Name: surgical procedures and methodology for a preclinical murine model of de novo mammary cancer metastasis
Uploaded: 2017-07-29T13:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Surgical Procedures and Methodology for a Preclinical Murine Model of De Novo Mammary Cancer Metastasis at JoVE.com

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><tbody><tr><td>Isofluorane</td><td>Piramal Healthcare</td><td>N/A</td><td>Prescription order</td></tr><tr><td>Collagenase A</td><td>Roche</td><td>11088793001</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>10104159001</td><td></td></tr><tr><td>DMEM</td><td>ThermoFisher</td><td>12634010</td><td></td></tr><tr><td>25 mL Pyrex bottle</td><td>Sigma-Aldrich</td><td>CLS139525</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Bio&amp;nbsp;</td><td>S11150</td><td></td></tr><tr><td>0.7 &amp;micro;m nylon strainer&amp;nbsp;</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>50 mL conical tube&amp;nbsp;</td><td>VWR</td><td>89039-658</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>Growth factor-reduced Matrigel&amp;nbsp;</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><td>Poly(vinylpyrrolidone)&amp;ndash;Iodine complex</td><td>Sigma-Aldrich</td><td>PVP1</td><td></td></tr><tr><td>29 gauge 0.3 mL insulin syringe</td><td>BD</td><td>324702</td><td></td></tr><tr><td>Small Vessel Cauterizer Kit</td><td>FST</td><td>18000-00</td><td></td></tr><tr><td>Wound clips</td><td>Texas Scientific</td><td>205016</td><td></td></tr><tr><td>AutoClip wound clip applier&amp;nbsp;</td><td>BD</td><td>427630</td><td></td></tr><tr><td>AutoClip wound clip remover&amp;nbsp;</td><td>BD</td><td>427637</td><td></td></tr><tr><td>Bromodeoxyuridine</td><td>Roche</td><td>10280879</td><td></td></tr><tr><td>Heparinized capillary tubes&amp;nbsp;</td><td>Fisher</td><td>22362566</td><td></td></tr><tr><td>Microtainer tubes with dipotassium EDTA&amp;nbsp;</td><td>BD</td><td>365974</td><td></td></tr><tr><td>20 mL syringe&amp;nbsp;</td><td>BD</td><td>309661</td><td></td></tr><tr><td>DPBS</td><td>Thermo-Fisher</td><td>14190-250</td><td></td></tr><tr><td>OCT-freezing medium&amp;nbsp;</td><td>VWR</td><td>25608930</td><td></td></tr><tr><td>Formalin, Buffered, 10% (Phosphate Buffer)</td><td>Fisher</td><td>SF100-4</td><td></td></tr><tr><td>23g needle</td><td>Fisher</td><td>14-826-6B</td><td></td></tr><tr><td>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 ...

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13.8K 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 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 ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

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. ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

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

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

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

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

Medicine

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, ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

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 ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

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

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

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

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

A Preclinical Murine Model of Hepatic Metastases

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy