This research was supported by a V Foundation V Scholar Award and NIH Grants DP2CA228043, R01CA227656 (to J.S. Blackburn) and NIH Training Grant T32CA165990 (to M.G. Haney).

All procedures described in this protocol have been approved by the University of Kentucky&#8217;s Institutional Animal Care and Use Committee (protocol 2015-2225). Patient samples were collected under University of Kentucky&#8217;s Institutional Review Board (protocol 44672). All animal experiments performed following this protocol must be approved by the user&#8217;s Institutional Animal Care and Use Committee.
1. Thawing Primary Patient Acute Lymphoblastic Leukemia Cells
<ol>
	<li>Thaw primary patient peripheral blood mononuclear cells (PBMCs) from frozen stock in a 37 &#176;C water bath. Immediately after cells have thawed, transfer cells in their freezing media (90% FBS + 10% dimethyl sulfoxide [DMSO]) to a 15 mL conical tube with slow pipetting, avoiding air bubbles. Add 10 mL of prewarmed 37 &#176;C thawing media (25% fetal bovine serum [FBS] in Iscove&#8217;s modified Dulbecco&#8217;s medium [IMDM]) dropwise (approximately 2&#8722;3 s per mL) to the cells in the 15 mL conical tube. 
	NOTE: PBMCs were collected from patient blood samples at the time of diagnosis. The buffy coat was separated by density centrifugation and cells were washed 2x in RPMI 1640 + 10% FBS. Cells were counted and 107 cells were frozen per cryovial in 1 mL of freezing media, and stored at -80 &#176;C.</li>
	<li>Centrifuge cells at 100 x g for 10 min and aspirate media from the cell pellet. Repeat the addition of thaw media, centrifugation, and aspiration one additional time to remove any residual DMSO.</li>
	<li>Resuspend cells in 5 mL of phosphate-buffered saline (PBS) and remove 10 &#956;L for counting on an automated cell counter or hemocytometer. Add 10 &#956;L of trypan blue to 10 &#956;L of cells removed for counting. Count number of cells per mL and record to later calculate the volume to resuspend cells in for xenografting (see step 2.5). 
	NOTE: Typically, 500 cells are xenografted per larval zebrafish. For example, 5 x 105 cells are needed to inject 1,000 zebrafish. Viability should be &#62;85% to use for xenografting. In this experiment, cell viability was 96% after thawing, assessed by trypan blue staining.</li>
</ol>
2. Fluorescently Labeling Cells with DiI
<ol>
	<li>Centrifuge the desired cell number in 5 mL of PBS at 200 x g for 5 min and aspirate supernatant. Stain a minimum of 2 x 106 cells in case of clumping or problems loading the needle during injection.</li>
	<li>Make 1,1&#8217;-dioctadecyl-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI) staining solution (5 mL of PBS containing 4 &#181;L per mL of DiI stain, Table of Materials) and resuspend the cell pellet in the staining solution. 
	NOTE: Cell density should not exceed 2 x 106 cells/mL when resuspended in the staining solution.</li>
	<li>Incubate cells at 37 &#176;C protected from light for 20 min, vortex gently, then incubate cells on ice for 15 min protected from light.</li>
	<li>Centrifuge cells at 200 x g for 5 min and aspirate supernatant. Wash cells with 5 mL of PBS, centrifuge at 200 x g for 5 min and aspirate supernatant. Repeat wash, centrifugation, and aspiration one additional time.</li>
	<li>Resuspend cells in 1 &#181;L of PBS per 250,000 live cells and transfer to a 1.5 mL microcentrifuge tube. Keep resuspended cells on ice in the dark and immediately continue to microinjections. 
	NOTE: This ensures that 500 patient cells are injected into the zebrafish with each injection pump of 2 nL volume.</li>
</ol>
3. Microinjecting Zebrafish Larvae
NOTE: Microinjections should be completed within 1&#8722;3 h of staining to improve viability of cells.
<ol>
	<li>Prior to staining cells and injecting, make agarose plates for injecting by pouring 25 mL of 3% agarose in 1x Tris/borate/EDTA (TBE) into a Petri dish and allow it to solidify. Store plates at 4 &#176;C for up to 2 weeks.</li>
