We thank Mr. Alhad Mahagaonkar for managing the University of Auckland zebrafish facility and the Biomedical Imaging Research Unit, School of Medical Sciences, University of Auckland, for assistance in time-lapse confocal microscopy. This work was supported by a Health Research Council of New Zealand project grant (14/105), a Royal Society of New Zealand Marsden Fund Project Grant (UOA1602) and an Auckland Medical Research Foundation Project Grant (1116012) awarded to J.W.A.

1. Preparation of Microinjection Needles
<ol>
	<li>Turn on a micropipette puller and set the following parameters (calibrated for the micropipette puller model listed in Table of Materials): Heat, 680; Pull, 75; Velocity, 40; Time, 55; Pressure: 530.</li>
	<li>Secure a borosilicate glass capillary into the micropipette puller and pull the capillary to make two needles. Repeat for as many needles as desired.</li>
</ol>
2. Cell Culture for Implantation
<s...

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144 (5), 646-674 (2011).</li><li>Huang, 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=Sunitinib+acts+primarily+on+tumor+endothelium+rather+than+tumor+cells+to+inhibit+the+growth+of+renal+cell+carcinoma.">Sunitinib acts primarily on tumor endothelium rather than tumor cells to inhibit the growth of renal cell carcinoma.</a> Cancer Research. 70 (3), 1053-1062 (2010).</li><li>Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tumor Angiogenesis Assays. , (2016).</li><li>Nicoli, S., Ribatti, D., Cotelli, F., Presta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+tumor+xenografts+induce+neovascularization+in+zebrafish+embryos.">Mammalian tumor xenografts induce neovascularization in zebrafish embryos.</a> Cancer Research. 67 (7), 2927-2931 (2007).</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> Journal of Pathology. 227 (4), 431-445 (2012).</li><li>Ablain, J., Durand, E. M., Yang, S., Zhou, Y., 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=A+CRISPR/Cas9+vector+system+for+tissue-specific+gene+disruption+in+zebrafish.">A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish.</a> Developmental Cell. 32 (6), 756-764 (2015).</li><li>Astin, J. W., 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=Vegfd+can+compensate+for+loss+of+Vegfc+in+zebrafish+facial+lymphatic+sprouting.">Vegfd can compensate for loss of Vegfc in zebrafish facial lymphatic sprouting.</a> Development. 141 (13), 2680-2690 (2014).</li><li>Yang, J. H., Hu, J., Wan, L., Chen, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barbigerone+inhibits+tumor+angiogenesis,+growth+and+metastasis+in+melanoma.">Barbigerone inhibits tumor angiogenesis, growth and metastasis in melanoma.</a> Asian Pacific Journal of Cancer Prevention. 15 (1), 167-174 (2014).</li><li>Zhang, 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=SKLB1002,+a+novel+potent+inhibitor+of+VEGF+receptor+2+signaling,+inhibits+angiogenesis+and+tumor+growth+in+vivo.">SKLB1002, a novel potent inhibitor of VEGF receptor 2 signaling, inhibits angiogenesis and tumor growth in vivo.</a> Clinical Cancer Research. 17 (13), 4439-4450 (2011).</li><li>Muthukumarasamy, K. 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=Identification+of+noreremophilane-based+inhibitors+of+angiogenesis+using+zebrafish+assays.">Identification of noreremophilane-based inhibitors of angiogenesis using zebrafish assays.</a> Organic &amp; Biomolecular Chemistry. 14 (5), 1569-1578 (2016).</li><li>Britto, D. 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=Macrophages+enhance+Vegfa-driven+angiogenesis+in+an+embryonic+zebrafish+tumor+xenograft+model.">Macrophages enhance Vegfa-driven angiogenesis in an embryonic zebrafish tumor xenograft model.</a> Disease Models &amp; Mechanisms. 11 (12), (2018).</li><li>Nicoli, S., Presta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish/tumor+xenograft+angiogenesis+assay.">The zebrafish/tumor xenograft angiogenesis assay.</a> Nature Protocols. 2 (11), 2918-2923 (2007).</li><li>Huang, H., Zhang, B., Hartenstein, P. A., Chen, J. N., Lin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NXT2+is+required+for+embryonic+heart+development+in+zebrafish.">NXT2 is required for embryonic heart development in zebrafish.</a> BMC Developmental Biology. 5, 7 (2005).</li><li>Lawson, N. D., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+embryonic+vascular+development+using+transgenic+zebrafish.">In vivo imaging of embryonic vascular development using transgenic zebrafish.</a> Developmental Biology. 248 (2), 307-318 (2002).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> Journal of Visualized Experiments. (25), (2009).</li><li>Nusslein-Volhard, C., Dahm, 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> Zebrafish: A Practical Approach. , (2002).