We thank Dr. Leonard I. Zon in whose laboratory these techniques were initially developed; Kristen Kwan and the late Chi-Bin Chien for gifts of plasmids used in this work; James Lister and David Raible for assistance with antibody staining; and James Neiswender for the microinjection video. This work was funded by NIH grant R00AR056899-03 to C.J.C.

1. Screening for Melanoma Onset Modifiers
<ol>
	<li>Create Gateway middle entry clones by PCR amplifying the full-length open reading frame of genes of interest (GOI) and recombining into pDONR 221 using BP clonase II (Invitrogen). Use Multisite Gateway technology (Invitrogen) to recombine p5E_mitfa, pME_GOI, Tol2kit #302 p3E_SV40polyA6 and miniCoopR5 to place genes of interest under the mitfa promoter in the miniCoopR vector (Figure 1A).</li>
	<li>Inject 25 picograms of each clone along with 25 picograms Tol2 transposase mRNA into one-cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)triply homozygous zebrafish embryos as previously described7. Incubate the injected embryos at 28.5 &deg;C. Remove any dead embryos at 24 hpf.</li>
	<li>Select transgenic animals with rescued melanocytes at 72 hpf by placing the Petri dish containing injected embryos on a dissecting microscope under incident light against a white background (Figure 1A).</li>
	<li>Transfer animals to 3 L tanks in the nursery of the zebrafish facility at 4 dpf as previously described8.</li>
	<li>At 2 months, select the animals with at least one area of melanocyte rescue greater than 4 mm2 (Figure 1A). There is a strong correlation between the degree of melanocyte rescue at 72 hpf and the degree of melanocyte rescue at 2 months. When embryos with melanocyte rescue are picked at 72 hpf, the majority of them (~80%) will have at least one area of melanocyte rescue greater than 4 mm2 at 2 months.</li>
	<li>Screen the selected animals weekly for the presence of tumors (Figure 1B). There is a strong correlation between histopathologic and morphologic changes. Transition from benign to malignant is recognized as a morphologic change when lesions become raised off the surface of the animal. Isolate tumor bearing animals for study.</li>
	<li>Draw melanoma-free survival curves with age in weeks on the abscissa and percent melanoma-free survival on the ordinate. Animals with melanocytes that express a gene of interest are compared to control animals that express EGFP (enhanced green fluorescent protein) in melanocytes (Figure 1C). The median tumor onset for animals injected with miniCoopR-EGFP is approximately 18 weeks. Determine whether the two curves are statistically different using a log rank test.</li>
</ol>
2. Tumor Invasion Assay
<ol>
	<li>Select isolated zebrafish that develop tumors dorsally, in a region between the posterior boundary of the hindbrain and anterior border of the dorsal fin (Figure 2A).</li>
	<li>Two weeks after melanoma onset euthanize the fish according to guidelines specified by the American Veterinary Medical Association Panel on Euthanasia and approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee (IACUC). Specifically, a fish is placed into a dish with 0.6 mg/ml tricaine until gill movement stops.</li>
	<li>Make an incision in the peritoneal cavity of the fish with a scalpel and then place the fish in 4% paraformaldehyde (PFA) overnight at 4 &deg;C for fixation.</li>
	<li>Treat with 0.5 M EDTA overnight at 4 &deg;C to decalcify.</li>
	<li>Fix with formalin in cassette overnight, dehydrate for 1 hr, clear with xylene 3 times for 1 hr and then treat with paraffin 2 times for 2 hr each at 60 &deg;C.</li>
	<li>Embed the tissue in paraffin in a mould and allow it to solidify for 30 min to 1 hr.</li>
	<li>Make 5 &mu;m sections, one every 50 &mu;m. The sections should be transverse and through the entire lesion so that the point of deepest invasion can be identified. Follow by hematoxylin and eosin staining.</li>
	<li>Assess tumor invasion by measuring whether tumor cells remain outside the collagenous stratum compactum, invade into the dorsal musculature or invade further into the spinal column (Figure 2B,C). Determine statistical difference between the two sets of samples.</li>
</ol>
3. Antibody Staining of Scale Melanocytes
<ol>
	<li>Make up PO4 buffer with 80 ml 0.1 M Na2HPO4 and 20 ml 0.1M NaH2PO4. Make sure the pH is 7.3. Make up 2x fix buffer containing 8.0 g sucrose, 0.15 ml 0.2M CaCl2 and 90 ml 0.2M PO4 buffer, pH 7.3. If necessary adjust the pH to 7.3 with NaOH or HCl then make up to 100 ml with PO4 buffer.</li>
	<li>Weigh up to 300 mg solid PFA and add to a microcentrifuge tube. Add 5 mM NaOH to a volume equal to 4.5 x mass, in mg, of PFA (e.g. 450 ul 5 mM NaOH to 100 mg PFA). Heat at 60-70 &deg;C with occasional shaking until the PFA is dissolved. Spin down remaining particulates and recover 20% PFA supernatant. Make fresh every time.</li>
	<li>Prepare fixation solution containing 1x fix buffer and 4% PFA.</li>
	<li>Anesthetize the fish in 0.17 mg/ml tricaine.</li>
	<li>Using incident light under a dissecting microscope pluck dorsal scales using sharp fine-tipped forceps, taking care not to damage the melanocyte-containing half of the scale (Figure 3A). Place scales directly from the forceps into 1 ml fixation solution and incubate at room temperature, while turning, for &ge; 2 hr. Transfer the fish into fish water and monitor the fish to confirm successful recovery.</li>
	<li>To bleach pigmented melanocytes wash fixed scales in PBST (1x PBS pH 7.4, 0.1% Triton X-100) 2 times for 5 min at room temperature. Add 1 ml freshly made bleach solution containing 0.4 ml 10% KOH and 0.15 ml 30% H2O2 in 5 ml with dH2O. Put on parafilm or cap locks to prevent gas from bursting the tubes open. Continue bleaching until pigment is gone (10 min is typical) (Figure 3B,C). Wash in PBST four times for 5 min at room temperature.</li>
	<li>Block for at least 30 min in block solution made up of 1x PBS pH 7.4, 0.2% Triton X-100, 2 mg/ml BSA, 1% DMSO, 0.02% NaN3 and 2% lamb serum. Leave out NaN3 if developing with HRP reaction.</li>
	<li>Incubate with primary antibody in block solution overnight at room temperature. You need to have at least 400 &mu;l of solution over the samples to keep them submerged.</li>
	<li>Wash the scales 3 times for 30 min in PBST at room temperature.</li>
	<li>Incubate with secondary antibody from 2 hr to overnight in block solution at room temperature.</li>
	<li>Stain for 10 min with PBST+ 0.1 &mu;M DAPI.</li>
	<li>Wash 3 times for 5 min with PBST.</li>
	<li>Mount the scales on a glass slide in a drop of vectashield so that the concave side of the scales faces down towards the slide and place a glass cover slip over them. Seal the edges of the slide with clear nail polish and observe the slides under a fluorescence microscope (Figure 3D,E,F,G).</li>
</ol>
4. Transplantation Assay
<ol>
	<li>Prepare recipient 2-3 month old casper9 fish by irradiating with 25 Gy of gamma irradiation one day prior to transplantation10. Allow the fish to recover in fish water.</li>
	<li>Euthanize a tumor-bearing fish as previously described (Section 2.2).</li>
	<li>Cut off the tumor and put it in a Petri dish with approximately 5 ml filter sterilized 0.9x PBS with 5% FBS. Dice the tumor with a razor while in solution.</li>
	<li>Working at room temperature, triturate with a P1000 pipette to get single cells. Make up the volume to 25 ml with filter sterilized 0.9x PBS with 5% FBS.</li>
	<li>Filter through 40 &mu;M mesh filter.</li>
	<li>Spin in an Eppendorf 5810R tabletop centrifuge at 1,500 rpm (453 rcf) for 10 min.</li>
	<li>Remove the supernatant and make cell suspension to roughly desired concentration (assume 107 cells for a 100 mm3 tumor).</li>
	<li>Calculate the exact cell number using a hemocytometer and dilute the cell suspension so that the final injection volume is approximately 5 &mu;l. For example, if 50,000 cells are to be injected, the cell suspension should be diluted to a final concentration of 10,000 cells/&mu;l. Flick the tube containing the cell suspension every few min to prevent clumping of the cells.</li>
	<li>Wash a 26s gauge (bevel tip) 701 N 10 &mu;l Hamilton syringe 2-3 times with 100% ethanol and 0.9x PBS. Load the syringe with 5 &mu;l of the cell suspension.</li>
	<li>Anesthetize the irradiated recipient fish in 0.17 mg/ml tricaine. Place fish on its side on a damp Kimwipe (Figure 4A).</li>
	<li>Stabilize the fish with one hand and insert the needle with the bevel facing up at a 45&deg; angle into the flank of the fish above the peritoneal cavity about halfway between the posterior boundary of the hindbrain and anterior border of the dorsal fin. Gently depress the plunger (Figure 4B).</li>
	<li>Allow the fish to recover in fresh fish water and observe the fish daily for tumor engraftment (Figure 4C). If engraftment has occurred, continued growth and disease development can also be observed.</li>
</ol>