	<li>Also prior to staining cells and injecting, dechorionate 2 dpf zebrafish using forceps under a dissecting microscope. For manual dechorionation, pull from opposite ends of the protective chorion of the zebrafish with forceps until the chorion tears and the zebrafish becomes unenveloped10. 
	NOTE: Dechorionation can also be performed by using enzymatic treatment with pronase, as previously described10. Casper (roy-/-;nacre-/-) zebrafish were used for these experiments11. Any zebrafish larval strain can be used for xenografting. If pigment interferes with imaging or visualization, propylthiouracil (PTU) treatment can be used to block melanin synthesis if optically clear zebrafish strains are not available12.</li>
	<li>Prechill needles at 4 &#176;C or on ice to prevent clumping of cells during microinjection. Load 5 &#181;L of stained cells into a chilled nonfilamentous borosilicate glass needle using microloader pipette tips. 
	NOTE: Microinjector and needle setup methods have been previously published13.</li>
	<li>Load the needle into the microinjector arm. Bevel the needle tip using a sterile razor blade. Measure the droplet size in mineral oil using a stage micrometer, keeping droplet volume consistently at 2 nL (~0.15 mm diameter) throughout.</li>
	<li>Use 350 &#181;L of 4 mg/mL tricaine-S to anesthetize ~30 dechorionated 2 dpf zebrafish in a Petri dish containing 25 mL of E3 media. After ~1 min transfer anesthetized larvae to a flat-surface injection plate (3% agarose in a Petri dish) and inject larvae with one pump of stained cells at the desired injection site (e.g., the yolk or the pericardium; see Figure 2A,B). 
	NOTE: Injection site should be chosen based on the goal of the experiment. The most common injection site is the yolk. To get cells circulating in the bloodstream, the pericardium, duct of Cuvier, perivitelline space, or retro-orbital space can be used as injection sites. Orthotopic injection sites can also be used, such as the brain.</li>
	<li>Wash larvae off the injection plate into a 10 cm2 Petri dish (30 larvae per plate) containing E3 media14 without methylene blue and incubate at 28 &#176;C for a 1 h recovery period. Continue injecting until a desired number of larvae have been injected. 
	NOTE: Ideally, inject 2&#8722;2.5-fold the number of larvae needed for experiments. There will be some die-off of larvae due to stress from injection and the increased incubation temperature. Typically, after practice with the technique, 800&#8722;1,500 zebrafish larvae can be injected by a single person within the 1&#8722;3 h when stained cells should be injected.</li>
	<li>Move plates of injected larvae to a 34 &#176;C incubator. Do not place the Petri dishes of larvae directly on a metal shelf in the incubator to prevent overheating of the E3 water. For example, place an empty Petri dish between the shelf and Petri dish of larvae to act as a buffer. Remove dead zebrafish larvae after 24 and 48 hours post injection (hpi).</li>
</ol>
4. Setting Up Drug Screen with Xenografted Zebrafish
<ol>
	<li>At 48 hpi, screen zebrafish larvae for fluorescence/tumor engraftment and health (Figure 2C,D). Remove any dead or malformed zebrafish and select zebrafish with similar engraftment (Figure 2C,D, 1&#8722;3 and 1&#8217;&#8722;3&#8217;). Remove unengrafted zebrafish (Figure 2C,D, 5 and 5&#8217;).
	<ol>
		<li>For yolk injected fish, remove fish where borders of the yolk cannot be seen around engrafted cell mass (Figure 2C, 4) as it makes quantification difficult. For pericardium injected fish, remove fish where injected cell mass encroaches into the yolk sac (Figure 2D, 4&#8217;).</li>
	</ol>
	</li>
	<li>Add zebrafish to a 96-well plate. To do this, cut the tip off of a 200 &#181;L pipette tip, just large enough for a 4 dpf zebrafish to fit through. Aspirate 150 &#181;L of E3 media with one zebrafish from the plate using a P200 pipette, and add to an empty well of a flat-bottom 96-well plate.</li>