</li><li>Benard, E. L., 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=Infection+of+zebrafish+embryos+with+intracellular+bacterial+pathogens.">Infection of zebrafish embryos with intracellular bacterial pathogens.</a> Journal of Visualized Experiments. (61), (2012).</li><li>Fior, R., 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=Single-cell+functional+and+chemosensitive+profiling+of+combinatorial+colorectal+therapy+in+zebrafish+xenografts.">Single-cell functional and chemosensitive profiling of combinatorial colorectal therapy in zebrafish xenografts.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (39), E8234-E8243 (2017).</li><li>Eng, T. C. Y., Astin, 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=Characterization+of+Zebrafish+Facial+Lymphatics.">Characterization of Zebrafish Facial Lymphatics.</a> Methods in Molecular Biology. 1846, 71-83 (2018).</li><li>Zhao, 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+novel+xenograft+model+in+zebrafish+for+high-resolution+investigating+dynamics+of+neovascularization+in+tumors.">A novel xenograft model in zebrafish for high-resolution investigating dynamics of neovascularization in tumors.</a> PloS One. 6 (7), e21768 (2011).</li><li>Nakamura, 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=KRN951,+a+highly+potent+inhibitor+of+vascular+endothelial+growth+factor+receptor+tyrosine+kinases,+has+antitumor+activities+and+affects+functional+vascular+properties.">KRN951, a highly potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, has antitumor activities and affects functional vascular properties.</a> Cancer Research. 66 (18), 9134-9142 (2006).</li><li>Okuda, K. 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=A+zebrafish+model+of+inflammatory+lymphangiogenesis.">A zebrafish model of inflammatory lymphangiogenesis.</a> Biology Open. 4 (10), 1270-1280 (2015).</li><li>Steinberg, F., Konerding, M. A., Streffer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+architecture+of+human+xenotransplanted+tumors:+histological,+morphometrical,+and+ultrastructural+studies.">The vascular architecture of human xenotransplanted tumors: histological, morphometrical, and ultrastructural studies.</a> Journal of Cancer Research and Clinical Oncology. 116 (5), 517-524 (1990).</li><li>Vermeulen, P. 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=Microvessel+quantification+in+primary+colorectal+carcinoma:+an+immunohistochemical+study.">Microvessel quantification in primary colorectal carcinoma: an immunohistochemical study.</a> British Journal of Cancer. 71 (2), 340-343 (1995).</li><li>Heilmann, 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=A+Quantitative+System+for+Studying+Metastasis+Using+Transparent+Zebrafish.">A Quantitative System for Studying Metastasis Using Transparent Zebrafish.</a> Cancer Research. 75 (20), 4272-4282 (2015).</li><li>Okuda, K. S., Lee, H. M., Velaithan, V., Ng, M. F., Patel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilizing+Zebrafish+to+Identify+Anti-(Lymph)Angiogenic+Compounds+for+Cancer+Treatment:+Promise+and+Future+Challenges.">Utilizing Zebrafish to Identify Anti-(Lymph)Angiogenic Compounds for Cancer Treatment: Promise and Future Challenges.</a> Microcirculation. 23 (6), 389-405 (2016).</li><li>Zhao, 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=Endothelial+Cords+Promote+Tumor+Initial+Growth+prior+to+Vascular+Function+through+a+Paracrine+Mechanism.">Endothelial Cords Promote Tumor Initial Growth prior to Vascular Function through a Paracrine Mechanism.</a> Scientific Reports. 6, 19404 (2016).</li><li>Goel, S., Wong, A. H., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+normalization+as+a+therapeutic+strategy+for+malignant+and+nonmalignant+disease.">Vascular normalization as a therapeutic strategy for malignant and nonmalignant disease.</a> Cold Spring Harbor Perspectives in Medicine. 2 (3), a006486 (2012).</li><li>Schmitt, C. E., Holland, M. B., Jin, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+vascular+networks+in+zebrafish:+an+introduction+to+microangiography.">Visualizing vascular networks in zebrafish: an introduction to microangiography.</a> Methods in Molecular Biology. , 59-67 (2012).</li></ol>

The first critical step in the protocol is the implantation of tumor cells. It is essential that cells are injected into a location that will allow the xenograft to implant successfully in the embryo without making the embryo edematous. An injection that is too anterior can allow the cells to move towards the heart, blocking the bloodstream and leading to edema, while an injection that is too posterior will result in a poorly implanted xenograft. An anterior injection is best avoided by inserting the needle through the y...