<ol>
	<li>Beroukhim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+somatic+copy-number+alteration+across+human+cancers.">The landscape of somatic copy-number alteration across human cancers.</a> Nature. 463, 899-905 (2010).</li><li>Curtin, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+sets+of+genetic+alterations+in+melanoma.">Distinct sets of genetic alterations in melanoma.</a> N. Engl. J. Med. 353, 2135-2147 (2005).</li><li>Lin, W. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+genomic+diversity+and+tumor+dependency+in+malignant+melanoma.">Modeling genomic diversity and tumor dependency in malignant melanoma.</a> Cancer Res. 68, 664-673 (2008).</li><li>Patton, E. E., 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=BRAF+mutations+are+sufficient+to+promote+nevi+formation+and+cooperate+with+p53+in+the+genesis+of+melanoma.">BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.</a> Curr. Biol. 15, 249-254 (2005).</li><li>Ceol, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histone+methyltransferase+SETDB1+is+recurrently+amplified+in+melanoma+and+accelerates+its+onset.">The histone methyltransferase SETDB1 is recurrently amplified in melanoma and accelerates its onset.</a> Nature. 471, 513-517 (2011).</li><li>Kwan, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev. Dyn. 236, 3088-3099 (2007).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide of the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>White, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2, 183-189 (2008).</li><li>Traver, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat. Immunol. 4, 1238-1246 (2003).</li></ol>

No conflicts of interest declared.

The miniCoopR method enables expression of genes of interest in zebrafish melanocytes. This approach takes advantage of the fact that the zebrafish mitfa gene acts cell-autonomously. For this reason, melanocytes rescued by the miniCoopR vector are certain to contain the minigene and any gene of interest to which it is physically coupled. Rescued melanocytes are clearly visible and can be obtained in animals that were injected as single-cell embryos. A specified degree of chimerism is selected, and animals surpas...

<table><tbody><tr><td>Gateway recombination reagents</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>miniCoopR</td><td></td><td></td><td>Reference&lt;sup&gt;5&lt;/sup&gt;</td></tr><tr><td>Mitfa antibody</td><td></td><td></td><td>Reference&lt;sup&gt;5&lt;/sup&gt;</td></tr><tr><td>FITC goat anti-rabbit IgG antibody</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Vectashield</td><td>Vector Labs</td><td>H-1000</td><td></td></tr><tr><td>&lt;em&gt;casper &lt;/em&gt;Zebrafish</td><td></td><td></td><td>Reference&lt;sup&gt;9&lt;/sup&gt;</td></tr><tr><td>701N 10 &amp;mu;l Syringe</td><td>Hamilton/Fisher</td><td>14-824</td><td></td></tr><tr><td>40 &amp;mu;M filter</td><td>BD Falcon/Fisher</td><td>352340</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>26140079</td><td></td></tr></tbody></table>

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.

One-cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos were injected with the miniCoopR vector containing the melanoma oncogene SETDB15 or EGFP, each under the control of the mitfa promoter. Embryos with melanocyte rescue were selected and allowed to mature. At 2 months of age animals with melanocyte rescue greater than 4 mm2 were selected. The animals were screened weekly for melanomas. Tumor incidence curves for the adults showed that the S...

Watch this Scientific Journal Video about Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model at JoVE.com

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

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 ...

screening-for-melanoma-modifiers-using-zebrafish-autochthonous-tumor

Research

JoVE Journal

Medicine

13.6K Views.  University of Massachusetts Medical School. 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.

Video: Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

Developmental Biology

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 ...

Cancer Research

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

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

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

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

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

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

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

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

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

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

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

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

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

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

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have been developed that permit engraftment of normal and malignant cells into irradiated, syngeneic, and immune compromised adult zebrafish. These models when coupled with optimized cell transplantation protocols allow for the rapid assessment of stem cell function, regeneration following injury, and cancer. Here, we present a method for cell transplantation of zebrafish adult skeletal muscle and embryonal rhabdomyosarcoma (ERMS), a pediatric sarcoma that shares features with embryonic muscle, into immune compromised adult rag2E450fs homozygous mutant zebrafish. Importantly, these animals lack T cells and have reduced B cell function, facilitating engraftment of a wide range of tissues from unrelated donor animals. Our optimized protocols show that fluorescently labeled muscle cell preparations from &#945;-actin-RFP transgenic zebrafish engraft robustly when implanted into the dorsal musculature of rag2 homozygous mutant fish. We also demonstrate engraftment of fluorescent-transgenic ERMS where fluorescence is confined to cells based on differentiation status. Specifically, ERMS were created in AB-strain myf5-GFP; mylpfa-mCherry double transgenic animals and tumors injected into the peritoneum of adult immune compromised fish. The utility of these protocols extends to engraftment of a wide range of normal and malignant donor cells that can be implanted into dorsal musculature or peritoneum of adult zebrafish.

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

Zebrafish have become a powerful tool for assessing development, regeneration, and cancer. More recently, allograft cell transplantation protocols have ...

Immunology and Infection

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 ...

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.

Test przeszczepu podskórnego: metoda przeszczepiania komórek czerniaka od dawcy do biorcy modelu danio pręgowanego

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

miniCoopR Vector-based Transgenesis: A Technique to Screen Melanoma-inducing Genes in Zebrafish Tumor 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 a method to screen for the melanoma modifying genes using a transgenic zebrafish tumor model. When injected with a melanoma-inducing miniCoopR vector, the zebrafish develops melanomas and can therefore be used to study the genetic factors affecting tumor development in zebrafish.

Transgeneza wektorowa miniCoopR: technika badań przesiewowych genów indukujących czerniaka w modelu guza danio pręgowanego

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