	<li>Dilute the drug to be tested in the required volume of E3 media, at 150 &#181;L per well. Prepare drug at 2-fold the desired concentration. For example, prepare 20 &#181;M of drug in E3 if the desired final concentration is 10 &#181;M, since half of the total volume in each well is comprised of the drug solution. For the DMSO control group, add DMSO at the same volume as the drug.</li>
	<li>Add 150 &#181;L of 2x diluted drug solution to each well containing zebrafish larvae in 150 &#181;L of E3, for a final volume of 300 &#181;L with 1x drug solution per well.</li>
	<li>Incubate the plate at 34 &#176;C. Check for dead zebrafish daily. After 2 days, if desired, refresh the drug by removing 200 &#181;L of liquid from each well of the 96-well plate and replacing with 200 &#181;L of 1x dilute drug solution or DMSO in E3 media. 
	NOTE: The best results were found after 3 days of drug treatment for this experiment; however, the length of drug treatment can vary between 2&#8722;4 days and may need to be optimized based on the experiment being done or drugs being used.</li>
</ol>
5. Imaging Xenografted Zebrafish Using a Fluorescence Microscope Equipped Imaging Unit and Automated Sampler Unit
<ol>
	<li>Prepare 1 L of fresh 4 mg/mL tricaine and 1.5 L of E3 media. Fill media bottle 1 with E3 media and media bottle 2 with tricaine.</li>
	<li>Remove all unwanted fluorescent channels in the imaging software and add the desired channel (DiI for this experiment). Check the desired fluorescent channel as an image will only be taken for the channels with a check mark. Also, select how the images will be taken (z-stacks, automation, loops in series, etc.). 
	NOTE: For this experiment, the focus was manually set for each fish imaged to obtain the highest number of images with optimal focus.</li>
	<li>Image the DMSO control fish before the drug-treated fish so that the appropriate exposure time can be set in the imaging software. Once the exposure is set, do not change the exposure time for the duration of the experiment. 
	NOTE: The focus can either be adjusted manually for each zebrafish to ensure there are no out of focus images, or for fully automated imaging, the same focus can be used between fish with out of focus images being discarded or fish reimaged prior to performing analysis. Furthermore, this experiment could be conducted by using any fluorescent imager followed by quantification of fluorescence using the ImageJ software.</li>
</ol>
6. Quantifying Fluorescence Using ImageJ
<ol>
	<li>Open ImageJ software.</li>
	<li>Go to File | Open and select the desired .czi file. The software will bring up an import options window.
	<ol>
		<li>For stack viewing select Hyperstacks, check Open Files Individually, check Autoscale, and check Split Channels. For the color option select Colorized.</li>
	</ol>
	</li>
	<li>Click Plugins | Macros | Record.</li>
	<li>Click Image | Adjust | Threshold. Select image type as red in the dropdown menu on the right side of the threshold window. Adjust the minimum threshold until the software is only highlighting areas with fluorescence (Figure 3A) and click Apply. The software will convert the photo to black and white, with the selected area in black. 
	NOTE: Use the same threshold for each image in the drug screen to keep results standardized and comparable.</li>
	<li>Click Analyze | Measure. The software will pull up a results window containing the fluorescent area for that image.</li>
	<li>Click Create on the Macro Recorder window. This will open up a new window with the code for the macro. Highlight all of the desired images for analysis and open as in step 6.2.</li>
	<li>Select Run on the window with the macro. The results window will now contain the area for each image. 
	NOTE: The image analysis can be done individually without recording and running a macro as well as by repeating the above steps for each image.</li>
	<li>Copy the measured data into a spreadsheet. Average the total fluorescence of all control (DMSO) samples. Calculate the percent difference using the following formula: &#8211;[(average DMSO area &#8211; experimental area)/average DMSO area] x 100% (Figure 3B).</li>
</ol>

