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
NOTE: When using this protocol, any mammalian cancer cell line can be used for implantation into zebrafish embryos as xenografts. However, there is much variation in the angiogenic response induced among different cell lines5,11,12. B16-F1 murine melanoma cells have been shown to induce a strong angiogenic response in zebrafish embryos11 and are therefore appropriate for use in this protocol.
<ol>
	<li>Grow B16-F1 cells at 37 &#176;C in a 75 cm2 flask using MEM-&#945; media supplemented with Fetal Bovine Serum (FBS) to a final concentration of 10% (v/v) and Penicillin /Streptomycin, each at a final concentration of 100 &#956;g/mL.</li>
	<li>Remove the media from a 95-100% confluent 75 cm2 flask of B16-F1 cells and wash the cells in 5 mL of room temperature phosphate-buffered saline (PBS).</li>
	<li>Remove the 5 mL PBS, add 2 mL of room-temperature 0.25% Trypsin/ ethylenediaminetetraacetic acid (EDTA) and incubate at 37 &#176;C for 60 s.</li>
	<li>Tap the side of the flask to determine whether the cells are beginning to lose their attachment to the bottom of the flask.</li>
	<li>Add 8 mL of room temperature MEM-&#945; with 10% FBS into the flask, pipette against the inside of the flask to bring any cells that remain adhered to the bottom of the flask into suspension and pipette the cell suspension into a 15 mL tube.</li>
	<li>Centrifuge the cell suspension at 800 x g, 4-8 &#176;C for 5 min, aspirate the media and proceed to labelling the cells with dye.</li>
</ol>
3. Labelling B16-F1 Cells with Fluorescent Dye
NOTE: In order to differentiate between the implanted tumor cells and other cells in the embryo, the tumor cells must be labelled with an appropriate fluorescent dye before implantation. This step can be skipped if the cells already express fluorescent reporters.
<ol>
	<li>Incubate 2 mL of serum-free MEM-&#945; media at 37 &#176;C.</li>
	<li>Prepare a stock solution of the chosen dye and dilute it in pre-incubated 2 mL of serum-free MEM-&#945; media to make a workable concentration (examples of dyes and concentrations appropriate for B16 F1 cells are provided in Table of Materials).</li>
	<li>Pipette 1 mL of the dye solution into the cell pellet (from step 2.6), mix thoroughly by pipetting, then add the other 1 mL of dye solution and mix.</li>
	<li>Incubate the cells and dye mixture at 37 &#176;C for 40 min, mixing by gentle shaking at 20 min.</li>
	<li>Centrifuge the cell suspension at 800 x g, 4-8 &#176;C for 5 min.</li>
	<li>Aspirate the supernatant and wash the labelled cells by pipetting with 5 mL of PBS.</li>
	<li>Centrifuge the cell suspension again at 800 x g, 4-8 &#176;C for 5 min, aspirate the supernatant and place the cells on ice until ready for implantation. 
	NOTE: The final tumor cell pellet should have a volume of 20-40 &#956;L.</li>
</ol>
4. Preparation of Embryos for Implantation
NOTE: Choose a transgenic zebrafish line that has fluorescently labelled blood vessels (e.g. kdrl:RFP, fli1a:EGFP, etc.)13,14.
<ol>
	<li>Two days prior to implantation, spawn the zebrafish as previously described and collect the embryos15. 
	NOTE: As we are not injecting these fish at an early stage, they do not need to be collected within 20 min of spawning as described by Rosen et al.15, but may be collected after a few hours of mating.</li>
	<li>Place the embryos in 100 mm culture dishes at a density of approximately 100 embryos/dish.</li>
	<li>Add 50 mL of E316 (supplemented with 5 &#956;L of a 1% w/v aqueous stock solution of methylene blue to prevent contamination) to each dish, clean out any dead embryos or debris and keep the dish in a dark incubator at 28 &#176;C until ready for injection.</li>
	<li>At 1 day post fertilization (dpf), add 1-phenyl-2-thiourea (PTU) to each dish to produce a final concentration of 30 mg/L PTU in E3. PTU prevents pigmentation, which can interfere with the ability to view the tumor cells and blood vessels.</li>
	<li>At 2 dpf, use needles or tweezers to manually dechorionate any embryos that are unhatched. Using a fluorescent microscope, select as many transgenic embryos as required for xenografting (50-200).</li>