Angiogenesis is one of the classic hallmarks of cancer and represents a target of anti-cancer therapy1,2. To study this process, xenograft models of cancer have been created by implanting mammalian tumor cells into animals such as mice3. A zebrafish xenograft model has also been developed, which involves the implantation of tumor cells into 2 days post fertilization (dpi) zebrafish which results in rapid growth of zebrafish blood vessels into the xenograft4.
This protocol describes an in vivo zebrafish embryo tumor xenograft model in which the angiogenic response can be accurately quantitated across the entire xenograft. This method allows the investigator to examine, in vivo, the molecular mechanisms that underpin the tumor angiogenic response. The genetic tractability of the zebrafish allows the host contribution to be interrogated, while selection of different tumor cell lines allows the tumor contribution to angiogenesis to also be examined5,6,7. In addition, as zebrafish larvae are permeable to small molecules, specific pathway inhibitors can be used or drug libraries can be screened to identify novel inhibitors of tumor angiogenesis8,9,10,11.
The zebrafish embryo xenograft model presents unique advantages compared with other mammalian xenograft models. Zebrafish xenografts are cheaper and easier to perform, large numbers of animals can be examined and live cell imaging allows detailed examination of cell behaviour4. Unlike other in vivo models, which require up to several weeks to observe significant vessel growth, angiogenesis in zebrafish xenografts can be observed within 24 h following implantation3,4. However, the lack of an adaptive immune system in embryonic zebrafish, while beneficial to maintaining the xenograft, means that the adaptive immune response and its contribution towards tumor angiogenesis cannot be examined. In addition, the lack of tumor stromal cells, the inability to orthotopically implant the tumor and the difference in maintenance temperature between zebrafish and mammalian cells are potential weaknesses of this method. Nonetheless, the amenability of this model for live imaging and the ability to accurately quantitate the angiogenic response makes it uniquely beneficial for studying the cellular processes that regulate tumor angiogenesis in vivo.

<table>
	<tbody>
		<tr>
			<td>Air cylinder</td>
			<td>BOC</td>
			<td>011G</td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>B16-F1 cells</td>
			<td>ATCC</td>
			<td></td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>BD Matrigel LDEV-free (extracellular matrix mixture)</td>
			<td>Corning</td>
			<td>356235</td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>Borosillicate glass capillaries</td>
			<td>Warner Instruments</td>
			<td>G100T-4</td>
			<td>OD=1.00 mm, ID=0.78 mm, Length =10 cm Cell injection</td>
		</tr>
		<tr>
			<td>Cell culture dish -35 mm diameter</td>
			<td>Thermofisher NZ</td>
			<td>NUN153066</td>
			<td>Fish husbandry</td>
		</tr>
		<tr>
			<td>Cell culture dish -100 mm diameter</td>
			<td>Sigma-Aldrich</td>
			<td>CLS430167-500EA</td>
			<td>Fish husbandry</td>
		</tr>
		<tr>
			<td>Cell culture flask 75 cm2</td>
			<td>In Vitro Technologies</td>
			<td>COR430641</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>CellTracker Green</td>
			<td>Invitrogen</td>
			<td>C2925</td>
			<td>Cell labelling, Stock concentration (10 mM in DMSO), working concentration (0.2 &#956;M in serum-free media)</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>Drug treatment, Cell labelling</td>
		</tr>
		<tr>
			<td>E3 Media (60x in 2 L of water) 34.8 g NaCl 
			 
