This work was supported by NCI Grants RO1CA157664 and RO1CA124515, and a Breast Cancer Alliance award. We would like to thank Jeffrey Faust, David Ambrose, and Scott Weiss from the Wistar Flow Cytometry core facility, James Hayden from the Wistar&#160;Imaging facility, and the entire staff of the Wistar Institute Animal Facility for their invaluable technical support.

All animal experiments were approved by the Wistar Institute Animal Care and Use Committee.
1. Generation and Maintenance of Transgenic Mice
<ol>
	<li>Breed LSL-K-rastm4Tyj 26 and Trp53tm1Brn 27 (obtained from NCI mouse models of human cancer consortium on a mixed background) to a full C57BL/6 background 28 by backcrossing at least 10 generations with C57BL/6 mice. To track tumor metastasis, breed B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (LSL-EYFP, obtained from The Jackson Laboratory on a full C57BL/6 background) with double transgenic LSL-K-rasG12D/+ p53loxP/loxP mice. 
	Note: Transgenic LSL-K-rasG12D/+ p53loxP/loxP mice have loxP sites flanking a transcriptionally silenced allele of oncogenic K-ras and the endogenous p53 locus, so that upon Cre-mediated excision, overexpression of an oncogenic K-ras mutant and ablation of p53 is achieved. 
	Note: The LSL-EYFP mouse contains a stop codon flanking a gene for enhanced yellow fluorescent protein (YFP) that upon Cre-mediated excision results in the expression of YFP in the tissues where the YFP stop cassette is excised.
	<ol>
		<li>Breed transgenic mice to obtain LSL-K-rasG12D/+ p53loxP/loxP mice or LSL-K-rasG12D/+ p53loxP/loxP LSL-EYFP mice for intraductal injections. 
		Note: Mice are bred as homozygous for p53loxP/loxP and heterozygous for LSL-K-rasG12D/+ because mice with a homozygous deletion of K-ras die in utero. Use na&#239;ve virgin females at least six-weeks old for intraductal injections. 
		Note: The primers for genotyping homozygous floxed p53 allele are p53-T010-fwd (5&#39;-AAGGGGTATGAGGGACAAGG-3&#39;) and p53-T011-rev (5&#39;-GAAGACAGAAAAGGGGAGGG-3&#39;). They produce a wild type allele at 391 bp and the p53 floxed allele at 461 bp29,30. 
		Note: The primers to detect the mutant form of K-ras are oIMR8273 (5&#39;-CGCAGACTGTAGAGCAGCG-3&#39;) and oIMR8274 (5&#39;-CCATGGCTTGAGTAAGTCTGC-3&#39;). They produce the mutant band detected at 600bp. 
		Note: For the YFP reporter triple transgenic mice, the primers to detect the ROSA cassette (5&#39;-AAGACCGCGAAGAGTTTGTC-3&#39;), the wild type allele (5&#39;-GGAGCGGGAGAAATGGATATG-3&#39;), and a shared allele (5&#39;-AAAGTCGCTCTGAGTTGTTAT-3&#39;) result in the amplification of bands at 320 bp for floxed allele and 600 bp for the wild type allele.</li>
	</ol>
	</li>
</ol>
2. Surgical Preparation
<ol>
	<li>Clean surgical materials with 75% EtOH and autoclave them before and after all injections.</li>
	<li>Perform surgery on a clean uncluttered laboratory bench in a sanitized room within an animal facility. Wipe down all surfaces including the stage and dials of the surgical microscope with a broad-spectrum disinfectant solution followed by 75% EtOH.</li>
	<li>Weigh and anesthetize mice by intraperitoneal injection of a mix of ketamine (80-100 mg/kg) and xylazine (8-10 mg/kg) in sterile saline.</li>
	<li>Gently place mice back into their cages undisturbed for 5 min while they go under anesthesia. During this time generate virus precipitates (see Protocol 3).</li>
	<li>Verify lack of response to pain by toe pinching. Gently cover the eyes of anesthetized mice with veterinary ointment to prevent excessive corneal drying.</li>
	<li>To prevent hypothermia, place anesthetized mice onto a heating pad set to low heat during the surgical procedure and until they begin to recover.</li>
	<li>For the management of pain, administer mice meloxicam subcutaneously at 1 mg/kg before the surgery and 24 hr after.</li>
</ol>
3. Generation of Virus Precipitates
CAUTION: Adenovirus vectors, although they have been modified and are unable to replicate, pose the risk of infection. Handle adenovirus with caution. All personnel should be appropriately trained according to the institution&#39;s guidelines for handling BSL2 agents After intraductal injection, dispose of adenovirus in accordance with BSL2 guidelines.
<ol>
	<li>Store adenovirus concentrated virus stocks at -80 &#176;C frozen in aliquots of 4 x 108 pfu each, sufficient for injecting 16 animals with 3 &#956;l of 2.5 x 107 pfu of adenovirus particles.</li>
	<li>Store adenovirus aliquots on dry ice until approximately 15-20 min before beginning the injections. 
	Note: Avoid repeated freeze thaw cycles, as virus titer drops significantly between each cycle. 
	Note: Adenovirus precipitates are formed by modifying a protocol described previously 31.</li>
	<li>Reconstitute 504 mg of MEM powder with 50 ml of sterile molecular grade water, supplement with 244 mg of sodium bicarbonate and filter in sterile conditions and store at 4 &#176;C.
	<ol>
		<li>Prepare the calcium chloride solution by adding 1.5 g of calcium chloride to 50 ml of molecular grade water and filter in sterile conditions and store at 4 &#176;C.</li>
		<li>Mix aliquots containing 4 x 108 pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 &#956;l. Add 34 &#181;l of MEM to the virus and gently mix. Then add 4 &#956;l of the CaCl2 solution, gently mix, and incubate at room temperature for 15-20 min.</li>
		<li>Store adenovirus on dry ice until ready to form precipitates. Avoid thawing of the adenovirus and storing on ice or room temperature for extended times, unless precipitates are formed. 
		Note: It is also possible to mix the sucrose, MEM and CaCl2 prior to the surgeries if it is not possible to thaw the adenovirus aliquot and begin making precipitates immediately after removal from -80 &#176;C. This aliquot of sucrose, MEM, and calcium can be saved on dry ice until ready to add the adenovirus. 
		Note: Virus particles are stable for approximately 1 hr.</li>
		<li>Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 &#956;l (2.5 x 107 pfu) of virus particles into the 10 &#956;l syringe and prepare the mouse for the intraductal injection.</li>
	</ol>
	</li>
</ol>
4. Intraductal Injection of Virus Particles
<ol>
	<li>Gently place the mouse on its back onto the illuminated stage of a clean dissection microscope. Illuminate the abdominal side with an extra light source and locate the left 4th or right 9th inguinal mammary gland by the small white patches of fur (visible on C57BL/6 females) surrounding each nipple.</li>
	<li>Rub the nipple gently with a sterile ethanol soaked cotton tipped applicator to clear hair away from the nipple and to sterilize the injection site. If they are difficult to locate, gently apply a thick layer of a depilatory cream or use shears to expose the nipples.</li>
	<li>Remove the keratin plug, a layer of dense dead skin cells, which is covering the nipple. Once the nipple is exposed, the keratin plug should be easily visible under the dissection microscope.</li>
	<li>Secure the nipple with fine surgical forceps and pull up with light force to remove the keratin plug.</li>
	<li>Stabilize the nipple between the forceps.</li>
	<li>Gently insert the needle between the forceps, cannulating the duct canal at 90&#176;. Enter the nipple slightly past the bevel of the needle (not more than 2 mm) to prevent penetration through the mammary tissue and into the serous membranes of the ventral body cavity.
	<ol>
		<li>Do not insert the needle too deep. To ensure proper depth of injection, gently pull the needle up after inserting it into the lumen of the duct, drawing the nipple up along the edges of the needle as it is pulled up. 
		Note: Visualization of the injection is difficult; therefore practice for this step is recommended using trypan blue.</li>
	</ol>
	</li>
	<li>When the needle is appropriately placed into the mammary duct, release the 3 &#181;l of virus precipitates (2.5 x 107 pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.</li>
</ol>
5. Recovery of Mice
<ol>
	<li>Place the mouse back onto the heating pad after the injection&#160;until it begins to recover from the anesthesia.</li>
	<li>Once the mouse is recovered, place it back into a clean cage and monitor for full recovery and movement.</li>
	<li>24 hr after the intraductal injection, subcutaneously administer meloxicam at 1 mg/kg.</li>
</ol>
6. Monitoring Tumor Progression
<ol>
	<li>Palpate the injected mammary gland at day 30 for enlargement and swelling.
	<ol>
		<li>Monitor tumor progression every 5-7 days once a swollen and enlarged mammary gland is observed.</li>
	</ol>
	</li>
	<li>Measure tumor volumes every 3-4 days for tumor growth kinetics once palpable tumors appear (approximately 50-60 days post adenoviral injection).</li>
	<li>Euthanize mice when tumor volumes exceed 10% of the body weight of the mice.</li>
</ol>

