Name: testing the vascular invasive ability of cancer cells in zebrafish (danio rerio)
Uploaded: 2016-11-03T15:00:00
Description: Watch this Scientific Journal Video about Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio at JoVE.com

We thank Peter Johnson of the Georgetown University Microscopy Core for assistance with imaging the zebrafish embryos. The Microscopy &amp; Imaging Shared Resource and the Zebrafish Shared Resource are partially supported by NIH/NCI grant P30-CA051008. This work was also supported by NIH/NCI CA71508 (AW) and CA177466 (AW).

Ethics Statement: Zebrafish embryos were generated according to an approved IACUC protocol. These experiments were carried out in compliance with recommendations by the Georgetown University Animal Care and Use Committee.
1. Organize Embryos for Injection and Create Stock Solutions
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	<li>Generate requisite zebrafish larvae to assess cancer cell vascular invasion.
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		<li>Set up pair-wise or group in-cross mating with Tg(kdrl:grcfp)zn1;mitfab692;ednrb1...

<ol>
	<li>Cummings, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+progression+of+breast+cancer:+insights+from+50+years+of+autopsies.">Metastatic progression of breast cancer: insights from 50 years of autopsies.</a> Am J Path. 232, 23-31 (2014).</li><li>Nguyen, D. X., Bos, P. D., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+from+dissemination+to+organ-specific+colonization.">Metastasis: from dissemination to organ-specific colonization.</a> Nat Rev Cancer. 9 (4), 274-284 (2009).</li><li>Reymond, N., d'&#193;gua, B. B., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crossing+the+endothelial+barrier+during+metastasis.">Crossing the endothelial barrier during metastasis.</a> Nat Rev Cancer. 13 (12), 858-870 (2013).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nat Med. 19 (11), 1423-1437 (2013).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Method Enzymol. 409, 625-642 (2006).</li><li>Rahim, S., &#220;ren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+real-time+electrical+impedance+based+technique+to+measure+invasion+of+endothelial+cell+monolayer+by+cancer+cells.">A real-time electrical impedance based technique to measure invasion of endothelial cell monolayer by cancer cells.</a> J Vis Exp. (50), (2011).</li><li>Jeon, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+3D+vascularized+organotypic+microfluidic+assays+to+study+breast+cancer+cell+extravasation.">Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation.</a> Proc Natl Acad Sci USA. 112 (1), 214-219 (2015).</li><li>Shin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+assay+for+simultaneous+culture+of+multiple+cell+types+on+surfaces+or+within+hydrogels.">Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.</a> Nat Protoc. 7 (7), 1247-1259 (2012).</li><li>Vargo-Gogola, T., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer:+one+size+does+not+fit+all.">Modelling breast cancer: one size does not fit all.</a> Nat Rev Cancer. 7 (9), 659-672 (2007).</li><li>Mohanty, S., Xu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+metastasis+assay.">Experimental metastasis assay.</a> J Vis Exp. (42), (2010).</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+lung.">Genes that mediate breast cancer metastasis to lung.</a> Nature. 436 (7050), 518-524 (2005).</li><li>Campbell, J. P., Merkel, A. R., Masood-Campbell, S. K., Elefteriou, F., Sterling, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+bone+metastasis.">Models of bone metastasis.</a> J Vis Exp. (64), e4260 (2012).</li><li>Arguello, F., Baggs, R. B., Frantz, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+experimental+metastasis+to+bone+and+bone+marrow.">A murine model of experimental metastasis to bone and bone marrow.</a> Cancer Res. 48 (23), 6876-6881 (1988).</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=Distinct+organ-specific+metastatic+potential+of+individual+breast+cancer+cells+and+primary+tumors.">Distinct organ-specific metastatic potential of individual breast cancer cells and primary tumors.</a> J Clin Invest. 115 (1), 44-55 (2005).</li><li>Bos, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+that+mediate+breast+cancer+metastasis+to+the+brain.">Genes that mediate breast cancer metastasis to the brain.</a> Nature. 459 (7249), 1005-1009 (2009).</li><li>Soares, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preclinical+murine+model+of+hepatic+metastases.">A preclinical murine model of hepatic metastases.</a> J Vis Exp. (91), e51677 (2014).</li><li>Kienast, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+imaging+reveals+the+single+steps+of+brain+metastasis+formation.">Real-time imaging reveals the single steps of brain metastasis formation.