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>

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</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><tbody><tr><td>Trp53&lt;sup&gt;tm1Brn &lt;/sup&gt;Transgenic mice&lt;br/&gt;K-ras&lt;sup&gt;tm4Tyj&lt;/sup&gt; Transgenic mice</td><td>Obtained from NCI mouse models of human cancer consortium</td><td></td><td>Mice were backcrossed ten times to a full C57BL/6 background</td></tr><tr><td>B6.129X1-&lt;p&gt;&lt;em&gt;Gt(ROSA)26Sor&lt;sup&gt;tm1(EYFP)Cos&lt;/sup&gt;&lt;/em&gt;/J&lt;/p&gt;Transgenic mice</td><td>Jackson Labs</td><td>006148</td><td></td></tr><tr><td>Primers p53&lt;sup&gt;loxP/loxP&lt;/sup&gt;</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-AAGGGGTATGAGGGACAAGG-3&#039;&lt;br/&gt; 5&#039;-GAAGACAGAAAAGGGGAGGG-3&#039;</td></tr><tr><td>Primers LSL-K-ras&lt;sup&gt;G12D/+&lt;/sup&gt;</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-CGCAGACTGTAGAGCAGCG-3&#039;&lt;br/&gt; 5&#039;-CCATGGCTTGAGTAAGTCTGC-3&#039;</td></tr><tr><td>Primers for LSL-EYFP to detect Rosa promoter</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-AAGACCGCGAAGAGTTTGTC-3&#039;&lt;br/&gt; 5&#039;-GGAGCGGGAGAAATGGATATG-3&#039;&lt;br/&gt; 5&#039;-AAAGTCGCTCTGAGTTGTTAT-3&#039;</td></tr><tr><td>Primers for detection of Mesothelin expression</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-TTGGGTGGATACCACGTCTG-3&#039;&lt;br/&gt; 5&#039;-CGGAGTGTAATGTTCTTCTGTC-3&#039;</td></tr><tr><td>Primers for detection of Progesterone Receptor expression</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-GCAATGGAAGGGCAGCATAA-3&#039;&lt;br/&gt; 5&#039;-TGGCGGGACCAGTTGAATTT-3&#039;</td></tr><tr><td>Primers for detection of Cytokeratin 8 expression</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-ATCAGCTCTTCCAGCTTTTCCC-3&#039;&lt;br/&gt; 5&#039;-GAAGCGCACCTTGTCAATGAAGG-3&#039;</td></tr><tr><td>Primers for detection of Erbb2 expression</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-ACCTGCCCCTACAACTACCT-3&#039;&lt;br/&gt; 5&#039;-AAATGCCAGGCTCCCAAAGA-3&#039;</td></tr><tr><td>Primers for detection of Estrogen Receptor A expression</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-ATGAAAGGCGGCATACGGAA-3&#039;&lt;br/&gt; 5&#039;-GCGGTTCAGCATCCAACAAG-3&#039;</td></tr><tr><td>Primers for detection of Estrogen Receptor B expression</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-ACCCAATGTGCTAGTGAGCC-3&#039;&lt;br/&gt; 5&#039;-TGAGGACCTGTCCAGAACGA-3&#039;</td></tr><tr><td>Primers for detection of B-Actin expression</td><td>Integrated DNA Technologies</td><td></td><td>5&#039;-GCCTTCCTTCTTGGGTATGG-3&#039;&lt;br/&gt; 5&#039;-CAGCTCAGTAACAGTCCGCC-3&#039;</td></tr><tr><td>Adenovirus-Cre</td><td>Gene Transfer Vector Core from the University of Iowa</td><td>Ad5CMVCre</td><td>Store aliquots of virus (4 x 10&lt;sup&gt;8&lt;/sup&gt; pfu/aliquot) at -80 &amp;#176;C to avoid repeated freeze thaw cycles.</td></tr><tr><td>Adenovirus-mCherry</td><td>Gene Transfer Vector Core from the University of Iowa</td><td>Ad5CMVmCherry</td><td>Store aliquots of virus (4 x 10&lt;sup&gt;8&lt;/sup&gt; pfu/aliquot) at -80 &amp;#176;C to avoid repeated freeze thaw cycles.</td></tr><tr><td>Hamilton syringe</td><td>Hamilton company</td><td>701RN</td><td>10 &amp;#956;l syringe, RN series.&lt;br/&gt;Autoclave before and after each use. Clean with PBS and 75% ethanol.</td></tr><tr><td>Custom needle</td><td>Hamilton company</td><td>7803-05</td><td>33 G 0.5 in long RN needle, with a 12&amp;#176; bevel. Autoclave before and after each use. Clean with PBS and 75% ethanol.</td></tr><tr><td>Surgical forceps</td><td>Dumont</td><td>52100-58</td><td>Dumostar No. 5 forceps. Clean with 75% ethanol after each use, followed by autoclaving</td></tr><tr><td>MEM powder</td><td>Cellgro</td><td>50 012 PB</td><td>Store at 4 &amp;#176;C in powder and reconstituted form</td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher</td><td>S233</td><td>Add to MEM and filter stearilize</td></tr><tr><td>Calcium Chloride</td><td>Sigma</td><td>C4901</td><td>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="Staining results: comparative analysis of vascular structures; diagram with regions marked TD, LD, MG." fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig1highres.jpg" src="/files/ftp_upload/51171/51171fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