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The authors have nothing to disclose.

In this study, we demonstrated a standardized method for thawing and injection of primary patient leukemia cells into zebrafish as a xenograft model. We also established a protocol for high-throughput drug screening of xenografted zebrafish using a fluorescence microscope equipped imaging unit and automated sampler unit. Previously, xenografts have been reported with human cell lines, and quantification of xenografted tumors in a high-throughput manner has been a challenge in the field. This method serves as a basis for ...

Xenografting of primary patient cancers or human cancer cell lines into model organisms is a widely used technique to study tumor progression and behavior in vivo, tumor response to drug treatment, and cancer cell interaction with the microenvironment, among others. Traditionally, cells are xenografted into immune-compromised mice, and this remains the standard in the field. However, this model system has several limitations, such as high cost, low replicate numbers, difficulties in accurately quantifying tumor burden in vivo, and the extended time that it takes for tumors to engraft and drug testing to be completed. In recent years, zebrafish have emerged as an alternate xenograft model, with the first being reported in 2005, with green fluorescent protein (GFP)-labeled human melanoma cell lines transplanted into blastula-stage embryos1,2. More recently, 2 day post-fertilization (dpf) zebrafish larvae have been used as xenograft recipients to allow for control of anatomic location of injection and for use in high resolution in vivo imaging of tumor interaction with the surrounding microenvironment3,4.
Zebrafish offer many advantages as a xenograft model. First, adult zebrafish can be housed and rapidly bred in large quantities at a relatively low cost. Each mating pair of adult zebrafish can produce hundreds of larval fish per week. Due to their small size, these larval zebrafish can be maintained in 96-well plates for high-throughput drug screening. Larvae do not have to be fed during the course of a typical xenograft experiment, as their yolk-sac provides the nutrients to sustain them for their first week of life. Furthermore, zebrafish do not have a fully functional immune system until 7 dpf, meaning that they do not require irradiation or immunosuppressive regimens prior to xenograft injection. Finally, optically clear zebrafish lines allow for high-resolution imaging of tumor-microenvironment interactions.
Perhaps the most promising application of zebrafish as a xenograft model is the ability to perform high-throughput drug screening on human cancer samples in a way that is not possible using any other model organism. Larvae absorb drugs from the water through the skin, enhancing the ease of drug administration5. Because animals are maintained in 96-well plates, typically in 100&#8722;300 &#956;L of water, screens require smaller drug quantities compared to mice. Currently, there are several different methods for standardization and quantification of the effect of drugs on human tumor burden in zebrafish, some of which are more practical than others for scaling-up single drug testing to high-throughput screening. For example, some groups dissociate fish into single cell suspensions, and quantify fluorescently labeled or stained tumor cells by imaging individual droplets of the suspension and quantifying fluorescence using a semi-automated ImageJ macro4. A semi-automated whole-larvae imaging method was developed in which larval fish were fixed in 96-well plates and imaged using an inverted fluorescent microscope before realignment of composite images and quantification of tumor cell foci6. Both of these assays are fairly labor-intensive methods for quantification, which has made truly high-throughput drug screening in zebrafish xenograft models impractical.
This issue has been addressed by the development of the Vertebrate Automated Screening Technology (VAST) Bioimager and Large Particle (LP) Sampler, a fluorescence microscope equipped imaging unit and automated sampler unit (Figure 1 and Table of Materials), which is a truly automated method for high-throughput imaging of zebrafish larvae7,8,9. With this unit, fish are anesthetized, sampled automatically from a 96-well plate, positioned in a capillary and rotated into the set orientation based on a preset user preference, imaged, and then either placed back into the same well of a new 96-well plate for further studies or discarded. Combining this imaging technology with zebrafish xenografts may allow for the possibility of personalized medicine that uses high-throughput drug screening of large drug compound libraries against individual patient tumors. Zebrafish xenografts also offer a large-scale and low-cost method for testing both toxicity and efficacy of novel compounds in vivo. Zebrafish can be used as a preliminary screening step before proceeding to mouse xenograft models.
We have developed a streamlined workflow for xenografting primary patient leukemia cells into zebrafish and performing high-throughput drug screens with automated imaging and quantification, which can be applied to any other primary patient tumor cells or cancer cell line. This workflow utilized a fluorescence microscope equipped imaging unit and automated sampler unit to improve upon current standardization and quantification methods and offers an automated alternative to previous, more labor-intensive methods of quantifying tumor mass in vivo.&#160;

<table><tbody><tr><td>10x TBE Liquid Concentrate</td><td>VWR</td><td>0658-5L</td><td></td></tr><tr><td>96-well plate, flat bottom</td><td>CELLTREAT</td><td>229195</td><td>VAST is compatible with a variety of standard or deep well 24, 48, or 96 well plates</td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP160-500</td><td></td></tr><tr><td>Borosilicate Glass Capillary without Filament</td><td>Sutter Instrument Company</td><td>B100-50-10</td><td></td></tr><tr><td>Dexamethasone</td><td>Enzo Life Sciences</td><td>BML-EI126-0001</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2438-5X10ML</td><td></td></tr><tr><td>E3 media</td><td></td><td>N/A</td><td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td></tr><tr><td>Femtotips Microloader Tips</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Fetal Bovine Serum (Premium Heat Inactivated)</td><td>Atlanta Biologicals</td><td>S11150H</td><td></td></tr><tr><td>ImageJ</td><td>FIJI</td><td>N/A</td><td>https://imagej.net/Fiji</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium</td><td>STEMCELL Technologies</td><td>36150</td><td></td></tr><tr><td>Large Particle (LP) Sampler</td><td>Union Biometrica</td><td>N/A</td><td>automated sampler unit http://www.unionbio.com/copas/features.aspx?id=8</td></tr><tr><td>Methotrexate</td><td>Sigma-Aldrich</td><td>A6770-10MG</td><td></td></tr><tr><td>Mineral Oil</td><td>Fisher Scientific</td><td>BP26291</td><td></td></tr><tr><td>Phosphate Buffered Saline (1x)</td><td>Caisson labs</td><td>PBL06-6X500ML</td><td></td></tr><tr><td>Stage Micrometer (400-Stage)</td><td>Hausser Scientific</td><td>400-S</td><td></td></tr><tr><td>Tricaine-S</td><td>Pentair Aquatic</td><td>TRS1</td><td></td></tr><tr><td>Trypan Blue</td><td>Thermo Fisher</td><td>T10282</td><td></td></tr><tr><td>VAST Bioimager</td><td>Union Biometrica</td><td>N/A</td><td>fluorescent equipped microscope imaging unit https://www.unionbio.com/vast/</td></tr><tr><td>Vincristine Sulfate</td><td>Enzo Life Sciences</td><td>BML-T117-0005</td><td></td></tr><tr><td>Vybrant DiI Stain</td><td>Thermo Fisher</td><td>V22885</td><td></td></tr></tbody></table>