</a> Nature Med. 16 (1), 116-122 (2010).</li><li>Hasegawa, H., Ushio, Y., Hayakawa, T., Yamada, K., Mogami, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+of+the+blood-brain+barrier+in+experimental+metastatic+brain+tumors.">Changes of the blood-brain barrier in experimental metastatic brain tumors.</a> J Neurosurg. 59 (2), 304-310 (1983).</li><li>Amatruda, J. F., Shepard, J. L., Stern, H. M., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+cancer+model+system.">Zebrafish as a cancer model system.</a> Cancer Cell. 1 (3), 229-231 (2002).</li><li>Feitsma, H., Cuppen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+cancer+model.">Zebrafish as a cancer model.</a> Molecular Cancer Res. 6 (5), 685-694 (2008).</li><li>Stoletov, K., Klemke, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catch+of+the+day:+zebrafish+as+a+human+cancer+model.">Catch of the day: zebrafish as a human cancer model.</a> Oncogene. 27 (33), 4509-4520 (2008).</li><li>Stoletov, K., 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=Visualizing+extravasation+dynamics+of+metastatic+tumor+cells.">Visualizing extravasation dynamics of metastatic tumor cells.</a> J Cell Sci. 123 (13), 2332-2341 (2010).</li><li>Kanada, M., Zhang, J., Yan, L., Sakurai, T., Terakawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell-initiated+extravasation+of+cancer+cells+visualized+in+zebrafish.">Endothelial cell-initiated extravasation of cancer cells visualized in zebrafish.</a> PeerJ. , (2014).</li><li>Teng, Y., Xie, X., Walker, S., White, D. T., Mumm, J. S., Cowell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+human+cancer+cell+metastasis+in+zebrafish.">Evaluating human cancer cell metastasis in zebrafish.</a> BMC Cancer. 13 (453), (2013).</li><li>Cross, L. M., Cook, M. A., Lin, S., Chen, J. -. N., Rubinstein, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+angiogenesis+drugs+in+a+live+fluorescent+zebrafish+assay.">Rapid analysis of angiogenesis drugs in a live fluorescent zebrafish assay.</a> Arterioscl Throm Vas. 23 (5), 911-912 (2003).</li><li>Sharif, G. 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=Cell+growth+density+modulates+cancer+cell+vascular+invasion+via+Hippo+pathway+activity+and+CXCR2+signaling.">Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling.</a> Oncogene. 34, 5879-5889 (2015).</li><li>Novoa, B., Figueras, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+model+for+the+study+of+inflammation+and+the+innate+immune+response+to+infectious+diseases.">Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases.</a> Adv Exp Med Biol. 946, 253-275 (2012).</li><li>Kitamura, T., Qian, B. -. Z., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cell+promotion+of+metastasis.">Immune cell promotion of metastasis.</a> Nature Rev Immunol. 15 (2), 73-86 (2015).</li><li>He, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil-mediated+experimental+metastasis+is+enhanced+by+VEGFR+inhibition+in+a+zebrafish+xenograft+model.">Neutrophil-mediated experimental metastasis is enhanced by VEGFR inhibition in a zebrafish xenograft model.</a> J Pathol. 227 (4), 431-445 (2012).</li><li>Tulotta, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+signaling+between+human+CXCR4+and+zebrafish+ligands+by+the+small+molecule+IT1t+impairs+the+formation+of+triple-negative+breast+cancer+early+metastases+in+a+zebrafish+xenograft+model.">Inhibition of signaling between human CXCR4 and zebrafish ligands by the small molecule IT1t impairs the formation of triple-negative breast cancer early metastases in a zebrafish xenograft model.</a> Dis Model Mech. 9 (2), 141-153 (2016).</li><li>Renshaw, S. A., Loynes, C. A., Trushell, D. M. I., Elworthy, S., Ingham, P. W., Whyte, M. K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+zebrafish+model+of+neutrophilic+inflammation.">A transgenic zebrafish model of neutrophilic inflammation.</a> Blood. 108 (13), 3976-3978 (2006).</li><li>Berens, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratin-associated+protein+5-5+controls+cytoskeletal+function+and+cancer+cell+vascular+invasion.">Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion.</a> Oncogene. , (2016).</li></ol>

This technique utilizes the zebrafish model to efficiently test the vascular invasive ability of cancer cells (see Figure 1). Here we applied the technique to a panel of breast cancer cell lines in order to provide a baseline onto which other investigators can then build their own studies (see Table 1; Figures 2 - 3). The observation that MDA-MB-231 cells readily invaded into the caudal region of zebrafish embryos would make this cell line ideal for testing agents that m...

