral middle line and along the upper and lower limbs using a pair of scissors. Lift the skin and expose the mammary glands.</li>
	<li>Remove the injected mammary gland from the skin using a pair of forceps and scissors. Also, remove an un-injected gland as a control.</li>
	<li>Place the mammary glands on glass slides and spread the glands to their original shape.</li>
	<li>Observe and image the glands under a fluorescent stereomicroscope.</li>
</ol>

<ol>
	<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. Eds Margot. , 259-270 (2000).</li><li>Gordon, J. W., Ruddle, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+and+stable+germ+line+transmission+of+genes+injected+into+mouse+pronuclei.">Integration and stable germ line transmission of genes injected into mouse pronuclei.</a> Science. 214 (4526), 1244-1246 (1981).</li><li>Costantini, F., Lacy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+a+rabbit+beta-globin+gene+into+the+mouse+germ+line.">Introduction of a rabbit beta-globin gene into the mouse germ line.</a> Nature. 294 (5836), 92-94 (1981).</li><li>Brinster, R. 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=Somatic+expression+of+herpes+thymidine+kinase+in+mice+following+injection+of+a+fusion+gene+into+eggs.">Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs.</a> Cell. 27 (1), 223-231 (1981).</li><li>Du, Z., 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=RCAS-TVA+in+the+mammary+gland:+an+in+vivo+oncogene+screen+and+a+high+fidelity+model+for+breast+transformation.">RCAS-TVA in the mammary gland: an in vivo oncogene screen and a high fidelity model for breast transformation.</a> Cell Cycle. 6 (7), 823-826 (2007).</li><li>Bu, 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=Mammary+precancerous+stem+and+non-stem+cells+evolve+into+cancers+of+distinct+subtypes.">Mammary precancerous stem and non-stem cells evolve into cancers of distinct subtypes.</a> Cancer Research. 79 (1), 61-71 (2019).</li><li>Bu, 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=Keratin+6a+marks+mammary+bipotential+progenitor+cells+that+can+give+rise+to+a+unique+tumor+model+resembling+human+normal-like+breast+cancer.">Keratin 6a marks mammary bipotential progenitor cells that can give rise to a unique tumor model resembling human normal-like breast cancer.</a> Oncogene. 30 (43), 4399-4409 (2011).</li><li>Haricharan, 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=Contribution+of+an+alveolar+cell+of+origin+to+the+high-grade+malignant+phenotype+of+pregnancy-associated+breast+cancer.">Contribution of an alveolar cell of origin to the high-grade malignant phenotype of pregnancy-associated breast cancer.</a> Oncogene. 33 (50), 5729-5739 (2014).</li><li>Bu, W., Zhang, X., Dai, H., Huang, S., 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=Mammary+cells+with+active+Wnt+signaling+resist+ErbB2-induced+tumorigenesis.">Mammary cells with active Wnt signaling resist ErbB2-induced tumorigenesis.</a> PLoS One. 8 (11), 78720 (2013).</li><li>Du, Z., 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=Introduction+of+oncogenes+into+mammary+glands+in+vivo+with+an+avian+retroviral+vector+initiates+and+promotes+carcinogenesis+in+mouse+models.">Introduction of oncogenes into mammary glands in vivo with an avian retroviral vector initiates and promotes carcinogenesis in mouse models.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (46), 17396-17401 (2006).</li><li>Siwko, S. K., 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=Lentivirus-mediated+oncogene+introduction+into+mammary+cells+in+vivo+induces+tumors.">Lentivirus-mediated oncogene introduction into mammary cells in vivo induces tumors.</a> Neoplasia. 10 (7), 653-662 (2008).</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> Journal of Mammary Gland Biology and Neoplasia. 14 (4), 401-404 (2009).</li><li>Russell, T. D., 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=Transduction+of+the+mammary+epithelium+with+adenovirus+vectors+in+vivo.">Transduction of the mammary epithelium with adenovirus vectors in vivo.</a> Journal of Virology. 77 (10), 5801-5809 (2003).</li><li>Wagner, S., Thresher, R., Bland, R., Laible, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated-virus-mediated+transduction+of+the+mammary+gland+enables+sustained+production+of+recombinant+proteins+in+milk.">Adeno-associated-virus-mediated transduction of the mammary gland enables sustained production of recombinant proteins in milk.</a> Scientific Reports. 5, 15115 (2015).</li><li>Naldini, 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=In+vivo+gene+delivery+and+stable+transduction+of+nondividing+cells+by+a+lentiviral+vector.">In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.