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 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...

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 ...

in-vivo-imaging-quantitation-host-angiogenic-response-zebrafish-tumor

Research

JoVE Journal

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture in a perfused microfluidic platform.

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

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 ...