Metastatic disease is a major cause of cancer mortality and many mechanisms that enable cancer cell dissemination remain to be discovered1. In order for a cancer cell to successfully metastasize, it must first invade through the stroma that surrounds a primary tumor, enter (intravasate) into the circulatory system, survive in transit, exit (extravasate) from the circulation, and lastly establish a viable colony at the distant organ site2. Intravasation and extravasation are thus crucial steps in the metastatic cascade, yet every cancer cell is not inherently adept at disrupting and migrating through endothelial junctions3. In fact, there are a series of unique selection pressures that surround cancer cell vascular invasion and the process can be further influenced by a variety of endogenous and exogenous factors4. For these reasons, techniques that probe the aggressive behavior of advanced stage cancer often focus on vascular invasive ability as a means to predict metastatic spread.
Various model systems exist to facilitate the study of cancer cell vascular invasion in vitro. The most used in vitro assays involve either transwell systems to assess cancer cell migration through an endothelial barrier5 or Electric Cell-Substrate Impedance Sensing (ECIS) technology to monitor the real-time disruption of an intact endothelial monolayer by cancer cells6. These assays typically lack the fluid dynamics and stromal factors that would otherwise impact cancer cell attachment to an endothelial wall. This issue is somewhat circumvented by perfusable vascular networks that arise from the 3D culture of endothelia with supporting stromal cells, and these 3D microfluidic systems now represent the forefront of current in vitro options7,8. Still, these approaches omit the robust microenvironment of a functional circulatory system and therefore only in part substitute for in vivo models.
The most widely used in vivo model of vascular invasion is the mouse, in which experimental metastasis assays are commonly performed because they occur on relatively short timescales and are generally indicative of metastatic ability9. These assays involve direct injection of cancer cells into circulation and therefore model the end stages of metastasis, namely extravasation and cancer cell colonization of organs. The experimental metastasis assays differ based on the site of cancer cell injection and the organs ultimately analyzed. In the first assay type, cancer cells are injected into the tail vein of mice and cancer cell seeding in the lungs is monitored10,11. The second assay involves performing intracardiac injections to direct metastatic seeding toward the bone microenvironment12-14, but also the brain15. In the third assay, cancer cells are injected into the spleen in order to permit colonization of the liver16 whereas the fourth delivery route into the carotid artery carries cancer cells to the brain17,18. Irrespective of the cancer cell delivery method, organ colonization is the accepted experimental endpoint and is generally determined via luminescence, histology, or PCR-based techniques. Despite the physiologic advantages of conducting experimental metastasis assays within a murine host, these experiments still require weeks to months to complete and analyze.
The zebrafish (Danio rerio) model has recently emerged as a new system to study cancer progression19,20, and allows for the assessment of cancer cell vascular invasion within a functional circulatory system over a much shorter timescale when compared with mice21-24. The method utilizes a transparent zebrafish strain that has its endothelia tagged with a green reef coral fluorescent protein reporter driven by the kdrl promoter, the zebrafish receptor for vascular endothelial growth factor25. In the assay, cancer cells are labeled with a red fluorescent marker and injected into the precardiac sinus of 2-day old embryos. Anywhere between 48 to 96 hr after the injection, cancer cells that have invaded out of the vasculature and into the caudal region of embryos can be scored efficiently on a fluorescent microscope. Here we apply the technique to a panel of commonly used human breast cancer cell lines to demonstrate stark differences in their vascular invasive ability. Furthermore, we demonstrate that changing the injection site to the embryo yolk sac allows for the study of heterogeneous cell interactions, as cancer cell populations can be differentially labeled with fluorescent dyes and injected into zebrafish embryos lacking fluorescent vasculature. In this latter assay, cancer cells that have invaded the yolk and intravasated into the vasculature are scored in the caudal region 24 to 48 hr after injection. Due to the effectiveness and convenience of this model, zebrafish are increasingly employed to rapidly test the vascular invasive ability of cancer cells under a physiologic setting.

