The authors thank Jo Hill for histopathology assistance, Dr. John Gleysteen for instruction in surgical technique, Tessa Diebel for videography assistance, all members of the Wong and Coussens laboratories for critical insight and discussions, and the OHSU Knight Cancer Institute for financial support. The authors acknowledge support from T32GM071388-10 and T32CA106195-11 to CEG, the NCI/NIH, the Department of Defense Breast Cancer Research Program, the Susan G Komen Foundation, the Breast Cancer Research Foundation, and a Stand Up To Cancer - Lustgarten Foundation Pancreatic Cancer Convergence Dream Team Translational Research Grant (SU2C-AACR-DT14-14) to LMC, a Women&#39;s Health Circle of Giving Foundation Award to MHW, and the Brenden-Colson Center for Pancreatic Health to MHW and LMC.

Animals used in the following protocol are covered by Oregon Health &#38; Science University&#39;s Institutional Animal Care and Use Committee (IACUC), which is designed to be compliant with the Animal Welfare Act regulations and Public Health Service (PHS) Policy.
Maintenance of sterile conditions: Sterilized instruments should be used and between mice, should be wiped clean with sterile gauze, rinsed in PBS followed by sterilization with disinfectant 70% ethanol for at least 15 minutes. A surgical cap, facemask, gown, and gloves should be worn for survival surgeries. Pre-operative preparation of the animal for survival surgeries is included in the following protocol. Refer to Table 1 for a list of reagents and equipment.
1. Isolation and Preparation of Single Cell Suspensions from Primary Mammary Tumors
<ol>
	<li>Anesthetize donor female 100 day old transgenic MMTV-PyMT (FVB/n) mice and maintain under continuous sedation by administering 2% isoflurane via an anesthesia mask. Confirm that the mouse is properly anesthetized with an absent foot pinch reflex.</li>
	<li>In a sterile setting, resect primary mammary tumors from 100-day old transgenic female MMTV-PyMT (FVB/n) mice by separating mammary tumor from overlying skin and surrounding adipose tissue and/or lymph nodes using sterile scissors. Euthanize the anesthetized mice by cervical dislocation.</li>
	<li>With sterile scissors or a scalpel, mince primary tumors manually into small pieces (~1.0 mm3). Place tumor pieces in 3.0 mg/mL collagenase A and 4.0 U/mL DNase I dissolved in DMEM. Use ~10 mL of the above digestion medium per 1.0 cm diameter tumor.
	<ol>
		<li>Perform digestion in a sterile 25 mL bottle with a sterile stir bar at ~125 rpm and 37 &#176;C for 40 min.</li>
	</ol>
	</li>
	<li>Stop the digestion by adding fetal bovine serum (FBS) to a final dilution of 10% and place the entire mixture on wet ice where it is maintained.</li>
	<li>Filter the digested tumor suspension through a 0.7 &#181;m nylon strainer into a 50 mL conical tube and discard any tumor remaining in the strainer. Centrifuge the supernatant at 300 x g at 4 &#176;C.</li>
	<li>Resuspend the pellet in 10 mL DMEM per 1.0 cm tumor and re-filter through a 0.7 &#181;m nylon strainer. Count cell concentration, then centrifuge at 300 x g at 4 &#176;C.</li>
	<li>Resuspend pellet in 10% dimethyl sulfoxide with 90% FBS at a concentration of 2 x 107 live cells/mL. Store single-cell suspensions of whole primary tumor at -80 &#176;C.</li>
</ol>
2. Orthotopic Injection of Mammary Tumor
<ol>
	<li>Partially thaw frozen primary tumor suspensions at 37 &#176;C until the frozen pellet can be released from the cryogenic tube into 20 mL DMEM and count the cells. Centrifuge at 300 x g at 4 &#176;C and resuspend cells in a 1:1 DMEM: growth factor-reduced solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma at a concentration of 1 x 107 cells/mL (see the Table of Materials).</li>
	<li>Place the anesthetized recipient female syngeneic mouse ventral side up and maintain under continuous sedation by administering 2% isoflurane via an anesthesia mask.</li>
	<li>Sterilize the right 4th mammary gland injection site using aerosolized 70% ethanol and applying Poly(vinylpyrrolidone)-Iodine with a sterile cotton swab.</li>
	<li>Inject 100 &#181;L (1 x 106 live cells from frozen primary tumor suspensions) bevel-side up into uncleared right 4th mammary gland of the 6 to 10 week-old female FVB/n mouse using a 29 G 0.3 mL insulin syringe.</li>
</ol>
3. Surgical Resection of Orthotopic Mammary Tumor
<ol>
	<li>38-60 days following tumor cell injection, euthanize mice not exhibiting orthotopic tumor volumes ranging between 172 to 450 mm3 in volume [length x (width2)/2] (about 75% of injected mice).</li>
	<li>Place anesthetized mice displaying proper tumor size ventral side up under continuous sedation by administering 2% isoflurane via an anesthesia mask and confirm the mouse is properly anesthetized with an absent foot pinch reflex. Apply vet ointment to the eyes for prevention of dryness while under sedation.</li>