</a> Science. 272 (5259), 263-267 (1996).</li><li>Coffin, J. M., Hughes, S. H., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Retroviruses. , (1997).</li><li>Mitani, K., Kubo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+as+an+integrating+vector.">Adenovirus as an integrating vector.</a> Current Gene Therapy. 2 (2), 135-144 (2002).</li><li>McCarty, D. M., Young, S. M., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+adeno-associated+virus+(AAV)+and+recombinant+AAV+vectors.">Integration of adeno-associated virus (AAV) and recombinant AAV vectors.</a> Annual Review of Genetics. 38, 819-845 (2004).</li><li>Bu, W., 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=Intraductal+injection+of+lentivirus+vectors+for+stably+introducing+genes+into+rat+mammary+epithelial+cells+in+vivo.">Intraductal injection of lentivirus vectors for stably introducing genes into rat mammary epithelial cells in vivo.</a> Journal of Mammary Gland Biology and Neoplasia. 25 (4), 389-396 (2020).</li><li>Dong, J., 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=Genetic+manipulation+of+individual+somatic+mammary+cells+in+vivo+reveals+a+master+role+of+STAT5a+in+inducing+alveolar+fate+commitment+and+lactogenesis+even+in+the+absence+of+ovarian+hormones.">Genetic manipulation of individual somatic mammary cells in vivo reveals a master role of STAT5a in inducing alveolar fate commitment and lactogenesis even in the absence of ovarian hormones.</a> Developmental Biology. 346 (2), 196-203 (2010).</li><li>Haricharan, 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=Mechanism+and+preclinical+prevention+of+increased+breast+cancer+risk+caused+by+pregnancy.">Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy.</a> eLife. 2, 00996 (2013).</li><li>Reddy, J. P., 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=Defining+the+ATM-mediated+barrier+to+tumorigenesis+in+somatic+mammary+cells+following+ErbB2+activation.">Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (8), 3728-3733 (2010).</li><li>Dong, J., 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=The+PR+status+of+the+originating+cell+of+ER/PR-negative+mouse+mammary+tumors.">The PR status of the originating cell of ER/PR-negative mouse mammary tumors.</a> Oncogene. 35 (31), 4149-4154 (2016).</li><li>Young, A., 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=Targeting+the+pro-survival+protein+BCL-2+to+prevent+breast+cancer.">Targeting the pro-survival protein BCL-2 to prevent breast cancer.</a> Cancer Prevention Research. 15 (1), 3-10 (2022).</li><li>Schlimgen, 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=Risks+associated+with+lentiviral+vector+exposures+and+prevention+strategies.">Risks associated with lentiviral vector exposures and prevention strategies.</a> Journal of Occupational and Environmental Medicine. 58 (12), 1159-1166 (2016).</li><li>Holloway, K. 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=Krt6a-positive+mammary+epithelial+progenitors+are+not+at+increased+vulnerability+to+tumorigenesis+initiated+by+ErbB2.">Krt6a-positive mammary epithelial progenitors are not at increased vulnerability to tumorigenesis initiated by ErbB2.</a> PLoS One. 10 (1), 0117239 (2015).</li><li>Holloway, K. 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=Targeting+oncogenes+into+a+defined+subset+of+mammary+cells+demonstrates+that+the+initiating+oncogenic+mutation+defines+the+resulting+tumor+phenotype.">Targeting oncogenes into a defined subset of mammary cells demonstrates that the initiating oncogenic mutation defines the resulting tumor phenotype.</a> International Journal of Biological Sciences. 12 (4), 381-388 (2016).</li><li>Hein, 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=Luminal+epithelial+cells+within+the+mammary+gland+can+produce+basal+cells+upon+oncogenic+stress.">Luminal epithelial cells within the mammary gland can produce basal cells upon oncogenic stress.</a> Oncogene. 35 (11), 1461-1467 (2015).</li><li>Annunziato, 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=In+situ+CRISPR-Cas9+base+editing+for+the+development+of+genetically+engineered+mouse+models+of+breast+cancer.">In situ CRISPR-Cas9 base editing for the development of genetically engineered mouse models of breast cancer.</a> EMBO Journal. 39 (5), 102169 (2020).</li><li>Annunziato, 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=Modeling+invasive+lobular+breast+carcinoma+by+CRISPR/Cas9-mediated+somatic+genome+editing+of+the+mammary+gland.">Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.</a> Genes &amp; Development. 30 (12), 1470-1480 (2016).</li></ol>