			1.6 g KCl 
			 
			5.8 g CaCl2&#183;2H2O 
			 
			9.78 g MgCl2&#183;6H2O adjust to pH 7.2 with NaOH</td>
			<td>In house [1]</td>
			<td></td>
			<td>Fish husbandry</td>
		</tr>
		<tr>
			<td>Ethyl-3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>Xenotransplantation, Imaging</td>
		</tr>
		<tr>
			<td>Filter tip 1000 &#956;L</td>
			<td>VWR</td>
			<td>732-1491</td>
			<td>Used during multiple steps</td>
		</tr>
		<tr>
			<td>Filter tip 200 &#956;L</td>
			<td>VWR</td>
			<td>732-1489</td>
			<td>Used during multiple steps</td>
		</tr>
		<tr>
			<td>Filter tip 10 &#956;L</td>
			<td>VWR</td>
			<td>732-1487</td>
			<td>Used during multiple steps</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Leica</td>
			<td>MZ16FA</td>
			<td>Preparation of embryos</td>
		</tr>
		<tr>
			<td>FBS (NZ origin)</td>
			<td>Thermofisher Scientific</td>
			<td>10091148</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Any commercial brand</td>
			<td></td>
			<td>Used during multiple steps</td>
		</tr>
		<tr>
			<td>Haemocytometer cell counting chamber Improved Neubauer</td>
			<td>HawksleyVet</td>
			<td>AC1000</td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>Heraeus Multifuge X3R Centrifuge</td>
			<td>Thermofisher Scientific</td>
			<td>75004500</td>
			<td>Cell culture, Cell labelling</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermofisher Scientific</td>
			<td>62249</td>
			<td>Cell labelling, Stock concentration (1 mg/ml in DMSO), working concentration (6 &#956;g/ml in serum-free media)</td>
		</tr>
		<tr>
			<td>Low Melting Point, UltraPure Agarose</td>
			<td>Thermofisher Scientific</td>
			<td>16520050</td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Methycellulose</td>
			<td>Sigma-Aldrich</td>
			<td>9004 67 5</td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>Methylene blue</td>
			<td>sigma-Aldrich</td>
			<td>M9140</td>
			<td>Fish husbandry</td>
		</tr>
		<tr>
			<td>Microloader 0.5-20 &#956;L pipette tip for loading microcapillaries</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Any commercial brand</td>
			<td></td>
			<td>Used during multiple steps</td>
		</tr>
		<tr>
			<td>Micropipette puller P 87</td>
			<td>Sutter Instruments</td>
			<td></td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>Microscope cage incubator</td>
			<td>Okolab</td>
			<td></td>
			<td>Time-lapse imaging</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Any commercial brand</td>
			<td></td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich</td>
			<td>M3516</td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>Minimal Essential Media (MEM) - alpha</td>
			<td>Thermofisher Scientfic</td>
			<td>12561056</td>
			<td>Cell Culture</td>
		</tr>
		<tr>
			<td>MPPI-2 Pressure Injector</td>
			<td>Applied Scientific Instrumentation</td>
			<td></td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>Narishige micromanipulator</td>
			<td>Narishige Group</td>
			<td></td>
			<td>Xenotransplantation</td>
		</tr>
		<tr>
			<td>Nikon D Eclipse C1 Confocal Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Imaging</td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td>Fish husbandry</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>S1 pipet filler</td>
			<td>Thermoscientific</td>
			<td>9501</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Serological stripette 10 mL</td>
			<td>Corning</td>
			<td>4488</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Serological stripette 25 mL</td>
			<td>Corning</td>
			<td>4489</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Serological stripette 5 mL</td>
			<td>Corning</td>
			<td>4485</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Serological stripette 2 mL</td>
			<td>Corning</td>
			<td>4486</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Terumo Needle 22 gauge</td>
			<td>Amtech</td>
			<td>SH 182</td>
			<td>Fish husbandry</td>
		</tr>
		<tr>
			<td>Tissue culture incubator</td>
			<td>Thermofisher Scientfic</td>
			<td>HeraCell 150i</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Tivozanib (AV951)</td>
			<td>AVEO Pharmaceuticals</td>
			<td></td>
			<td>Drug treatment</td>
		</tr>
		<tr>
			<td>Transfer pipette 3 mL</td>
			<td>Mediray</td>
			<td>RL200C</td>
			<td>Fish husbandry</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA (0.25% )</td>
			<td>Life Technologies</td>
			<td>T4049</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Fine Science Tools</td>
			<td>11295-10</td>
			<td>Fish husbandry</td>
		</tr>
		<tr>
			<td>Volocity Software (v6.3)</td>
			<td>Improvision/Perkin Elmer</td>
			<td></td>
			<td>Image analysis</td>
		</tr>
	</tbody>
</table>

in vivo imaging and quantitation of the host angiogenic response in zebrafish tumor xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

By imaging an individual xenograft at 6, 24 and 48 hpi, the angiogenic response at different timepoints can be calculated as shown in Figure 1A-C. The largest angiogenic response is observed between 24-48 h post implantation, with the maximum levels of graft vascularization seen around 2 dpi (Figure 1A-C). A time-lapse movie of a typical angiogenic response to a B16-F1 xenograft from 20.75 hpi un...