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.

Following the protocol described above, zebrafish were xenografted in the yolk and pericardium with primary patient PBMCs that were originally isolated from a T-cell acute lymphoblastic leukemia (T-ALL) patient at diagnosis and banked as a viable, frozen sample. At 48 hpi, xenografted fish were screened for fluorescently labeled tumor cells (Figure 2C,D) and treated with chemotherapy (dexamethasone or vincristine) or DMSO. Fish were imaged at 7 dpi, after 3 days on drug trea...

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Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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 ...

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Research

JoVE Journal

Cancer Research

7.9K Views.  University of Kentucky. Onze zebrafish xenograft drug screening workflow zorgt voor geautomatiseerde beeldvorming en kwantificering van menselijke kankercellen en hun reactie op medicamenteuze behandeling. Het kan worden gebruikt om snel te testen van de reactie van kankercellen van een geduld op bekende geneesmiddelen of om nieuwe antikanker verbindingen te identificeren. Met behulp van zebravis xenograaf modellen en drug screening zorgt voor Inviva replicant nummers die voorheen alleen verkrijgbaar waren met in vi...

Video: Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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 ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Medicine

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 ...

Establishment of Zebrafish Patient-Derived Xenografts from Pancreatic Cancer for Chemosensitivity Testing

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms of screening, prevention, and treatment; however, preclinical models that predict the chemosensitivity profile of cancer patients are still lacking. To fill this gap, an in vivo patient-derived xenograft model was developed and validated. The model was based on zebrafish (Danio rerio) embryos at 2 days post fertilization, which were used as recipients of xenograft fragments of tumor tissue taken from a patient's surgical specimen. 
It is also worth noting that bioptic samples were not digested or disaggregated, in order to maintain the tumor microenvironment, which is crucial in terms of analyzing tumor behavior and the response to therapy. The protocol details a method for establishing zebrafish-based patient-derived xenografts (zPDXs) from primary solid tumor surgical resection. After screening by an anatomopathologist, the specimen is dissected using a scalpel blade. Necrotic tissue, vessels, or fatty tissue are removed and then chopped into 0.3 mm x 0.3 mm x 0.3 mm pieces. 
The pieces are then fluorescently labeled and xenotransplanted into the perivitelline space of zebrafish embryos. A large number of embryos can be processed at a low cost, enabling high-throughput in vivo analyses of the chemosensitivity of zPDXs to multiple anticancer drugs. Confocal images are routinely acquired to detect and quantify the apoptotic levels induced by chemotherapy treatment compared to the control group. The xenograft procedure has a significant time advantage, since it can be completed in a single day, providing a reasonable time window to carry out a therapeutic screening for co-clinical trials.

Preclinical models aim to advance the knowledge of cancer biology and predict treatment efficacy. This paper describes the generation of zebrafish-based patient-derived xenografts (zPDXs) with tumor tissue fragments. The zPDXs were treated with chemotherapy, the therapeutic effect of which was assessed in terms of cell apoptosis of the transplanted tissue.

Cancer is one of the main causes of death worldwide, and the incidence of many types of cancer continues to increase. Much progress has been made in terms ...

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 ...

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

Biology

Subcutaneous Transplantation Assay: A Method to Transplant Melanoma Cells from a Donor to a Recipient Zebrafish Model

Source: Iyengar, S. et al. <a href="https://www.jove.com/v/50086"> Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model</a>. J. Vis. Exp.(2012)
This video describes the method for transplantation of tumor cells from the melanoma-bearing donor zebrafish model to an immunocompromised recipient Casper zebrafish. It helps to visualize tumor engraftment and proliferation in the recipient fish.

Source: Iyengar, S. et al.  Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model. J. Vis. Exp.(2012)
This video describes the ...

JoVE Encyclopedia of Experiments

Skin Cancer

Animal model studies

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 ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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