Both the authors declare no conflicts of interest.

This article demonstrates the viral intraductal injection technique for introducing genes into mouse mammary epithelial cells for modeling sporadic breast cancer. Usually, mice of at least 5 weeks or older are injected so that the oncogenic process starts after the mammary gland is developed. Besides, the nipple opening of mice younger than 5 weeks old is often too small for injection. On the other hand, the nipples of very old mice are sometimes degenerated, and transection may fail to reveal a ductal opening. It is als...

Epithelial cells of mammary glands play a major role in the function of these glands and are the major cell of origin of breast cancer. Studies of mammary gland biology and tumorigenesis frequently need the delivery of gene(s) of interest into these cells. Each mouse mammary gland comprises a ductal tree lined by epithelial cells with a single opening at the tip of the nipple. This structure makes the mammary epithelial cells easily accessible to viral vectors, which can be delivered into the lumen of a ductal tree via intraductal injection1.
The technique of mammary intraductal injection was originally used for much larger animals such as goats, rabbits, and rats1. For a much smaller animal such as mice, intraductal injection needs many delicate tools and more practices of the operators. There are two approaches for mouse intraductal injection. One is up-the-teat injection1. Another one is the direct injection of the primary duct of #3 or #4 mammary gland after surgical exposure1. Since the first one is non-invasive and faster once the operator has been well-trained, this technique is more commonly used and will be described in detail in this article.
Compared to the widely used traditional transgenic mouse models, in which the gene of interest is introduced at the stage of fertilized eggs through microinjection2,3,4, gene delivery through the intraductal virus injection method has many advantages, including: (1) it avoids the time-consuming process of making a transgenic mouse line for each gene of interest; (2) it avoids potential impairment on the normal development of mammary glands imposed by the gene of interest; (3) it introduces the gene of interest at any desired time after birth; (4) it can easily co-introduce more than one gene of interest; (5) it better mimics the natural tumorigenic process because the infected and thus oncogene-carrying cells are surrounded by normal cells; and (6) in combination with the TVA (tumor virus A, an avian cell surface protein and the receptor for retrovirus RCAS vector) technology5, the gene of interest can be introduced into a specific cell population to study the cell origin of tumorigenesis and to conduct cell lineage-tracing assays in the mammary glands6,7,8,9.
Any vectors derived from retrovirus10, lentivirus11,12, adenovirus13, and adenovirus-associated virus (AAV)14 may be used for intraductal delivery of genetic materials. Retrovirus and lentivirus vectors integrate into the host genome permanently; thus, they introduce genes of interest stably into mammary epithelial cells. While lentivirus can integrate into the genome of any cell it encounters15, the efficient genomic integration of retrovirus needs the proliferation of the target cells16. The adenoviral and AAV vectors do not integrate into the genome of infected cells and, therefore, only transiently express the gene of interest17,18. This feature can be an advantage when the gene of interest needs to be expressed for only a short amount of time, such as Cre, for deleting a floxed tumor suppressor gene.
Lentivirus, adenovirus, and AAV infect any mouse cells they encounter. But since luminal epithelium is largely insulated from the underlying basal layer, which is further separated from the stroma by the basement membrane, intraductal injection limits the infection largely to luminal epithelial cells, the primary cell of origin of breast cancer. Within this luminal epithelial layer, there are also distinct cell subtypes, including stem cells, progenitor cells, and several groups of differentiated cells. To infect specific cell subsets within the luminal cell population, the TVA technology may be used, with which avian leukosis virus-derived RCAS vectors5,10 or pseudotyped lentiviral vectors11 selectively infect the cells that express TVA in mice that carry a tva transgene under the control of a cell type-specific promoter, such as a promoter that is active only in stem cells6 or certain progenitors6,7 or alveolar cells8 or Wnt-pathway active cells9.
This protocol presents the technique of introducing genes of interest into mammary epithelial cells through intraductal injection of a viral vector. The detection of expression of the introduced genes and the resulting hyperplastic lesions and tumors are then demonstrated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-HA antibody</td>
			<td>Covance</td>
			<td>MMS-101P</td>
			<td>Dilution: 1 : 1000</td>
		</tr>
		<tr>
			<td>Artificial Tears</td>
			<td>Covetrus</td>
			<td>NDC 11695-0832-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma</td>
			<td>B5525</td>
			<td>microwave radiation for 45 seconds at power high of 1250W microwave oven</td>
		</tr>
		<tr>
			<td>FACSCantoII</td>
			<td>BD Biosciences</td>
			<td>V96100899</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16 FA</td>
			<td></td>
		</tr>
		<tr>
			<td>FUCGW lenti-virus</td>
			<td>Self-made</td>
			<td>N/A</td>
			<td>See reference # 12</td>
		</tr>
		<tr>
			<td>FVB/N</td>
			<td>The Jackson Laboratory</td>
			<td>JAX:001800</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton needle</td>
			<td>Hamilton</td>
			<td>91033</td>
			<td>autoclaved</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>201000</td>
			<td>autoclaved</td>
		</tr>
		<tr>
			<td>LED magnifying lamp</td>
			<td>Intertek</td>
			<td>3165273</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissection spring scissor</td>
			<td>Roboz</td>
			<td>RS-5621</td>
			<td>autoclaved</td>
		</tr>
		<tr>
			<td>MMTV-tva</td>
			<td>Self-made</td>
			<td></td>
			<td>See reference # 10</td>
		</tr>
		<tr>
			<td>RCAS-Neu (HA)</td>
			<td>Self-made</td>
			<td>N/A</td>
			<td>See reference # 10</td>
		</tr>
		<tr>
			<td>Rodent Comboanesthetic III</td>
			<td>Veterinary Pharmacy</td>
			<td>Veterinary prescription</td>
			<td>37.6 mg/mL ketamine, 1.92 mg/mL xylazine, and 0.38 mg/mL acepromazine</td>
		</tr>
	</tbody>
</table>

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.