	<li>Spray 70% ethanol to sterilize the surgical area surrounding the primary tumor, then apply Poly(vinylpyrrolidone)-Iodine with a sterile cotton swab. 
	NOTE: Hair removal at the surgical site resulted in skin excoriations and/or infections for ~5% mice and was therefore excluded from this step (data not shown).</li>
	<li>As shown in Figure 1C, make an initial skin incision using blunt scissors medial-caudal to the tumor.</li>
	<li>Next, make a superior excision of the skin (Figure 1C) medial to the tumor. Pay attention to the need to cauterize any vasculature feeding the tumor located on the skin before extending the incision.</li>
	<li>Continue the skin incision laterally (posterior to the tumor), then make a superior skin excision (lateral to tumor), and medial excision (superior to tumor) (Figure 1C).</li>
	<li>After the skin has been excised circumferentially around the tumor (Figure 1C), lift overlying skin attached to the tumor using forceps while blunt dissecting the tumor away from the abdominal wall musculature and keeping the mammary glands intact.</li>
	<li>Identify (by blunt dissection) and cauterize large vessels running through the 4th and 5th mammary glands.</li>
	<li>Excise approximately half of the 4th and 5th mammary glands at the cauterization site to free the tumor, overlying skin, and segments of the mammary glands (Figure 1C).</li>
	<li>In the event of bleeding, identify actively bleeding vessels and immediately cauterize them. If more than 250 &#181;L of blood is lost, exclude the mouse from study and euthanize it.</li>
	<li>Close excision sites with wound clips using a wound clip applier (Figure 1C). 
	NOTE: Remove the wound clips 10 days post-surgery with a wound clip remover.</li>
	<li>Administer warm sterile saline subcutaneously (4% of the animal&#39;s body weight) and keep the animal warm with a heat lamp. Check the animals every 5-10 min during recovery from anesthesia. 
	NOTE: Administration of either bupivacaine or lidocaine increased post-operative mortality (data not shown). Mice that showed signs of pain (hunching, unwilling to move, failure to groom) 1 h after surgery were euthanized.
	<ol>
		<li>Once the mouse has fully recovered, return it to the company of other mice.</li>
	</ol>
	</li>
</ol>
4. Isolation and Processing of Blood and Lung for Flow Cytometry and Histology
<ol>
	<li>At the study endpoints, prepare mice for various histopathologic assessments if desired. 90 min before euthanasia, give each mouse an intraperitoneal injection of bromodeoxyuridine (BrdU, 50 &#181;g/g mouse weight) at a concentration of 6.25 &#181;g/&#181;L in 1x PBS. 
	NOTE: Frozen stocks of dissolved bromodeoxyuridine are used within 1 month after preparation.</li>
	<li>10 min before euthanasia, with the mouse anesthetized, collect retroorbital blood (&#62;500 &#181;L) using heparinized capillary tubes, and subsequently transfer to tubes coated with dipotassium EDTA held on ice.</li>
	<li>To remove lungs and remaining mammary tissue, make a midline incision with scissors from the lower abdomen to the mouth to expose the thoracic and peritoneal cavities (Figure 2A), and peel the skin laterally to also expose the remaining right 4th and 5th mammary glands.</li>
	<li>Excise the remaining mammary gland tissue and examine it to rule out primary tumor regrowth by assessing serial sectioned formalin-fixed paraffin-embedded (FFPE) tissue by hematoxylin and eosin (H&#38;E) staining (Supplementary Figure 1).</li>
	<li>To remove lungs, open the abdominal wall to the diaphragm with scissors, then euthanize the animal by cutting the abdominal aorta to drain blood prior to perfusion of lungs (Figure 2B-2C).</li>
	<li>Cut the diaphragm along the rib cage from an abdominal approach making sure to avoid the lung and heart. Then, expose the thorax by cutting through the lateral sides of the rib cage (Figure 2D).</li>
	<li>Using a 23-gauge needle on a 20 mL syringe, perfuse lungs with ~5.0 mL DPBS (~10 mL/min) through the right ventricle of the heart until the lungs turn entirely white (Figure 2E-2F). Immediately cut off the heart from the main vessels so blood does not re-perfuse into the lung.</li>
	<li>For lung tissue to be fixed and processed for histopathologic assessments, inject ~1.0 mL 10% formalin at 4 &#176;C into the exposed trachea bevel side up toward the lungs using a 23-gauge needle (Figure 2G-2H). Cease injection once lungs are completely expanded and filled with fixative (Figure 2I).</li>
	<li>Excise lung lobes from trachea, and emersion fix lung tissue in neutral-buffered formalin for subsequent paraffin embedding or OCT-freezing medium, per standard histopathologic procedures.</li>
	<li>Quantitatively evaluate metastatic burden in lungs by serial sectioning of FFPE lung tissue and evaluate microtome sections (100 &#181;m, thirteen sections), by H&#38;E staining (Figure 1B).</li>
</ol>