<ol>
	<li>Youlden, 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=The+descriptive+epidemiology+of+female+breast+cancer:+an+international+comparison+of+screening,+incidence,+survival+and+mortality.">The descriptive epidemiology of female breast cancer: an international comparison of screening, incidence, survival and mortality.</a> Cancer Epidemiol. 36, 237-248 (2012).</li><li>Siegel, R., Naishadham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2012.">Cancer statistics, 2012.</a> Cancer J. Clin. 62, 10-29 (2012).</li><li>Sorlie, T., 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=Gene+expression+patterns+of+breast+carcinomas+distinguish+tumor+subclasses+with+clinical+implications.">Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.</a> Proc. Natl. Acad. Sci. U.S.A. 98, 10869-10874 (2001).</li><li>Gatza, 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=A+pathway-based+classification+of+human+breast+cancer.">A pathway-based classification of human breast cancer.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 6994-6999 (2010).</li><li>Bastien, 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=PAM50+breast+cancer+subtyping+by+RT-qPCR+and+concordance+with+standard+clinical+molecular+markers.">PAM50 breast cancer subtyping by RT-qPCR and concordance with standard clinical molecular markers.</a> BMC Med. Genom. 5, 44 (2012).</li><li>Montagna, 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=Heterogeneity+of+triple-negative+breast+cancer:+histologic+subtyping+to+inform+the+outcome.">Heterogeneity of triple-negative breast cancer: histologic subtyping to inform the outcome.</a> Clin. Breast Cancer. 13, 31-39 (2013).</li><li>Karrison, T. G., Ferguson, D. J., Meier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dormancy+of+mammary+carcinoma+after+mastectomy.">Dormancy of mammary carcinoma after mastectomy.</a> J. Natl. Cancer Inst. 91, 80-85 (1999).</li><li>Rakha, 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=The+prognostic+significance+of+lymphovascular+invasion+in+invasive+breast+carcinoma.">The prognostic significance of lymphovascular invasion in invasive breast carcinoma.</a> Cancer. 118, 3670-3680 (2012).</li><li>Kim, I., Baek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+breast+cancer+metastasis.">Mouse models for breast cancer metastasis.</a> Biochem. Biophys. Res. Commun. 394, 443-447 (2010).</li><li>Vargo-Gogola, T., Rosen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer:+one+size+does+not+fit+all.">Modelling breast cancer: one size does not fit all.</a> Nat. Cancer. 7, 659-672 (2007).</li><li>Archer, T., 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=Steroid+hormone+receptor+status+defines+the+MMTV+promoter+chromatin+structure+in+vivo.">Steroid hormone receptor status defines the MMTV promoter chromatin structure in vivo.</a> J. Steroid Biochem. Mol. Biol. 53, 421-429 (1995).</li><li>Cato, A., Henderson, D., Ponta, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hormone+response+element+of+the+mouse+mammary+tumour+virus+DNA+mediates+the+progestin+and+androgen+induction+of+transcription+in+the+proviral+long+terminal+repeat+region.">The hormone response element of the mouse mammary tumour virus DNA mediates the progestin and androgen induction of transcription in the proviral long terminal repeat region.</a> EMBO J. 6, 363-368 (1987).</li><li>Schoenenberger, C., Zuk, A., Groner, B., Jones, W., Andres, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+the+endogenous+whey+acidic+protein+(Wap)+gene+and+a+Wap-myc+hybrid+gene+in+primary+murine+mammary+organoids.">Induction of the endogenous whey acidic protein (Wap) gene and a Wap-myc hybrid gene in primary murine mammary organoids.</a> Dev. Biol. , 327-337 (1990).</li><li>Li, Y., 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=Deficiency+of+Pten+accelerates+mammary+oncogenesis+in+MMTV-Wnt-1+transgenic+mice.">Deficiency of Pten accelerates mammary oncogenesis in MMTV-Wnt-1 transgenic mice.</a> BMC Mol. Biol. 2, 2 (2001).</li><li>Martelli, 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=In+vivo+imaging+of+lymph+node+migration+of+MNP-+and+(111)In-labeled+dendritic+cells+in+a+transgenic+mouse+model+of+breast+cancer+(MMTV-Ras).">In vivo imaging of lymph node migration of MNP- and (111)In-labeled dendritic cells in a transgenic mouse model of breast cancer (MMTV-Ras).</a> Mol. Imaging Biol. 14, 183-196 (2012).</li><li>Klover, P. 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=Loss+of+STAT1+from+mouse+mammary+epithelium+results+in+an+increased+Neu-induced+tumor+burden.">Loss of STAT1 from mouse mammary epithelium results in an increased Neu-induced tumor burden.</a> Neoplasia. 12, 899-905 (2010).</li><li>Moody, S. 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=Conditional+activation+of+Neu+in+the+mammary+epithelium+of+transgenic+mice+results+in+reversible+pulmonary+metastasis.">Conditional activation of Neu in the mammary epithelium of transgenic mice results in reversible pulmonary metastasis.