Representative data are presented here to demonstrate successful intraductal injection, successful viral infection, and the impact of the delivered genes on mammary tumorigenesis. The amount of virus injected must be tailored to the purpose of each experiment. To illustrate how extensively the mammary duct tree can be infected, a large amount of virus carrying genes that can be imaged, such as GFP, needs to be used. On the other hand, to mimic the natural spontaneous tumorigenesis, a small amount of virus carrying an onc...

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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.

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 ...

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Research

JoVE Journal

Cancer Research

1.2K Views.  Baylor College of Medicine. The present protocol describes intraductal injection of viral vectors via the teat to deliver genes of interest into the mammary epithelial cells.

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

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study end-points. Thus, studies focused on elucidating mechanisms of late-stage de novo metastasis are compromised, as are studies examining efficacy of anti-cancer therapies targeting mediators of metastasis in the adjuvant setting. Numerous murine mammary cancer models have been developed via targeted expression of dominant oncoproteins to mammary epithelial cells yielding models variably mimicking histopathologic and transcriptome-defined breast cancer subtypes common in women1. While much has been learned regarding the biology of mammary carcinogenesis with these models, their utility in identifying molecules regulating growth of late-stage metastasis are compromised as mice are typically euthanized at earlier time points due to significant primary tumor burden. Moreover, since a significant percentage of women diagnosed with breast cancer receive adjuvant therapy after surgical resection of primary tumors and prior to presence of detectable metastatic disease, preclinical models of de novo metastasis are urgently needed as platforms to evaluate new therapies aimed at targeting metastatic foci. To address these deficiencies, we developed a murine model of de novo mammary cancer metastasis, wherein primary mammary tumors are surgically resected, and metastatic foci subsequently develop over a 115 day post-surgical period. This long latency provides a tractable model to identify functionally significant regulators of metastatic progression in mice lacking primary tumor, as well as a model to evaluate preclinical therapeutic efficacy of agents aimed at blocking functionally significant molecules aiding metastatic tumor survival and growth.

Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis

Pre-clinical models evaluating adjuvant therapy targeting breast cancer metastasis are lacking. To address this, we developed a murine model of de novo pulmonary mammary adenocarcinoma metastasis, wherein therapies administered in the adjuvant setting (post surgical resection of primary tumors) can be evaluated for efficacy in impacting previously seeded pulmonary metastases.

A rate-limiting aspect of transgenic mouse models of mammary adenocarcinoma is that primary tumor burden in mammary tissue typically defines study ...

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

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, ...

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 ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Rat Mammary Epithelial Cell Transplantation into the Interscapular White Fat Pad

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary epithelium using transplantation techniques takes advantage of the hormonal environment provided by the adolescent rodent host. These studies are ideally suited to explore the impact of various biological manipulations on mammary gland development and dissect many aspects of mammary gland biology. A common, but limiting, feature is that transplanted epithelial cells are strongly influenced by the surrounding stroma and outcompeted by endogenous epithelium; to utilize native mammary tissue, the abdominal-inguinal white fat pad must be cleared to remove host mammary epithelium prior to the transplantation. A major obstacle when using the rat model organism is that clearing the developing mammary tree in post-weaned rats is not efficient. When transplanted into gland-free fat pads, donor epithelial cells can repopulate the cleared host fat pad and form a functional mammary gland. The interscapular fat pad is an alternative location for these grafts. A major advantage is that it lacks ductal structures yet provides the normal stroma that is necessary to promote epithelial outgrowth and is easily accessible in the rat. Another major advantage of this technique is that it is minimally invasive, because it eliminates the need to cauterize and remove the growing endogenous mammary tree. Additionally, the interscapular fat pad contains a medial blood vessel that can be used to separate sites for grafting. Because the endogenous glands remain intact, this technique can also be used for studies comparing the endogenous mammary gland to the transplanted gland. This paper describes the method of mammary epithelial cell transplantation into the interscapular white fat pad of rats.

This article describes a transplantation method to graft donor rat mammary epithelial cells into the interscapular white fat pad of recipient animals. This method can be used to examine host and/or donor effects on mammary epithelium development and eliminates the need for pre-clearing, thereby extending the usefulness of this technique.

As early as the 1970s, researchers have successfully transplanted mammary epithelial cells into the interscapular white fat pad of rats. Grafting mammary ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

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 ...