<ol>
	<li>Fantozzi, A., Christofori, G. <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+of+breast+cancer+metastasis.">Mouse models of breast cancer metastasis.</a> Breast Cancer Res. 8, 212 (2006).</li><li>Howlader, N. N. A., Krapcho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SEER Cancer Statistics Review 1975-2008. , (2011).</li><li>National Comprehensive Cancer Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Breast Cancer (Version 3.2014). , (2016).</li><li>Kataja, V., Castiglione, M., Group, E. G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locally+recurrent+or+metastatic+breast+cancer:+ESMO+clinical+recommendations+for+diagnosis,+treatment+and+follow-up.">Locally recurrent or metastatic breast cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up.</a> Ann Oncol. 19 Suppl 2, 11-13 (2008).</li><li>Margolese, R. G., Hortobagyi, G. N., Buchholz, T. A., Kufe, D. W., Pollock, R. E., Weichselbaum, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Management of Metastatic Breast Cancer. Holland-Frei Cancer Medicine. , (2003).</li><li>Guy, C. T., Cardiff, R. D., Muller, W. J. <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+mammary+tumors+by+expression+of+polyomavirus+middle+T+oncogene:+a+transgenic+mouse+model+for+metastatic+disease.">Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease.</a> Mol Cell Biol. 12, 954-961 (1992).</li><li>Kouros-Mehr, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA-3+links+tumor+differentiation+and+dissemination+in+a+luminal+breast+cancer+model.">GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model.</a> Cancer Cell. 13, 141-152 (2008).</li><li>Minn, A. 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=Genes+that+mediate+breast+cancer+metastasis+to+the+lung.">Genes that mediate breast cancer metastasis to the lung.</a> Nature. 436, 518-524 (2005).</li><li>Qian, B. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLT1+signalling+in+metastasis-associated+macrophages+activates+an+inflammatory+signature+that+promotes+breast+cancer+metastasis.">FLT1 signalling in metastasis-associated macrophages activates an inflammatory signature that promotes breast cancer metastasis.</a> J Exp Med. 212 (9), 1433-1448 (2015).</li><li>Verkooijen, H. 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=Patients'+refusal+of+surgery+strongly+impairs+cancer+survival.">Patients' refusal of surgery strongly impairs cancer survival.</a> Ann Surg. 242 (2), 276-280 (2005).</li><li>Peinado, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-metastatic+niches:+organ-specific+homes+for+metastasis.">Pre-metastatic niches: organ-specific homes for metastasis.</a> Nat Rev Cancer. 17 (5), 302-317 (2017).</li></ol>

The authors have no disclosures of conflict with data presented herein.