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 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 and 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-α (Figure 2C).
<img alt="Mouse tumor growth study; chart, analysis, and gel electrophoresis; metastasis progression results." fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig2highres.jpg" src="/files/ftp_upload/51171/51171fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
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 β-actin, mesothelin, cytokeratin-8, Her2/neu, progesterone receptor, estrogen receptor-α, and estrogen receptor-β. <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 αβ and γδ 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="Tumor progression analysis with lymph node charts, flow cytometry data, imaging of tissue samples." fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig3highres.jpg" src="/files/ftp_upload/51171/51171fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
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="Flow cytometry analysis; dot plots show CD markers (CD3, CD45, γδTCR, GR1) for cell differentiation." fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig4highres.jpg" src="/files/ftp_upload/51171/51171fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Immune infiltrates in mouse transgenic breast tumors. Mouse breast tumors were homogenized and stained for CD45, CD3, γδTCR, 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+ γδ+ (γδ T cells, 13), CD3+ γδnegative (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="Mammalian skin lesion analysis, numbered anatomical diagram, mouse model experiment results." fo:content-width="5in" fo:src="/files/ftp_upload/51171/51171fig5highres.jpg" src="/files/ftp_upload/51171/51171fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
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

23.3K Views.  Wistar Institute.  Activering van latente mutaties met adenovirus-Cre in de borstklier ductale systeem een ​​klinisch relevante metastatische borstkanker. Opneming van een YFP promotor maakt het volgen van distale metastatische tumorcellen. Dit model is nuttig om latente metastase, anti-tumor immuniteit, en voor het ontwerpen van nieuwe immunotherapieën behandeling van borstkanker bestuderen. 

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

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 ...

Cancer Research

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

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 ...

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

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

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

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

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

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

Segmental Adeno-Cre Infection: A Technique to Generate Isolated Colorectal Cancer using Genetically Engineered Mouse Models

Source: Betzler, A. M., et al. <a href="https://www.jove.com/v/55952"> A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer.</a>&#160;J. Vis. Exp. (2017).
This video describes the generation of a colorectal mouse model by segmental adeno-cre infection. The infected colon segment leads to tumorigenesis within this segment of the mucosa, resulting in adenomas ultimately progressing to invasive and metastatic carcinoma. This model is an attractive platform for studies of cancer biology and preclinical therapeutic trials.

Source: Betzler, A. M., et al.  A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer.&#160;J. Vis. Exp. (2017).
This video describes the ...

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

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