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			<td height="21" style="height:21px;width:491px;">0.05% Trypsin-EDTA</td>
			<td style="width:316px;">Life Technologies</td>
			<td style="width:321px;">25300-054</td>
			<td style="width:125px;"></td>
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			<td height="21" style="height:21px;">100mm Dishes</td>
			<td>Corning Incorporated</td>
			<td>3160-100</td>
			<td></td>
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			<td height="21" style="height:21px;">5 3/4&#34; Disposable Pastur Pipets, borosilicate Glass</td>
			<td>Fisher Brand</td>
			<td>13-678-20B</td>
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			<td height="21" style="height:21px;">60 mm Dish</td>
			<td>Corning Incorporated</td>
			<td>3160-60</td>
			<td></td>
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			<td height="21" style="height:21px;">Agarose, Low Melting</td>
			<td>Fisher</td>
			<td>BP165-25</td>
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			<td height="21" style="height:21px;">Agarose, Molecular Grade</td>
			<td>Bioline</td>
			<td>BIO-41026</td>
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			<td height="21" style="height:21px;">Capillary Glass, Standard, 1.2MM x 0.68MM, 4&#34;</td>
			<td>A-M Systems, Inc</td>
			<td>627000</td>
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			<td height="21" style="height:21px;">David Kopf 700C Vertical Pipette Puller</td>
			<td>Hofstra Group</td>
			<td>3600</td>
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			<td>11995-065</td>
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			<td height="21" style="height:21px;">Electrode Storage Jar, 1.0MM</td>
			<td>World Precision Instruments, Inc</td>
			<td>E210</td>
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			<td height="21" style="height:21px;">Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine, MS-222)</td>
			<td>Fluka</td>
			<td>A5040</td>
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			<td>113</td>
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			<td height="21" style="height:21px;">Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
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			<td height="21" style="height:21px;">Fisherbrand Transfer Pipettes</td>
			<td>ThermoFisher Scientific</td>
			<td>13-711-7M</td>
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			<td height="21" style="height:21px;">Gel Loading Pipet Tips</td>
			<td>Fisher Brand</td>
			<td>02-707-181</td>
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			<td height="21" style="height:21px;">Glass Bottom Dishes (12.0 mm)</td>
			<td>ThermoFisher Scientific</td>
			<td>150680</td>
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			<td height="21" style="height:21px;">Glass Depression Slide</td>
			<td>VWR</td>
			<td>470005-634</td>
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			<td height="21" style="height:21px;">Instant Ocean Salt, Sea Salt</td>
			<td>Pentair</td>
			<td>IS50</td>
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			<td height="21" style="height:21px;">Latex Rubber Bulbs, 2mL, Pack of 72</td>
			<td>Heathrow Scientific</td>
			<td>HS20622B</td>
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			<td height="21" style="height:21px;">SP8 Confocal Microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Narishige</td>
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			<td height="21" style="height:21px;">Eclipse E600</td>
			<td>Nikon</td>
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			<td height="21" style="height:21px;">PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
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			<td height="21" style="height:21px;">Penicillin-G Potassium</td>
			<td>Fisher Biotech</td>
			<td>BP914-100</td>
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			<td height="21" style="height:21px;">Petri Plates, 100mm x 15mm</td>
			<td>Fisher Brand</td>
			<td>&#160;FB0875713</td>
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			<td height="21" style="height:21px;">Picospritzer II</td>
			<td>General Valve Corporation</td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
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			<td height="21" style="height:21px;">Streptomycin Sulfate</td>
			<td>Fisher Biotech</td>
			<td>BP910-50</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiI</td>
			<td>ThermoFisher Scientific</td>
			<td>V22885</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiO</td>
			<td>ThermoFisher Scientific</td>
			<td>V22886</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Zebrafish&#160;</td>
			<td>Georgetown Zebrafish Shared Resources</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and HCC18-6 cells, which were mantained in RPMI + 10% FBS.</td>
			<td></td>
		</tr>
	</tbody>
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testing the vascular invasive ability of cancer cells in zebrafish (danio rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Here we tested the vascular invasive ability of commonly used breast cancer cell lines in a zebrafish embryo model (Figure 1). Rigorous criteria were employed in scoring extravasation for these different cell lines, where positive events were only counted if the cancer cells had clearly extravasated, this being done chiefly to limit any false-positives that could arise from scoring cellular debris.
Our analysis ...

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Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

Research

JoVE Journal

Cancer Research

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