Modifications and troubleshooting:
When blunt dissecting tumor away from the abdominal wall, the tumor may remain adherent to the abdominal wall. This was observed in &#60;5% of mice injected with tumor (data not shown). For mice with tumors adherent to the abdominal wall, the mouse should be euthanized as resection is difficult without primary tumor regrowth.
Limitations of model/technique:
Whereas other...

Women in North America have a ~12% lifetime risk of developing breast cancer2; a majority of these individuals will have primary tumors removed via surgery, and depending on cancer subtype, will then receive targeted, endocrine, chemo- and/or radiation therapy in the adjuvant setting3. Examples include, women diagnosed with hormone receptor-positive cancers receiving anti-estrogen therapies and women with HER2-positive tumors receiving HER2-targeted therapies with radiation/chemotherapy, whereas no targeted therapies are yet available for triple negative tumors3. Despite advances in radiation, chemotherapy, personalized and hormone-based therapies that supplement surgical resection, disease recurs in 30-70% of women diagnosed with stage II or III disease4, as therapies are largely ineffective in eradicating metastatic disease in distant organs, including lung, bone, brain and/or liver5. This is especially significant given that when metastatic disease occurs in the absence of primary tumor regrowth, this implies that disseminated malignant cells were likely already present in secondary organs at the time of definitive surgery. Thus therapies able to eradicate or slow growth of metastatic tumors are urgently needed.
While de novo mouse models of mammary carcinogenesis have been remarkably informative in revealing mechanisms regulating neoplastic progression1, existing models also have several limitations. One of these is the fact that de novo transgenic models typically develop primary tumors in multiple mammary glands, wherein primary tumor burden limits duration of studies. While primary tumor cell escape and metastatic seeding likely occur early in neoplastic progression in these models, frank development of metastatic tumors occurs late, and depending on the mouse model and strain background, is often partially penetrant1. This further limits the utility of de novo models for discovery of molecules regulating metastasis in secondary organs, and for evaluating preclinical efficacy of therapeutics in the adjuvant setting.
To circumvent these limitations, we developed a de novo autochthonous model of mammary carcinoma metastasis to lungs. Parental transgenic females (i.e., MMTV-PyMT on the FVB/n strain background for studies described herein) bearing late-stage de novo mammary tumors are aged to ~100 days6, at which point their primary tumors are surgically resected and enzymatically dissociated into single cell suspensions. Suspensions (1 x 106 cells) are in turn orthotopically explanted into 6-7-week-old recipient syngeneic female mice, where single primary mammary tumors develop over a 38 to 60 day period (Figure 1A). At a defined tumor size (172 to 450 mm3), recipient mice are anesthetized and primary tumors are surgically resected such that tumor regrowth at the surgical site is minimized, consistent with surgery in women (Supplementary Figure 1). On the FVB/n strain background, mice develop histologically-detectable metastatic foci in lungs with 45% penetrance by ~115 days post-surgery (Figure 1B). With this extended latency of metastatic tumor growth, the model is uniquely positioned for adjuvant therapy delivery, and for elucidating and evaluating underlying biology influencing metastatic progression following surgical removal of primary tumors.

<table border="0" cellpadding="0" cellspacing="0" width="718">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:292px;">Isofluorane</td>
			<td style="width:140px;">Piramal Healthcare</td>
			<td style="width:119px;">N/A</td>
			<td style="width:167px;">Prescription order</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase A</td>
			<td>Roche</td>
			<td>11088793001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>ThermoFisher</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 mL Pyrex bottle</td>
			<td>Sigma-Aldrich</td>
			<td>CLS139525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Atlanta Bio&#160;</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.7 &#181;m nylon strainer&#160;</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tube&#160;</td>
			<td>VWR</td>
			<td>89039-658</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Growth factor-reduced Matrigel&#160;</td>
			<td>BD</td>
			<td>354230</td>
			<td>Growth factor-reduced solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly(vinylpyrrolidone)&#8211;Iodine complex</td>
			<td>Sigma-Aldrich</td>
			<td>PVP1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">29 gauge 0.3 mL insulin syringe</td>
			<td>BD</td>
			<td>324702</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small Vessel Cauterizer Kit</td>
			<td>FST</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wound clips</td>
			<td>Texas Scientific</td>
			<td>205016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoClip wound clip applier&#160;</td>
			<td>BD</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AutoClip wound clip remover&#160;</td>
			<td>BD</td>
			<td>427637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromodeoxyuridine</td>
			<td>Roche</td>
			<td>10280879</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparinized capillary tubes&#160;</td>
			<td>Fisher</td>
			<td>22362566</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtainer tubes with dipotassium EDTA&#160;</td>
			<td>BD</td>
			<td>365974</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 mL syringe&#160;</td>
			<td>BD</td>
			<td>309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS</td>
			<td>Thermo-Fisher</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT-freezing medium&#160;</td>
			<td>VWR</td>
			<td>25608930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formalin, Buffered, 10% (Phosphate Buffer)</td>
			<td>Fisher</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23g needle</td>
			<td>Fisher</td>
			<td>14-826-6B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FVB/n mouse</td>
			<td>Jackson Laboratories</td>
			<td>001800</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Greater than 75% of recipient mice receiving 1 x 106 cells from primary mammary tumors derived from MMTV-PyMT mice, develop single mammary adenocarcinomas ranging in size from 172 to 450 mm3 within 38-60 days (data not shown). Mice eligible for randomization are then enrolled into study groups following surgical resection of primary tumors as shown (Figure 1C). Primary tumor regrowth was identified in less than 2% of mice that underwent ...