	<li>Prior to implantation, anaesthetize the selected embryos in a solution of 300 &#956;g/mL Tricaine in E3 in order to prevent movement during the injection procedure.</li>
	<li>Fill a 35 mm dish with a 2% methylcellulose in E3 solution to a quarter of the volume of the dish.</li>
	<li>Use a transfer pipette to place approximately 50 embryos (minimizing the volume of E3 solution also transferred) onto the methylcellulose.</li>
	<li>Use a Microloader pipette tip to arrange the embryos so that they are all oriented vertically, with the heads to the top and with the left side facing up. 
	NOTE: Steps 4.7-4.9 can also be carried out by arranging the embryos on an agarose block as previously described17, but in our experience, the embryos are more easily arranged when arrayed in methylcellulose.</li>
</ol>
5. Perivitelline Injection of Mammalian Cancer Cells into 2 dpf Embryos
NOTE: To ensure the cells clump together as a graft when implanted, the cells must be mixed together with an extracellular matrix mixture (ECM). We have described the steps of making such a cell/ECM mixture when using an ECM mixture referenced in the Table of Materials. If an alternative matrix is used, the steps should be adjusted accordingly.
<ol>
	<li>Divide a stock of ECM into 100 &#956;L aliquots in 500 &#956;L tubes and store at -20 &#176;C until needed.</li>
	<li>Thaw out an aliquot of ECM and add 100 &#956;L of PBS to dilute the mixture to 50% (v/v). 
	NOTE: Diluted 50% ECM can be stored at 4 &#176;C for up to 1 month.</li>
	<li>Mix the 50% ECM well with the B16-F1 cell pellet (from step 3.7) by pipetting and stirring, to produce a mixture of cells/ECM in a 2:1 ratio and store the mixture on ice.</li>
	<li>Using a microcapillary pipette, take up 3-10 &#956;L of cells.</li>
	<li>Carefully insert the pipette tip into a needle and eject the B16-F1/matrix mixture into the end of the needle.</li>
	<li>Insert the needle into the needle-holder and tilt at a 45&#176; angle to the dish.</li>
	<li>Break the tip of the needle using tweezers to make a hole large enough for the cells to be ejected from the needle without squeezing the cells.</li>
	<li>Turn on the pressurized air cylinder attached to the injection apparatus and turn the injector on &#8220;continuous&#8221; mode momentarily to push the cells to the tip of the needle.</li>
	<li>Remove the needle out of the dish and replace the dish with a hemocytometer.</li>
	<li>Place a drop of oil on the hemocytometer and inject the needle once onto this drop.</li>
	<li>Count the number of cells ejected with a single pulse using the hemocytometer and calibrate the needle to eject approximately 150 cells per pulse by adjusting pulse duration. 
	NOTE: Ejecting this quantity of cells per pulse will require several pulses in order to implant a successful xenograft. This will give the researcher more control over the final size of the tumor xenograft by allowing them to moderately adjust the number/location of pulses to make each xenograft larger or smaller as they see fit.</li>
	<li>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. 
	NOTE: Position the needle so that it enters the embryonic yolk sac anterior to the final location where the cells will be ejected. This will ensure that the cells will be ejected in a direction away from the heart, decreasing the likelihood of injecting the cells into the common cardinal vein.</li>
	<li>Carefully push the tip of the needle forward a little until it has created a space (perivitelline space) between the epidermis and the yolk sac membrane and then pulse the injector to eject some of the cell mixture into the perivitelline space.</li>
	<li>Repeat the pulses until 500-800 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, then remove the needle from the embryo. 
	NOTE: If the cells block the needle or are damaged during the injection, purge the needle by momentarily switching to &#8220;continuous&#8221; mode and then back to &#8220;pulse&#8221;. This should unblock the needle and allow the cells to be ejected freely and undamaged.</li>