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In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

This protocol describes how transgenic zebrafish larvae can be used to provide an in vivo quantitative assay of tumor xenograft vascularization. The main advantage of this technique over previous techniques is that it allows the investigator to accurately quantitate changes in the levels of xenograft vascularization. The key step is to ensure that the tumor cells are injected correctly into zebrafish larvae so that subsequent imaging and quantitation steps are not compromised.After growing and labeling B16-F1 cells with fluorescent dye, start preparing embryos for implantation. Fill a 35-millimeter dish with a 2%methylcellulose in E3 solution to a quarter of the volume of the dish. Using a transfer pipette, place approximately 50 previously prepared transgenic embryos onto the methylcellulose while minimizing the volume of E3 solution transferred.Using a microcapillary pipette tip, arrange the embryos so that they are all oriented vertically with their heads to the top, tails to the bottom, and the embryo with its left side up. Add 50%ECM to a previously prepared B16-F1 cell pellet to produce a mixture of cells to ECM in a two-to-one ratio. Mix well by pipetting and stirring, and store the mixture on ice.Use a microcapillary pipette to take up three to 10 microliters of cells. Carefully insert the pipette tip into a previously prepared microinjection needle, and then eject the B16-F1 matrix mixture into the end of the needle. Insert the needle into the needle holder, and tilt at a 45-degree angle to the dish.Use tweezers to break the tip of the needle to make a hole large enough for the cells to be ejected from the needle without squeezing the cells. Turn on the pressurized air cylinder attached to the injection apparatus, and then momentarily turn the injector on continuous mode for no more than one second to push the cells to the tip of the needle. Point the needle towards the yolk sac of an embryo, and push it through the yolk sac in a ventral direction until the tip of the needle has emerged from the yolk sac and is pushing the embryonic epidermis on the ventral side of the embryo, just posterior to the heart.Carefully and slightly push the tip of the needle forward until it has created a small space between the epidermis and the yolk sac membrane. Pulse the injector to eject some of the cell mixture into this perivitelline space. Carefully look at the size, shape, and location of the xenograft as it is being implanted, and adjust the pulse number and position of the needle accordingly to ensure a correctly implanted xenograft.Repeat the pulses until 500 to 800 cells have been injected into the perivitelline space, creating a visible bulge that extends at least half of the way along the bottom of the yolk sac. Remove the needle from the embryo. After injecting all the embryos, use a microcapillary pipette tip to push all the embryos together so that they can be pipetted out with as little methylcellulose as possible.Transfer the embryos into a recovery dish containing E3 with PTU and methylene blue. Then gently pipette E3 around the embryos to wash them, and then incubate at 34 degrees Celsius. Then image anesthetized embryos using a confocal microscope.Use a laser channel appropriate for the tumor cells to determine a volume to be imaged, allowing for at least one or two optical sections either side of the graft. Use section intervals that are around five micrometers apart to create a Z stack. Use two-channel imaging to image both the blood vessels and the tumor xenograft.Image the tumor xenograft and the blood vessels for one larva, and repeat these steps for each larva to be imaged. To begin with quantitation of the angiogenic response to the zebrafish xenograft, transfer previously created Z stack confocal image files of a tumor xenograft to a new folder on the 3D image analysis software. To create the Tumor Volume protocol for measuring tumor xenograft volume, go to the Measurement tab on the top menu of the window, and then from the list of protocols that appears drag the protocol named Find Objects to the space that is titled Drag Tasks Here To Make Measurements.After ensuring that this protocol is set to measure objects in the tumor channel, drag the protocols named Clip To ROIs and Make ROI From Population from the list of protocols to the Drag Tasks Here To Make Measurements space, making sure that these two commands apply to the objects identified by Find Objects. Prior to calibrating the settings of this protocol, go to the dropdown menu above the Mode label at the top left of the window, and select the Extended Focus view. Deselect the non-tumor channels in the pane on the far right by clicking the black circle under each channel heading so that only the tumor channel