</a> Cancer Cell. 2, 451-461 (2002).</li><li>Banerji, 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=Sequence+analysis+of+mutations+and+translocations+across+breast+cancer+subtypes.">Sequence analysis of mutations and translocations across breast cancer subtypes.</a> Nature. 486, 405-409 (2012).</li><li>Miyakis, S., Sourvinos, G., Spandidos, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+mutation+of+the+ras+family+genes+in+human+breast+cancer.">Differential expression and mutation of the ras family genes in human breast cancer.</a> Biochem. Biophys. Res. Commun. 251, 609-612 (1998).</li><li>Malaney, S., Daly, 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=The+ras+signaling+pathway+in+mammary+tumorigenesis+and+metastasis.">The ras signaling pathway in mammary tumorigenesis and metastasis.</a> J. Mammary Gland Biol. Neoplasia. 6, 101-113 (2001).</li><li>Loboda, 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=A+gene+expression+signature+of+RAS+pathway+dependence+predicts+response+to+PI3K+and+RAS+pathway+inhibitors+and+expands+the+population+of+RAS+pathway+activated+tumors.">A gene expression signature of RAS pathway dependence predicts response to PI3K and RAS pathway inhibitors and expands the population of RAS pathway activated tumors.</a> BMC Med. Genomics. 3, 26 (2010).</li><li>von Lintig, F. 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=Ras+activation+in+human+breast+cancer.">Ras activation in human breast cancer.</a> Breast Cancer Res. Treat. 62, 51-62 (2000).</li><li>Downward, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+RAS+signalling+pathways+in+cancer+therapy.">Targeting RAS signalling pathways in cancer therapy.</a> Nat. Rev. Cancer. 3, 11-22 (2003).</li><li>Eckert, L. 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=Involvement+of+Ras+activation+in+human+breast+cancer+cell+signaling,+invasion,+and+anoikis.">Involvement of Ras activation in human breast cancer cell signaling, invasion, and anoikis.</a> Cancer Res. 64, 4585-4592 (2004).</li><li>Hollestelle, A., Elstrodt, F., Nagel, J., Kallemeijn, W., Schutte, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-3-OH+kinase+or+RAS+pathway+mutations+in+human+breast+cancer+cell+lines.">Phosphatidylinositol-3-OH kinase or RAS pathway mutations in human breast cancer cell lines.</a> Mol. Cancer Res. 5, 195-201 (2007).</li><li>Jackson, 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=Analysis+of+lung+tumor+initiation+and+progression+using+conditional+expression+of+oncogenic+K-ras.">Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.</a> Genes Dev. 15, 3243-3248 (2001).</li><li>Jonkers, 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=Synergistic+tumor+suppressor+activity+of+BRCA2+and+p53+in+a+conditional+mouse+model+for+breast+cancer.">Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.</a> Nat. Genet. 29, 418-425 (2001).</li><li>Scarlett, U., 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=Ovarian+cancer+progression+is+controlled+by+phenotypic+changes+in+dendritic+cells.">Ovarian cancer progression is controlled by phenotypic changes in dendritic cells.</a> Exp. Med. 209, 495-506 (2012).</li><li>Vooijs, M., Jonkers, J., Berns, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+efficient+ligand-regulated+Cre+recombinase+mouse+line+shows+that+LoxP+recombination+is+position+dependent.">A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent.</a> EMBO Rep. 2, 292-297 (2001).</li><li>Young, N., Crowley, D., Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+cancer+mutations+reveals+critical+timing+of+p53+loss+in+sarcomagenesis.">Uncoupling cancer mutations reveals critical timing of p53 loss in sarcomagenesis.</a> Cancer Res. 71, 4040-4047 (2011).</li><li>Dinulescu, 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=Role+of+K-ras+and+Pten+in+the+development+of+mouse+models+of+endometriosis+and+endometrioid+ovarian+cancer.">Role of K-ras and Pten in the development of mouse models of endometriosis and endometrioid ovarian cancer.</a> Nat. Med. 11, 63-70 (2005).</li><li>Landua, J., Visbal, A., Lewis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+preparing+fluorescent+and+neutral+red-stained+whole+mounts+of+mouse+mammary+glands.">Methods for preparing fluorescent and neutral red-stained whole mounts of mouse mammary glands.</a> J. Mammary Gland Biol. Neoplasia. 14, 411-415 (2009).</li><li>Barham, W., Sherrill, T., Connelly, L., Blackwell, T. S., Yull, F. E. <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+LPS+as+a+mouse+model+of+mastitis:+signaling+visualized+via+an+NF-kappaB+reporter+transgenic.">Intraductal injection of LPS as a mouse model of mastitis: signaling visualized via an NF-kappaB reporter transgenic.</a> J. Vis. Exp. , e4030 (2012).</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></ol>