	<li>Continue to do the same for all the embryos in the dish, loading a new needle with tumor cells when required.</li>
	<li>After injecting all the embryos, remove the needle and push all the embryos together using a microcapillary pipette tip so that they can be pipetted out with as little methylcellulose as possible.</li>
	<li>Transfer the embryos into a recovery dish containing E3 (with PTU and methylene blue) and wash them by gently pipetting E3 around the embryos. 
	NOTE: At this point, the xenografted embryos can be treated with drugs by separating them into different dishes and adding a drug solution to one dish and a control solution (such as the drug vehicle) to the other dish.</li>
	<li>Incubate the embryos at 34 &#176;C and perform twice daily checks to remove dead or oedematous embryos from the dish. 
	NOTE: 34 &#176;C was found to be the optimal temperature for xenograft vascularization and has also been used by other studies employing the zebrafish embryonic xenograft angiogenesis model18. The xenografts can be imaged any time up to 48 hpi (hours post-implantation).</li>
</ol>
6. Live Imaging
<ol>
	<li>At 48 hpi, identify 3-5 healthy embryos without oedema. Anaesthetize them in 150 &#956;g/mL Tricaine and mount them laterally in 1% Low Melting Point (LMP) agarose as previously described19. 
	NOTE: As the agarose is solidifying, use a microcapillary pipette tip to keep the embryos positioned laterally.</li>
	<li>Turn on the confocal microscope, the microscope controller, the lasers and the computer software for controlling the microscope.</li>
	<li>Place the dish on a confocal microscope. Using a 20x magnification, water dipping objective lens, position the xenograft in the center of the field using brightfield imaging.</li>
	<li>Select excitation lasers that are appropriate for detecting the dye used to label the tumor cells and the fluorophore used to label the zebrafish blood vessels.</li>
	<li>Adjust the gain for each laser to a level that allows the detection of both the tumor cells and the blood vessels. 
	NOTE: Once confocal settings have been established, ensure these are kept unchanged throughout the experiment so that comparisons can be made between xenografts.</li>
	<li>Using the laser channel appropriate for the tumor cells, determine a volume to be imaged that includes the entire volume of the xenograft, allowing for at least one or two optical sections either side of the graft. Use section intervals that are around 5 &#956;m apart to create a z-stack. 
	NOTE: It is essential that the z-stack should contain the entire volume of the xenograft.</li>
	<li>Using two channel imaging, image both the tumor xenograft and the blood vessels. Repeat steps 6.6-6.7 for each larva to be imaged.</li>
</ol>
7. Time Lapse Imaging
NOTE: This model is highly suited to imaging dynamic cellular processes due to its transparency and the availability of zebrafish transgenics that fluorescently label different cell types. This makes time-lapse imaging a key application of this model.
<ol>
	<li>Turn on the environmental chamber and set to 34 &#176;C at least 2 h before imaging. If the chamber is not humidified, add dishes of water.</li>
	<li>Anaesthetize 3-5 embryos (using 120 &#956;g/mL Tricaine at 2 dpf and 100 &#956;g/mL Tricaine at 3 dpf) and mount them laterally in 0.8% LMP agarose for time-lapse imaging according to the previously described protocol19. 
	NOTE: As the agarose is solidifying, use a microcapillary pipette tip to keep the embryos positioned laterally.</li>
	<li>Set up the equipment and image the xenograft as described in steps 6.2-6.7, acquiring z-stacks of the xenograft at 10 min intervals. 
	NOTE: The embryo must be maintained at 34 &#176;C as it is being imaged and the E3 on top of the larvae in agar must be checked and supplemented occasionally to ensure that it does not dry out.</li>
</ol>
8. Quantitation of the Angiogenic Response to the Zebrafish Xenograft
NOTE: The following steps use 3D image analysis software. Specific steps will vary depending on the software used.
<ol>
	<li>Transfer the z-stack confocal image files of a tumor xenograft to a new folder on the 3D Image Analysis software. 