can be seen.Then use the Freehand tool at the top of the window to draw a region of interest, or ROI, around all of the tumor volume. In the panel below the image, select the Summary label, and then set the Display option to show population two. To calibrate, on the Find Objects task, click on the asterisk to open the settings window.Adjust the threshold using intensity, and determine the lower threshold until only the tumor cells are selected and not the background fluorescence. Determine the minimum object size so that only intact cells are measured, as opposed to smaller items of cell debris, by setting the minimum object size as 100 cubic micrometers. Save this protocol as Tumor Volume by clicking the Measurements tab on the top menu and clicking Save Protocol.Use the same settings for measuring all xenografts. To measure the volume of the xenograft-associated blood vessels, create a new protocol by going to the top menu and then clicking on the Measurements tab and then Clear Protocol. Drag the Find Objects and Clip To ROIs tasks into the space that is titled Drag Tasks Here To Make Measurements for this protocol.Ensure that the first command is set to measure objects in the blood vessel channel and that the following command is set to apply to the objects identified by the first command. Then deselect the non-vessel channels in the far-right pane so that only the blood vessels can be seen. Determine the settings for the Vessel Volume protocol in a similar manner as for the Tumor Volume protocol previously described, and save this protocol as Vessel Volume.Clear the protocol, and draw an ROI around the xenograft, taking care not to include any non-tumor autofluorescence. To measure the sum of volume of the objects in the tumor channel inside this ROI, click the Measurements tab, select the Restore Protocol command, choose the Tumor Volume protocol, and click Restore. Using the new ROI made by the Tumor Volume protocol, click the Measurements tab and select the Restore Protocol command, choose the Vessel Volume protocol, and click on the sum row to calculate the total volume of objects in the vessel channel inside this ROI.Divide the vessel volume by the tumor volume, and multiply the answer by 100 to obtain a percentage value of graft vascularization. By imaging an individual xenograft at six, 24, and 48 hours post-implantation, the angiogenic response at different time points can be calculated, with the largest angiogenic response between 24 to 48 hours post-implantation and the maximum levels of graft vascularization seen around 48 hours. Confocal time-lapse imaging of a B16-F1 xenograft implanted into a two days post-fertilization zebrafish embryo shows GFP-labeled blood vessels growing through the xenograft, forming a twisting network with vessels of irregular size and morphology typical of the abnormal vascular network seen in mammalian tumors.Xenografts incubated in the VEGFR inhibitor show great vessel reduction compared to control xenografts incubated in DMSO. In control embryos, there was an expansive vascular network stretching across the entire xenograft region, the typical angiogenic response observed following implantation of B16-F1 cells. When quantified, angiogenic response shows a clear reduction in graft vascularization in the VEGFR inhibitor-treated xenografts when compared to controls.The tumor channel shows a mass of B16-F1 cells fluorescently labeled below the yolk sac, which displays autofluorescence. Therefore, an ROI must carefully be drawn around the xenograft, taking care not to include any of the autofluorescence from the yolk sac. Tumor Volume protocol identified all the B16-F1 cells in the ROI, measured their volume, and clipped the ROI to their volume.Tumor Vessel Volume Protocol in this new clipped ROI allowed the identification of all the vessels associated with the mass of B16-F1 cells and measured their volume. The values from the Tumor Volume and Tumor Vessel Volume protocols can be used to give a measure of graft vascularization. The genetic tractability and permeability of zebrafish allow investigation of high signaling pathways in tumor angiogenesis, and it can also act as a screening platform to identify anti-angiogenic compounds.

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Research

JoVE Journal

Cancer Research

6.8K visualizações.  University of Auckland. Este protocolo descreve como as larvas transgênicas de zebrafish podem ser usadas para fornecer um ensaio quantitativo in vivo da vascularização do xenoenxerto tumoral. A principal vantagem dessa técnica em relação às técnicas anteriores é que permite ao pesquisador quantificar com precisão as mudanças nos níveis de vascularização do xenoenxerto. O passo fundamental é garantir que as células tumorais sejam injetadas corretamente em larvas de zebrafish para que as etapas subseq...