The authors declare no competing financial interests.

The success of this procedure hinges on proper technique during intraductal injections, which will be difficult for untrained experimenters. Experienced researchers in our laboratory typically obtain breast tumor masses 79% of the times they administer adenoviral injections. Problems with the injection can result in significantly delayed, variable, or absent tumor development. If the needle is inserted too deep or at an inappropriate angle, the ductal canal may be missed. It is important to enter the nipple slightly past the bevel of the needle (not more than 2 mm), to prevent penetration through the mammary tissue and into the serous membranes of the ventral body cavity. Also, too shallow placement of the needle or the injection of greater than 3 ml of virus precipitates can result in spillage of viral prep outside the mammary gland and the induction of unintentional tumors. One way to overcome these issues is to insert the needle into the nipple slightly deeper than 3 mm, and slowly draw the syringe back up out of the duct until 2 mm from the tip of the bevel. This will ensure that mammary tissue is targeted instead of the muscle surrounding the peritoneal cavity of mice (Figure 1A). This will also stretch the nipple along the edges of the syringe so that when the virus is expelled into the duct, there is no spillage and loss of viral precipitates.
Visualization of the injection is difficult and practice for this step is recommended. We have observed an increase in successful injections following practice, resulting in a higher penetrance of tumor development. Because this technique uses nonlactating virgin females, it is critical to remove the keratin plug covering the nipple to reveal the underlying duct canal. We recommend practicing this step by injecting trypan blue or some other sterile traceable dye inside of the duct and preparing whole mammary mounts to confirm targeting of the ductal tree. Additionally, other protocols describing intraductal injection of reagents have been published33,34, which may be useful for developing proper technique. Issues with viral preparation or infection of the ductal lumen can also be investigated by using a mCherry expressing adenovirus. Nontransgenic mice can be used for each of these purposes until the injection technique is optimized.
Although any mammary gland can be used to initiate tumors, we have achieved the most consistent growth rates by targeting mammary gland 4 or 9, which we believe is because it is easier to perform proper injections on these glands, resulting in more efficient targeting of the duct. The proximity of the left 4th or right 9th inguinal mammary glands to the draining inguinal lymph node is also useful to examine anti-tumor immune responses during different temporal points of tumor progression. To model and track latent metastasis to distal sites, transgenic mice were crossed with LSL-EYFP mice. As the tumor progresses, vasculature connecting the tumor to the axillary lymph node will become slightly engorged at approximately 5 weeks, before the tumor begins to grow exponentially (Figure 3A). Eventually, after 7-8 weeks, lymphovascular invasion will result in tumor growth within the axillary lymph node (Figures 3A and 3B). Using reporter mice and Cre-loxP technology, incorporation of YFP creates a platform to track tumor cells metastasizing to distal sites throughout tumor progression. This can facilitate studies aimed at elucidating the cellular and epigenetic mechanisms that promote latent metastasis. In our hands, breast tumor cell lines expressed cytokeratin-8, mesothelin, estrogen receptor-&#945;, and Her2/neu, confirming targeting of the ductal epithelium. However, depending upon the mutations induced and due to the difficulty and the variability of injections, we recommend histological characterization of tumors once the model is well established in the laboratory.
Because breast cancer is such a deadly and pervasive disease1,2, it is important to use animal models that accurately recapitulate the complex interplay between tumor and host. Here we describe a fully backcrossed C57BL/6 murine model of breast tumor. First, by inducing tumors from native cells, we allow the tumor to evolve naturally in a full immune microenvironment. The immune microenvironment in the advanced mouse breast tumors recapitulates the populations of &#945;&#946; and &#947;&#948; T cells, myeloid derived suppressor cells, and macrophages commonly observed in human breast cancer (Figure 4). As we have published previously using adenovirus-Cre to induce ovarian tumors, we found that the viral injection had negligible effect on the corresponding tumor infiltrates and tumor progression28. Second, endocrine independent expression of oncogenes ensures that tumor cells have persistently high levels of target gene expression. Third, by taking advantage of latent mutations, we can control the timing of tumorigenesis to facilitate precise temporal tracking of tumor evolution. Applications of this model include research on tumor cell biology, studies on factors in the tumor microenvironment, anti-tumor immune responses, and even efficacy evaluation of new therapeutics. Through the availability of the Cre-loxP system, this technique can be used as a platform for investigating a diverse array of additional mutations in the initiation and progression of breast tumors. We hope that the use of this model will enhance the understanding of breast cancer biology and eventually lead to new therapeutics aimed at treating metastatic breast cancer.