	NOTE: In order to quantitate the levels of tumor vascularisation, Measurement Protocols must be created with settings calibrated so that they can be used to measure either Tumor Volume or Vessel Volume for all xenografts.</li>
	<li>To create the &#8220;Tumor Volume&#8221; protocol for measuring the volume of the tumor xenografts, go to the &#8220;Measurement&#8221; tab on the top menu and then drag the protocols named &#8220;Find Objects&#8221; from the list of protocols that appears on the left-hand side of the window to the space that is titled &#8220;Drag tasks here to make measurements&#8221;.</li>
	<li>Ensure that this protocol is set to measure objects in the tumor channel, and then drag the protocols named &#8220;Clip to ROIs&#8221; and &#8220;Make ROI from Population&#8221; from the list of protocols to the &#8220;Drag tasks here to make measurements&#8221; space. Ensure that these two commands apply to the objects identified by &#8220;Find Objects.&#8221;&#160;</li>
	<li>Prior to calibrating the settings of this protocol, go to the dropdown menu above the &#8220;Mode&#8221; label at the top left of the window and select the &#8220;Extended Focus&#8221; view.&#160;</li>
	<li>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. Use the &#8220;Freehand&#8221; tool at the top of the window to draw a &#8220;region of interest&#8221; (ROI) around all of the tumor volume. 
	NOTE: Take care to ensure that none of the tumor is left out of the ROI. This will provide a region on which to perform the protocol as you are calibrating the settings.</li>
	<li>Select the &#8220;Summary&#8221; label in the panel below the image, and set the &#8220;Display&#8221; option to show &#8220;Population 2&#8221;. To calibrate, click on the asterisk on the Find Objects task, which will open the settings window. Adjust the &#8220;Threshold&#8221; using &#8220;Intensity&#8221; and determining the &#8220;Lower&#8221; threshold until only the tumor cells are selected and not the background fluorescence.&#160; 
	NOTE: This is important so that only the fluorescence from the tumor cells in the selected ROI (drawn in 8.5) is detected and none of the background fluorescence is measured.</li>
	<li>Determine the &#8220;Minimum object size&#8221; so that only intact cells are measured as opposed to smaller items of cell debris (e.g. by setting the &#8220;Minimum object size&#8221; as 100 &#956;m3). Save this protocol as &#8220;Tumor Volume&#8221; by clicking the Measurements tab on the top menu and clicking &#8220;Save Protocol&#8221;, and use the same settings for measuring all xenografts.</li>
	<li>To measure the volume of the xenograft-associated blood vessels, you will need to make a new protocol. Go to the top menu, click on the Measurements tab and click &#8220;Clear Protocol&#8221;. Drag the &#8220;Find Objects&#8221; and &#8220;Clip to ROIs&#8221; tasks into the space that is titled &#8220;Drag tasks here to make measurements&#8221; for this protocol.&#160;</li>
	<li>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.</li>
	<li>Determine the settings for the &#8220;Vessel Volume&#8221; in a similar manner as for the &#8220;Tumor Volume&#8221; protocol and save this protocol as &#8220;Vessel Volume&#8221; (see 8.6-8.7).</li>
	<li>Clear the protocol and draw an ROI around the xenograft, taking care not to include any non-tumor autofluorescence. Measure the sum of volume of the objects in the tumor channel inside this ROI by clicking the Measurements tab, selecting the &#8220;Restore Protocol&#8221; command and choosing the &#8220;Tumor Volume&#8221; protocol.</li>
	<li>Using the new ROI made by the &#8220;Tumor Volume&#8221; protocol, use the &#8220;Vessel Volume&#8221; protocol to measure the total volume of objects in the vessel channel inside this ROI by clicking the Measurements tab, selecting the &#8220;Restore Protocol&#8221; command and choosing the &#8220;Vessel Volume&#8221; protocol.</li>
	<li>Divide vessel volume by the tumor volume and multiply the answer by 100 to obtain a percentage value of graft vascularisation.</li>
	<li>Repeat steps 8.11 to 8.13 for all the other xenografts.</li>
</ol>