Watch this Scientific Journal Video about Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis at JoVE.com

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.

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 ...

surgical-procedures-methodology-for-preclinical-murine-model-de-novo

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Cancer Research

13.7K Views.  Oregon Health & Science University. 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.

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

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of acute leukemia. However, the nature of this disease has not been well documented in the literature. Here, we provide a technical protocol for characterizing C1498 cells in vitro and for determining the nature of the induced leukemia in vivo. The first part of this procedure is focused on determining the hematopoietic lineage and the stage of differentiation of cultured C1498 cells. To achieve this, multi-parametric flow cytometric staining is used to detect hematopoietic cell markers. Immunofluorescence microscopy, cytochemistry and a May-Gr&#252;nwald Giemsa staining are then performed to assess the expression of myeloperoxidase, the activity of esterases and cellular morphology, respectively. The second part of this protocol is dedicated to describing the leukemia disease that is induced in vivo. The latter can be achieved by determining the frequencies of leukemic and inherent cells in the blood, hematopoietic organs (e.g., bone marrow and spleen) and non-lymphoid tissues (e.g., the liver and lungs) using specific staining and flow cytometry analyses. The nature of the leukemia is then confirmed using May-Gr&#252;nwald Giemsa staining and staining for specific esterases in the bone marrow. Here, we present the results that were obtained using this protocol in age-matched C1498- and PBS-injected mice.

This manuscript provides a technical procedure that can be used to characterize C1498 cell cultures in vitro and the acute leukemia induced in mice after their injection. Phenotypic and functional analyses are performed using flow cytometry, immunofluorescence microscopy, cytochemistry and May-Gr&#252;nwald Giemsa staining.

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Operating Procedures of the Electrochemotherapy for Treatment of Tumor in Dogs and Cats

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane permeabilization by means of &#34;electroporation&#34;. To achieve the &#34;electroporation&#34; of the cells, electric pulses are generated by a generator and delivered to the target tissue by the use of electrodes. Electroporation is a physical method which is used to introduce molecules, like cytostatic drugs, into the cells that could not pass the cell membrane on their own. In electrochemotherapy, currently, cisplatin and bleomycin are clinically used. Electrochemotherapy antitumor effectiveness is high, for example up to 100% complete response of canine mast cell tumors smaller than 2 cm3 was achieved. Additionally, electrochemotherapy can be used for the treatment of inoperable tumors. One of the important characteristics of electrochemotherapy is that it can be effective as a one-time treatment only. However, in the case of failure or partial tumor response it can be repeated several times with equal or improved effectiveness. Electrochemotherapy is already a standard treatment for cutaneous and subcutaneous tumors of various histologies in human and veterinary oncology. Furthermore, several clinical studies exploiting electrochemotherapy for deep-seated tumors are on-going.

Electrochemotherapy is the local treatment of solid tumors. The main mechanism is electroporation-mediated permeabilization of the tumor cells' membrane which enables increased internalization of cytostatics like bleomycin and cisplatin. Thus, antitumor effectiveness is potentiated at the site of the application of electric pulses to the tumors.

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane ...

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 ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...