A correction was made to <a href="/video/51171">Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer</a>. Protocol sections 3.1, 3.3.2, 3.4, and 4.7 were updated. The materials table was also updated.
Protocol section 3.1 was updated from:
3.1) Store adenovirus concentrated virus stocks at -80&#176;C frozen in aliquots of 4 x 108 pfu each, sufficient for injecting 16 animals with 3 ml of 2.5 x 107 pfu of adenovirus particles.
to:
3.1) Store adenovirus concentrated virus stocks at -80&#176;C frozen in aliquots of 4 x 108 pfu each, sufficient for injecting 16 animals with 3 &#181;l of 2.5 x 107 pfu of adenovirus particles.
Protocol section 3.3.2 was updated from:
Mix aliquots containing 4 x 108 pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 ml. Add 34 ml of MEM to the virus and gently mix. Then add 4 ml of the CaCl2 solution, gently mix, and incubate at room temperature for 15-20 min.
to:
Mix aliquots containing 4 x 108 pfu adenovirus-Cre with sufficient 3% sucrose in sterile water for a final volume of 10 &#181;l. Add 34 &#181;l of MEM to the virus and gently mix. Then add 4 &#181;l of the CaCl2 solution, gently mix, and incubate at room temperature for 15-20 min.
Protocol section 3.4 was updated from:
Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 ml (2.5 x 107 pfu) of virus particles into the 10 ml syringe and prepare the mouse for the intraductal injection.
to:
Prior to each injection, gently flick the tube to make sure virus particles are mixed. Draw up 3 &#181;l (2.5 x 107 pfu) of virus particles into the 10 &#181;l syringe and prepare the mouse for the intraductal injection.
Protocol section 4.7 was updated from:
When the needle is appropriately placed into the mammary duct, release the 3 ml of virus precipitates (2.5 x 107 pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.
to:
When the needle is appropriately placed into the mammary duct, release the 3 &#181;l of virus precipitates (2.5 x 107 pfu of adenovirus-Cre) by gently plunging the syringe with the thumb of the hand holding the syringe. The nipple should slightly inflate as the liquid is added.
Item 15 in the Materials table under Comments was updated from:
10ml syringe, RN series Autoclave before and after each use. Clean with PBS and 75% ethanol.
to:
10&#181;l syringe, RN series Autoclave before and after each use. Clean with PBS and 75% ethanol.