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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 authors have nothing to disclose.

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 cm&lt;sup&gt;2&lt;/sup&gt;</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 &amp;mu;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&lt;br/&gt; &lt;br/&gt; 1.6 g KCl&lt;br/&gt; &lt;br/&gt; 5.8 g CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H2O&lt;br/&gt; &lt;br/&gt; 9.78 g MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;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 1,000 &amp;mu;L</td><td>VWR</td><td>732-1491</td><td>Used during multiple steps</td></tr><tr><td>Filter tip 200 &amp;mu;L</td><td>VWR</td><td>732-1489</td><td>Used during multiple steps</td></tr><tr><td>Filter tip 10 &amp;mu;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 &amp;mu;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 &amp;mu;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 G</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...

Watch this Scientific Journal Video about In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts at JoVE.com

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 ...

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Cancer Research

6.8K Views.  University of Auckland. 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.

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

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.

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.

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

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a peri-vascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development. 
Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

Here, we present a protocol to establish versatile orthotopic and ectopic zebrafish xenograft models for ocular melanoma to assess the growth kinetics of the primary tumor, dissemination, extravasation and distant, peri-vascular metastasis formation and the effect of chemical inhibition thereon.

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Utilizing Larval Zebrafish for Efficient In Vivo Tumor Studies

A Simple, Rapid, and Effective Method for Tumor Xenotransplantation Analysis in Transparent Zebrafish Embryos

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we present larval zebrafish as a transplant model that has numerous advantages over murine models, including ease of handling, low expense, and short experimental duration. Moreover, the absence of an adaptive immune system during larval stages obviates the need to generate and use immunodeficient strains. While established protocols for xenotransplantation in zebrafish embryos exist, we present here an improved method involving embryo staging for faster transfer, survival analysis, and the use of flow cytometry to assess disease burden. Embryos are staged to facilitate rapid cell injection into the yolk of the larvae and cell marking to monitor the consistency of the injected cell bolus. After injection, embryo survival analysis is assessed up to 7 days post injection (dpi). Finally, disease burden is also assessed by marking transferred cells with a fluorescent protein and analysis by flow cytometry. Flow cytometry is enabled by a standardized method of preparing cell suspensions from zebrafish embryos, which could also be used in establishing the primary culture of zebrafish cells. In summary, the procedure described here allows a more rapid assessment of the behavior of tumor cells in vivo with larger numbers of animals per study arm and in a more cost-effective manner.

Author Spotlight: Advancing Cancer Therapeutics Using Tumor Xenotransplantation in Zebrafish

We describe a protocol for xenotransplantation into the yolk of transparent zebrafish embryos that is optimized by a simple, rapid staging method. Post-injection analyses include survival and assessing the disease burden of xenotransplanted cells by flow cytometry.

In vivo studies of tumor behavior are a staple of cancer research; however, the use of mice presents significant challenges in cost and time. Here, we ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Medicine

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

Zebrafish Xenograft Assay: A Method to Assess the Anti-cancer Efficacy of Therapeutics on Tumorigenicity in Zebrafish In Vivo

Source: Lee, J. Y et al.,<a href="https://www.jove.com/v/57490/preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids">Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.</a>&#160;J. Vis. Exp. (2018)
This video describes the zebrafish xenograft assay to assess in vivo tumor formation and study the effect of therapeutic agents.

Source: Lee, J. Y et al.,Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish ...