Name: surgical procedures and methodology for a preclinical murine model of de novo mammary cancer metastasis
Uploaded: 2017-07-29T13:00:00
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 su...

<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>

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.

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			<td>BD</td>
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			<td>Jackson Laboratories</td>
			<td>001800</td>
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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 (Video) | JoVE

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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