A correction was made to <a href="/video/51171">Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer</a>. Protocol sections 3.1, 3.3.2, 3.4, and 4.7 were updated. The materials table was also updated.

Erratum: 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 the most commonly occurring malignancy in women throughout the world1,2 and the second leading cause of cancer-related deaths2. Complex genetic3,4, histological5, and clinical phenotypes6 are used to characterize the various subtypes of breast cancer and often are used as a means to predict survival. Analysis of a large cohort of women with breast cancer indicated that most (approximately 80%) of the patients that died had recurred within 10 yr post removal of the primary tumor7. For a majority of invasive breast carcinomas, lymphovascular invasion has been shown to be strongly correlated to a poor outcome and more aggressive clinical course of disease8.
Because of the genetic and phenotypic complexity of breast cancer, there is no animal model that recapitulates the entire course of disease. Human breast tumor cell lines have been frequently used as xenograft or orthotopic9 models of invasive and metastatic breast cancer in immune deficient mice. Although informative, these models occur in the absence of immune pressure and because it is a cross species graft, distort the effects of the entire tumor microenvironment. Inducible genetic mutations driven by mammary specific promoters such as murine mammary tumor virus (MMTV) and whey acidic protein (WAP) have contributed a tremendous amount of knowledge about the genetic nature of breast cancer. However, the tissue specific expression of these promoters is compromised by their responsiveness to the endocrine system10-16, resulting in the variable expression of induced genetic mutations that do not mirror the expression of oncogenes typically overexpressed in human breast cancer. To overcome endocrine control of MMTV driven expression of oncogenes, Moody et al. generated a conditional, doxycycline inducible model overexpressing Neu in the breast epithelium17. This model is useful for deinducing Neu after tumor formation to study regression and recurrence, but requires constant doxycycline administration for consistent, long-term oncogene expression. A comprehensive discussion of the many relevant breast tumor models available can be found in the review by Vargo-Gogola et al.10
Our goal was to develop a mouse model of traceable breast cancer on a full C57BL/6 background that, after the permanent induction of mutational events, models the formation of a nascent tumor in the presence of immune pressure. We introduced an adenovirus expressing Cre-recombinase into the mammary ducts of transgenic mice containing floxed alleles of Tp53, and an oncogenic form of K-ras, and YFP. Cre expression ablates Tp53, a frequently mutated gene in many breast cancers18 and induces an oncogenic allele of K-ras in addition to YFP expression specifically in the mammary ductal epithelium. Although mutations in K-ras are infrequent in breast cancer, occurring in only 6.5% of breast cancer patients19,20, the overexpression of upstream kinases such as Her2/neu and EGFR result in constitutive activation of the Ras signaling pathway in human breast tumors21-23. Activation of the Ras signaling pathway in many breast tumor cell lines has also been reported24,25. We will describe the initiation of tumor formation and the technique of intraductal injection of an adenovirus expressing Cre-recombinase into sexually mature, virgin female mice. This model of breast cancer develops overt lesions that grow exponentially after about 8 weeks of slow tumor progression, with lymphovascular invasion and metastasis to the axillary lymph node by 7-8 weeks. Because these mice are on a full C57BL/6 background and YFP-expressing tumor cells are traceable in distal lymph nodes, this model provides a relevant tool to study the cellular and immunological mechanisms of latent metastasis and will help to develop novel therapeutic approaches for the treatment of metastatic ductal breast cancer.

<table border="0" cellpadding="0" cellspacing="0" width="825">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="80">
			<td height="80" style="height:80px;width:251px;">Trp53tm1Brn transgenic mice 
			Krastm4Tyj transgenic mice</td>
			<td style="width:145px;">Obtained from NCI mouse models of human cancer consortium</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">Mice were backcrossed ten times to a full C57BL/6 background</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:251px;">B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J Transgenic mice</td>
			<td style="width:145px;">Jackson labs</td>
			<td style="width:141px;">006148</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers p53loxp/loxp</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-AAGGGGTATGAGGGACAAGG-3&#39; 
			5&#39;-GAAGACAGAAAAGGGGAGGG-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers LSL-K-ras G12D/+</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-CGCAGACTGTAGAGCAGCG-3&#39; 
			5&#39;-CCATGGCTTGAGTAAGTCTGC-3&#39;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Primers for LSL-EYFP to detect Rosa promoter</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-AAGACCGCGAAGAGTTTGTC-3&#39; 
			5&#39;-GGAGCGGGAGAAATGGATATG-3&#39; 
			5&#39;-AAAGTCGCTCTGAGTTGTTAT-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Mesothelin expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-TTGGGTGGATACCACGTCTG-3&#39; 
			5&#39;-CGGAGTGTAATGTTCTTCTGTC-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Progesterone Receptor expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-GCAATGGAAGGGCAGCATAA-3&#39; 
			5&#39;-TGGCGGGACCAGTTGAATTT-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Cytokeratin 8 expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-ATCAGCTCTTCCAGCTTTTCCC-3&#39; 
			5&#39;-GAAGCGCACCTTGTCAATGAAGG-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Erbb2 expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-ACCTGCCCCTACAACTACCT-3&#39; 
			5&#39;-AAATGCCAGGCTCCCAAAGA-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Estrogen Receptor A expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-ATGAAAGGCGGCATACGGAA-3&#39; 
			5&#39;-GCGGTTCAGCATCCAACAAG-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			Estrogen Receptor B expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-ACCCAATGTGCTAGTGAGCC-3&#39; 
			5&#39;-TGAGGACCTGTCCAGAACGA-3&#39;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Primers for detection of 
			B-Actin expression</td>
			<td style="width:145px;">Integrated DNA Technologies</td>
			<td style="width:141px;"></td>
			<td style="width:289px;">5&#39;-GCCTTCCTTCTTGGGTATGG-3&#39; 
			5&#39;-CAGCTCAGTAACAGTCCGCC-3&#39;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Adenovirus-CRE</td>
			<td style="width:145px;">Gene Transfer Vector Core from the University of Iowa</td>
			<td style="width:141px;">Ad5CMVCre</td>
			<td style="width:289px;">Store aliquots of virus (4 x 108 pfu/aliquot) at -80C to avoid repeated freeze thaw cycles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Adenovirus-mCherry</td>
			<td style="width:145px;">Gene Transfer Vector Core from the University of Iowa</td>
			<td style="width:141px;">Ad5CMVmCherry</td>
			<td style="width:289px;">Store aliquots of virus (4 x 108 pfu/aliquot) at -80C to avoid repeated freeze thaw cycles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Hamilton syringe</td>
			<td style="width:145px;">Hamilton company</td>
			<td style="width:141px;">701RN</td>
			<td style="width:289px;">10ml syringe, RN series 
			Autoclave before and after each use. Clean with PBS and 75% ethanol.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:251px;">Custom needle</td>
			<td style="width:145px;">Hamilton company</td>
			<td style="width:141px;">7803-05</td>
			<td style="width:289px;">33 gauge 0.5 inch long RN needle, with a 12 degree bevel. Autoclave before and after each use. Clean with PBS and 75% ethanol.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:251px;">Surgical forceps</td>
			<td style="width:145px;">Dumont</td>
			<td style="width:141px;">52100-58</td>
			<td style="width:289px;">Dumostar No5 forceps. Clean with 75% ethanol after each use, followed by autoclaving</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">MEM powder</td>
			<td style="width:145px;">Cellgro</td>
			<td style="width:141px;">50 012 PB</td>
			<td style="width:289px;">Store at 4C in powder and reconstituted form</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">Sodium Bicarbonate</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:141px;">S233</td>
			<td style="width:289px;">Add to MEM and filter stearilize</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:251px;">Calcium Chloride</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:141px;">C4901</td>
			<td style="width:289px;">Minimum 96%, anhydrous</td>
		</tr>
	</tbody>
</table>

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.

Successful targeting of the mammary ductal tree can be visualized by preparing whole mounts of the mammary gland as previously described32 after injection of trypan blue (to verify proper injection technique (Figure 1A) or an adenovirus expressing mCherry (to verify proper viral preparation and infection of ductal epithelial cells, Figure 1B).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig1highres.jpg" src="/files/ftp_upload/51171/51171fig1.jpg" /> 
Figure 1. Intraductal targeting of mammary glands by injection with trypan blue or adenovirus-mCherry. A) A whole mount, as previously reported32, of the mammary gland after injection of mammary gland #4 with trypan blue was prepared 3 hr post injection to visualize/confirm targeting of the entire ductal tree. Images are 4X magnification. B) Infection of the ductal epithelium with adenovirus by injecting 2.5 x 107 pfu of adenovirus expressing mCherry. Mice were injected intraductally, and 4 days post-injection, a whole mount of the mammary gland was prepared to confirm viral infection of the ductal tree. Image is 4X&#160;magnification. MG is mammary gland, LD is the lactiferous, or main duct, and TD is the terminal duct. <a href="https://www.jove.com/files/ftp_upload/51171/51171fig1highres.jpg" target="_blank">Click here to view larger image.</a>
When tumors are induced in p53loxP/loxP LSL-K-rasG12D/+ transgenic mice, tumors will not be apparent until around day 40 when the mammary glands will become enlarged and swollen. It is necessary to begin palpations when this is observed, monitoring tumor growth every 5-7 days. In our hands, hardening of the mammary gland always precedes the onset of tumor development. Tumors will progress slowly for an additional 2 weeks. Beginning around day 56, tumors will begin to grow exponentially (Figure 2B). At this point, it is critical to measure tumor volumes every 3 days if kinetic studies are desired because there will be slight mouse to mouse variability in tumor progression, which is normal (Figures 2A&#160;and&#160;3A). Large abdominal masses will be apparent by day 80 (Figure 2A, 2B, and 3A), after which mice should be euthanized if tumors exceed more than 10% of their body weight. cDNA analysis of three clones from a tumor-bearing mouse revealed expression of mesothelin, cytokeratin-8, Her2/neu, and estrogen receptor-&#945; (Figure 2C).
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig2highres.jpg" src="/files/ftp_upload/51171/51171fig2.jpg" /> 
Figure 2. Tumor development in p53loxP/loxP LSL-K-rasG12D/+ mice injected intraductally with adenovirus-Cre. A) Two examples of tumors 80 days after injection with adenovirus expressing Cre. Mice were given 2.5 x 107 PFU of adenovirus expressing Cre and 80 days post tumor-initiation, large palpable masses can be visualized protruding from the abdominal side of the animal. B) Typical tumor kinetics and palpation schedule for tumors induced in p53loxP/loxP LSL-K-rasG12D/+ mice. C) Characterization of three tumor cell clones derived from the same homogenized tumor of a p53loxP/loxP LSL-K-rasG12D/+ mouse. RNA was extracted and cDNA synthesized for RT-PCR analysis using primers specific to &#946;-actin, mesothelin, cytokeratin-8, Her2/neu, progesterone receptor, estrogen receptor-&#945;, and estrogen receptor-&#946;. <a href="https://www.jove.com/files/ftp_upload/51171/51171fig2highres.jpg" target="_blank">Click here to view larger image.</a>
Similar to the cellular microenvironment in human breast cancer, we have observed infiltration of &#945;&#946; and &#947;&#948; T cells as well as myeloid derived suppressor cells and macrophages into the tumors (Figure 4). Vasculature draining to the axillary lymph node will begin to engorge before tumors have grown to encompass the entire mammary tissue where the injection was performed (Figure 3A). Lymphovascular invasion and metastasis of tumor cells can be tracked by crossing LSL-K-rasG12D/+ p53loxP/loxP mice with LSL-EYFP mice. After Cre-mediated excision, tumor cells expressing YFP (both high and low) are detected in the tumor and can be traced metastasizing to the draining axillary lymph node (Figure 3B). Metastasis to the distal axillary lymph node was confirmed by successfully culturing a tumor cell line from this site in a tumor-bearing LSL-K-rasG12D/+ p53loxP/loxP mouse (data not shown).
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig3highres.jpg" src="/files/ftp_upload/51171/51171fig3.jpg" /> 
Figure 3. Formation of tumors and latent metastasis to the axillary lymph node. A) Example of three advanced breast tumors with different rates of tumor progression. A solid mass, indicated by the arrowhead, forms and eventually grows to the size of the entire abdominal mammary tissue. The tumor stays confined to the mammary tissue and is not observed to invade or attach to the muscle covering the peritoneal cavity. There is evident engorgement of the superficial epigastric vein between the inguinal and axillary lymph nodes, denoted by white arrowheads. After 7-8 weeks, the axillary lymph node begins to become enlarged due to lymphovascular invasion of the tumor cells, indicated by arrow. B) Metastasis of YFP positive tumor cells can be visualized in the axillary lymph node by flow cytometry. LSL-K-rasG12D/+ p53loxP/loxP LSL-EYFP mice were used to induce tumors and activation of YFP by intraductal delivery of adenovirus-Cre. To verify lymphovascular invasion of tumor cells into the axillary lymph node, 80 days post adenoviral injection, the indicated lymph nodes and organs were harvested and stained for lymphocyte markers and examined for YFP expression. CL represents contralateral nontumor draining lymph node. Results represent gating on CD45 negative tumor cells, indicating the tumor cells are invading the distal axillary lymph node. Numbers represent percent YFP positive cells from total population. <a href="https://www.jove.com/files/ftp_upload/51171/51171fig3highres.jpg" target="_blank">Click here to view larger image.</a>
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig4highres.jpg" src="/files/ftp_upload/51171/51171fig4.jpg" /> 
Figure 4. Immune infiltrates in mouse transgenic breast tumors. Mouse breast tumors were homogenized and stained for CD45, CD3, &#947;&#948;TCR, CD11b and GR1. Numbers represent percent of positive leukocytes in entire tumor (63.5), total CD3+ (46.7), total CD3 negative (40.1), total CD3+ &#947;&#948;+ (&#947;&#948; T cells, 13), CD3+ &#947;&#948;negative (24), total GR1 high CD11b (MDSC, 28.6) and total CD11b GR1 low (macrophages, 18.5). <a href="https://www.jove.com/files/ftp_upload/51171/51171fig4highres.jpg" target="_blank">Click here to view larger image.</a>
Due to the anatomy of the mouse and technique of intraductal injections, we find targeting of mammary glands 4 and 9 (Figure 5) yields the most consistent results and reliable injections. However, any gland can be targeted depending upon the preference of the technician performing the surgery.
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig5highres.jpg" src="/files/ftp_upload/51171/51171fig5.jpg" /> 
Figure 5. Numbering of mammary ducts. Mammary ducts 4 and 9 are highlighted in red on the diagram and indicated with arrows on the mouse. In our hands, we found that injections were easiest to perform on these mammary ducts, however all other mammary tissues that we targeted developed tumors with similar kinetics. <a href="https://www.jove.com/files/ftp_upload/51171/51171fig5highres.jpg" target="_blank">Click here to view larger image.</a>

Watch this Scientific Journal Video about Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer at JoVE.c

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

initiation-metastatic-breast-carcinoma-targeting-ductal-epithelium

Research

JoVE Journal

Medicine

23.0K Views.  Wistar Institute. 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.

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

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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 ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...