This work was supported by a grant R01 CA240323 (S.Z.) from the National Cancer Institute; a grant W81XWH-17-1-0498 (S.Z.) from the United States Department of Defense (DoD); a V Scholar award from the V Foundation for Cancer Research (S.Z.) and a Platform Grant from the Mayo Center for Biomedical Discovery (S.Z.); and supports from the Mayo Clinic Cancer Center and Center for Individualized Medicine (S.Z.).

<ol>
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The authors declare that they have no competing financial interests.

Zebrafish has been commonly used in research for the past few decades, especially in cancer research, for obvious reasons, such as its ease of maintenance, robust reproduction, and clear advantages for in vivo imaging1,28. The zebrafish model can be easily manipulated embryonically due to their external fertilization and development, which complements well to mammalian model organisms, such as rats and mice, for large-scale genetic studies1,2,3. Moreover, the zebrafish genome has high-level similarities to the human genome. Comparing the detailed annotations of the zebrafish genome to the human reference genome, about 70% of human genes have been shown to obtain a zebrafish orthologue29. In addition, other uses of zebrafish in research have been extended to drug discovery and patient avatars for individualized cancer therapy30,31. Accumulating studies have shown that tumors induced in zebrafish are similar to human tumors at the histological and molecular levels, which can aid in dissection of tumor initiation, progression, and heterogeneity32,33. Together, the zebrafish demonstrates tremendous potential as a valuable animal model that can be used to study metastasis and elucidate possible oncogenic pathways involved in disease pathogenesis.
Using the transgenic zebrafish model with overexpression of both LMO1 and MYCN, the cooperation between these two oncogenes in NB tumorigenesis and metastasis has been clearly demonstrated. With today&#39;s availability of powerful live imaging techniques, tumor screens can be easily performed biweekly and metastasis can be traced over time. The widespread metastasis can also be further confirmed by paraffin sectioning and antibody staining. Strikingly, the metastases detected in the MYCN;LMO1 zebrafish correlate well with the common sites of metastasis seen in high-risk NB cases, such as in the bone marrow, lymph nodes, orbit, and lung34,35, further supporting zebrafish as a feasible and beneficial model for metastasis study.
Moreover, due to relatively small body size, the whole zebrafish can be sectioned though completely, which is yet another clear advantage of this model, allowing thorough characterization of the primary tumor along with the metastases in other regions of the body. The picrosirius red staining of tumor sections has clearly highlighted the collagen networks in fish tumors and demonstrates the increased stiffness of extracellular matrix in tumors with LMO1 overexpression. Although this technique is not unique to zebrafish, its application together with the high-throughput compound screening on zebrafish embryos that are genetically modified or transplanted with tumor cells might provide a novel means in screening for effective compounds that could target extracellular matrix remodeling, which is a critical process involved in tumor cell metastasis.
Stable transgenic zebrafish models are very helpful for us to understand the contribution of candidate oncogenes to tumor development in vivo, although it can be challenging and laborious to develop these lines. Creating a stable transgenic line requires a long period of time since multiple generations must be acquired before line propagation. Furthermore, propagating these lines can be tedious, and strategic mating plans may be needed. For example, the productivity of a MYCN transgenic fish is often markedly reduced once the tumor has developed, and homozygous MYCN transgenic fish do not survive well into adulthood. Therefore, to better maintain the MYCN transgenic fish line, it is recommended to outcross the heterozygous non-tumor-bearing MYCN transgenic fish at a younger age with WT or other genetically engineered fish lines, such as the LMO1 transgenic fish line. To overcome the challenges in developing and maintaining stable transgenic fish lines, mosaic transient transgenesis may be an alternative approach to rapidly and effectively assess the contribution of a single gene or combination of genes to the tumor initiation and progression in primarily injected fish. In addition, the mosaic pattern of transgene integration in the primary injected fish may better mimic the disease pathogenesis, especially those induced by somatic events36.
However, like any other animal model used in research to study cancer, the zebrafish also has its disadvantages. For example, antibodies specifically against zebrafish proteins remain largely underdeveloped, although several antibodies against neuroblastoma marker genes-such as tyrosine hydroxylase, synaptophysin, and HuC-are fortunately working well in zebrafish6,13. To combat this issue, many vendors have begun to test their products and predict the potential of their antibodies in cross-reacting with zebrafish proteins. More information about validated antibodies can also been found in the zebrafish information network (ZFIN). With these efforts, more and more antibodies that can specifically detect zebrafish proteins will soon become available to the zebrafish community. Another challenge of using zebrafish as a genetic model to dissect the interplay of complex signaling pathways in NB pathogenesis is its partially duplicated genome. Such genome duplication, which occurred in the natural ancestry of zebrafish37,38, can often lead to more than one variant of zebrafish homologues to humans. This can cause an evolved gain of novel gene functions or unique expression patterns in the animal model39. Therefore, when studying genes with potential roles in tumor suppression, it may be necessary for multiple alleles of the duplicated genes to be knocked out at the same time to demonstrate their tumor suppression function, which can be a potentially time-consuming and a technically-challenging endeavor.
Nevertheless, the transgenic zebrafish model can faithfully recapitulate all stages of tumor metastasis in vivo, and possesses clear advantages for genetic analysis and real-time imaging of tumor dissemination. This model system, therefore, offers a unique tool for us to address many daunting questions in the field, such as what the molecular and cellular events underlying the multistep process of tumor dissemination and metastasis in vivo are, when the NB cells disseminate from primary tumors, and how the tumor microenvironment contributes to NB metastasis. With the robustness of zebrafish for drug screening and testing, the fish model would also provide a valuable means for the evaluation of efficacy of novel agents and inhibitors to prevent or treat metastatic NB.

Zebrafish has been widely used and applied to several areas of research, especially in cancer. This model provides many advantages-such as its robust reproduction, cost-effective maintenance, and versatile visualization of tumor growth and metastasis-all of which make zebrafish a powerful tool to study and investigate the cellular and molecular bases of tumorigenesis and metastasis. New techniques for large-scale genome mapping, transgenesis, genes overexpression or knockout, cell transplantation, and chemical screens have immensely augmented the power of the zebrafish model1. During the past few years, many zebrafish lines have been developed to study tumorigenesis and metastasis of a variety of human cancers, including but not limited to leukemia, melanoma, rhabdomyosarcoma, and hepatocellular carcinoma2,3,4,5. Additionally, the first zebrafish model of neuroblastoma (NB) was generated by overexpressing MYCN, an oncogene, in the peripheral sympathetic nervous system (PSNS) under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. With this model, it was further demonstrated that activated ALK can synergize with MYCN to accelerate tumor onset and increase tumor penetrance in vivo6.
NB is derived from the sympathoadrenal lineage of the neural crest cells, and is a highly metastatic cancer in children7. It is responsible for 10% of pediatric cancer-related deaths8. Widely metastasized at diagnosis, NB can be clinically presented as tumors primarily originating along the chain of the sympathetic ganglia and the adrenal medulla of PSNS9,10. MYCN amplification is commonly associated with poor outcomes in NB patients11,12. Moreover, LMO1 has been identified as a critical NB susceptibility gene in high-risk cases13,14. Studies found that the transgenic coexpression of MYCN and LMO1 in the PSNS of the zebrafish model not only promotes earlier onset of NB, but also induces widespread metastasis to the tissues and organs that are similar to sites commonly seen in patients with high-risk NB13. Very recently, another metastatic phenotype of NB has also been observed in a newer zebrafish model of NB, in which both MYCN and Lin28B, encoding an RNA binding protein, are overexpressed under control of the d&#946;h promoter16.
The stable transgenic approach in zebrafish is often used to study whether overexpression of a gene of interest could contribute to the normal development and disease pathogenesis14,15. This technique has been successfully used to demonstrate the importance of multiple genes and pathways to NB tumorigenesis6,16,17,18,19,20. This paper will introduce how the transgenic fish line that overexpresses both MYCN and LMO1 in the PSNS was created and how it was demonstrated that the cooperation of these two oncogenes accelerate the onset of NB tumorigenesis and metastasis13. First, the transgenic line that overexpresses EGFP-MYCN under control of the d&#946;h promoter (designated MYCN line) was developed by injecting the d&#946;h-EGFP-MYCN construct into one-cell stage of wild-type (WT) AB embryos, as previously described6,17. A separate transgenic line that overexpresses LMO1 in the PSNS (designated LMO1 line) was developed by coinjecting two DNA constructs, d&#946;h-LMO1 and d&#946;h-mCherry, into WT embryos at the one-cell stage13. It has been previously demonstrated that coinjected double DNA constructs can be cointegrated into the fish genome; therefore, LMO1 and mCherry are coexpressed in the PSNS cells of the transgenic animals. Once the injected F0 embryos reached sexual maturity, they were then out-crossed with WT fish for the identification of positive fish with transgene(s) integration. Briefly, the F1 offspring were first screened by fluorescent microscopy for mCherry expression in the PSNS cells. The germline integration of LMO1 in mCherry-positive fish was further confirmed by genomic PCR and sequencing. After successful identification of each transgenic line, the progeny of heterozygous MYCN and LMO1 transgenic fish were interbred to generate a compound fish line expressing both MYCN and LMO1 (designated MYCN;LMO1 line). Tumor-bearing MYCN;LMO1 fish were monitored by fluorescent microscopy biweekly for the evidence of metastatic tumors in the regions distant to the primary site, interrenal gland region (IRG, zebrafish equivalent of human adrenal gland)13. To confirm the metastasis of tumors in MYCN;LMO1 fish, histological and immunohistochemical analyses were applied.

<table><tbody><tr><td>3,3&amp;rsquo;-Diaminobenzidine (DAB) Vector Kit</td><td>Vector</td><td>SK-4100</td><td></td></tr><tr><td>Acetic Acid</td><td>Fisher Scientific / Acros Organic</td><td>64-19-7</td><td></td></tr><tr><td>Agarose GP2</td><td>Midwest Scientific</td><td>009012-36-6</td><td></td></tr><tr><td>Anti-Tyrosine Hydroxylase (TH) Antibody</td><td>Pel-Freez</td><td>P40101</td><td></td></tr><tr><td>Avidin/Biotin Blocking Kit</td><td>Vector</td><td>SP-2001</td><td></td></tr><tr><td>BOND Intense R Detection</td><td>Leica Biosystems</td><td>DS9263</td><td></td></tr><tr><td>BOND primary antibody diluent</td><td>Leica Biosystems Newcastle, Ltd.</td><td>AR9352</td><td></td></tr><tr><td>BOND-MAX IHC instrument</td><td>Leica Biosystems Newcastle, Ltd.</td><td>N/A</td><td>fully automated IHC staining system</td></tr><tr><td>CH211-270H11 BAC clone</td><td>BACPAC resources center (BRFC)</td><td>N/A</td><td></td></tr><tr><td>Compound microscope equipped with DP71 camera</td><td>Olympus</td><td>AX70</td><td></td></tr><tr><td>Cytoseal XYL (xylene based mounting medium)</td><td>Richard-Allan Scientific</td><td>8312-4</td><td></td></tr><tr><td>Eosin</td><td>Leica</td><td>3801601</td><td>ready-to-use (no preparation needed)</td></tr><tr><td>Ethanol</td><td>Carolina</td><td>86-1263</td><td></td></tr><tr><td>Expand Long Template PCR System</td><td>Roche Applied Science, IN</td><td>11681834001</td><td></td></tr><tr><td>Gateway BP Clonase II enzyme mix</td><td>Invitrogen, CA</td><td>11789-020</td><td></td></tr><tr><td>Gateway LR Clonase II enzyme mix</td><td>Invitrogen, CA</td><td>11791-100</td><td></td></tr><tr><td>Goat anti-Rb secondary antibody (Biotinylated)</td><td>Dako</td><td>E0432</td><td></td></tr><tr><td>Hematoxylin Solution, Harris Modified</td><td>Sigma Aldrich Chemical Company Inc. / SAFC</td><td>HHS-32-1L</td><td></td></tr><tr><td>HRP Avidin D</td><td>Vector</td><td>A-2004</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Aqua Solutions</td><td>4360-1L</td><td></td></tr><tr><td>Hydrogen Peroxide, 3%</td><td>Fisher Scientific</td><td>H324-500</td><td></td></tr><tr><td>I-SceI enzyme</td><td>New England Biolabs, MA</td><td>R0694L</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Teknova, Inc.</td><td>K2150</td><td></td></tr><tr><td>Kimberly-Clark Professional Kimtech Science Kimwipes</td><td>Fisher Scientific</td><td>34133</td><td></td></tr><tr><td>Lithium Carbonate</td><td>Sigma Aldrich Chemical Company Inc. / SAFC</td><td>554-13-2</td><td></td></tr><tr><td>Microtome for sectioning</td><td>Leica Biosystems</td><td>RM2255</td><td></td></tr><tr><td>One Shot TOP10 Chemically Competent&amp;nbsp;E. coli</td><td>Invitrogen</td><td>C404006</td><td></td></tr><tr><td>p3E-polyA</td><td>&amp;nbsp;Dr. Chi-Bin Chien, Univ. of Utah</td><td>N/A</td><td>a generous gift&lt;br/&gt; (Please refer to webpage http://tol2kit.genetics.utah.edu/index.php/Main_Page to obtain material, which is freely distrubted as described.)</td></tr><tr><td>Parafin wax</td><td>Surgipath Paraplast</td><td>39603002</td><td>Parrafin to parafin</td></tr><tr><td>Paraformaldehyde</td><td>Alfa Aesar</td><td>A11313</td><td></td></tr><tr><td>pDEST vector (modified destination vector containing I-SceI recognition sites)</td><td>Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germany</td><td>N/A</td><td>a generous gift</td></tr><tr><td>pDONR 221 gateway donor vector</td><td>Thermo Fisher Scientific</td><td>12536-017</td><td></td></tr><tr><td>pDONRP4-P1R donor vector</td><td>&amp;nbsp;Dr. Chi-Bin Chien, Univ. of Utah</td><td>N/A</td><td>a generous gift</td></tr><tr><td>Phenol red, 0.5%</td><td>Sigma Aldrich</td><td>&amp;nbsp;P0290</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS), 10X</td><td>BioRad</td><td>1610780</td><td></td></tr><tr><td>Picrosirrius red stain kit</td><td>Polysciences</td><td>24901-250</td><td></td></tr><tr><td>pME-mCherry</td><td>Addgene</td><td>26028</td><td></td></tr><tr><td>Proteinase K, recombinant, PCR Grade</td><td>Roche</td><td>21712520</td><td></td></tr><tr><td>QIAprep Spin MiniPrep Kit</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>RDO Rapid Decalcifier</td><td>Apex Enginerring</td><td>RDO04</td><td></td></tr><tr><td>Sodium Azide (NaN3)</td><td>Sigma Aldrich</td><td>26628-22-8</td><td></td></tr><tr><td>Stereo fluorescence microscope</td><td>Leica</td><td>MZ10F</td><td></td></tr><tr><td>Stereoscopic fluorescence microscope equipped with a digital sight DS-U1 camera for imaging</td><td>Nikon</td><td>SMZ-1500</td><td></td></tr><tr><td>Taq DNA Polymerase</td><td>New England Biolabs, MA</td><td>M0273L</td><td></td></tr><tr><td>Tissue-Tek VIP&amp;reg;&amp;nbsp;6 AI Vacuum Infiltration Processor</td><td>Sakura</td><td>N/A</td><td>Model #: VIP-6-A1</td></tr><tr><td>Tricaine-S</td><td>Western Chemical Incorporated</td><td>20513</td><td></td></tr><tr><td>Xylene</td><td>Thermo Fisher Scientific</td><td>X3P1GAL</td><td></td></tr></tbody></table>

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.

To determine whether LMO1 synergizes with MYCN to affect NB pathogenesis, transgenic constructs that drive expression of either LMO1 (d&#946;h:LMO1 and d&#946;h:mCherry) or MYCN (d&#946;h:EGFP-MYCN) in the PSNS cells under control of the d&#946;h promoter were injected into zebrafish embryos13. As illustrated in Figure 1A, after the development of stable transgenic lines and validation of their genotypes, heterozygous MYCN and LMO1 fish were interbred. Their offspring were sorted for MYCN (EGFP+) or LMO1 (mCherry+) expression at 1 dpf or 3-4 dpf, respectively. MYCN overexpression has been shown to suppress PSNS development6. Thus, the EGFP-MYCN expression is more prominent in the non-PSNS dopaminergic neuronal cells at 1 dpf6, such as the cranial ganglia (CA), arch-associated catecholaminergic neurons (AAC), and medulla oblongata (MO). Due to the instability of EGFP-MYCN protein, the EGFP signal becomes dimmer after 2 days. In contrast, the mCherry expression is prominent in both PSNS cells, such as the superior cervical ganglion and non-PSNS dopaminergic neuronal cells, which includes CA, AAC, and MO, from 3 dpf and onwards6. Hence, the offspring of MYCN and LMO1 mating are sorted&#160;at 1 dpf for MYCN (EGFP+) and at 3 dpf for LMO1 (mCherry+). The sorted fish were separated into different genotypic groups, as follows: i) MYCN, ii) LMO1, iii) MYCN;LMO1, and iv) WT, and raised in the identical conditions. Beginning at 4 wpf, the offspring were screened biweekly for the evidence of tumors by fluorescent microscopy. Fluorescent positive tumor masses were detected in the tissues and organs distant from the primary tumor site in the compound transgenic fish with overexpression of both MYCN and LMO1, but not in the transgenic fish with expression of MYCN alone (Figure 1B).
To further verify the metastasis in these transgenic animals, tumor-bearing MYCN and MYCN;LMO1 transgenic fish at 5 to 9 months of age were subjected for paraffin sectioning and immunohistochemical analyses with an antibody against the neuroblastoma marker, TH. The representative results for a MYCN;LMO1 fish is presented in Figure 2. H&#38;E staining of the sagittal sections showed that the primary tumor arose from the interrenal gland region, the zebra&#64257;sh equivalent of the human adrenal gland, which is the most common site of primary disease in neuroblastoma patient12,22 (Figures 2A,B). Composed of tiny, undifferentiated, and round cancerous cells with hyperchromatic nuclei, the tumor often formed layers that were histologically similar to the human neuroblastomas as described previously6 (Figures 2A,B). Consistent with the observation by fluorescent microscopy (Figure 1B), widespread tumor masses distant from the inter-renal gland were detected in multiple regions, including: distal portion of the kidney (the primary adult hematopoietic niche of zebrafish and comparable to the mammalian bone marrow23) (Figure 2A,C), orbit (Figures 2A,D), gill (analogous to the mammalian lung24) (Figures 2A,E), spleen (analogous to the mammalian lymph node25) (Figure 2A and 2F) and inner wall of atrial chamber of heart (Figures 2G). Many of these metastases recapitulate the common sites of metastases seen in patients with high-risk neuroblastoma, which include the bone marrow, lymph node, orbit region and lung13,23,24,25. Further immunohistochemistry with the antibody against TH confirmed the PSNS neuroblast lineage of tumor cells at the primary tumor and all the metastatic sites (Figures 2H-M).
In an effort to better understand the mechanisms underlying the synergy between MYCN and LMO1 in accelerating tumor metastatic spread, it was discovered that the expression levels of a panel of genes involved in tumor cell to extracellular matrix interaction were significantly elevated in the fish tumors with overexpression of both MYCN and LMO113. To examine whether the extracellular matrix was indeed affected by the altered gene expression in LMO1-overexpressing tumors leading to enhanced tumor dissemination or migration, the paraf&#64257;n sections from MYCN-only and MYCN;LMO1 tumors were stained with Picrosirius red (PSR), a highly selective stain for collagen &#64257;bers, to assess collagen deposition and extracellular matrix (ECM) stiffness26,27. As demonstrated in Figure 3A-E, there were signi&#64257;cantly amplified amounts and increased thickness of PSR-stained collagen &#64257;bers found in MYCN;LMO1 tumors, when compared to the tumors only expressing MYCN alone. Together, these results demonstrate that the overexpression of LMO1 can remodel the tumor ECM to increase its stiffness, and therefore, facilitating tumor cell dissemination.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62416/62416fig01v2.jpg" /> 
Figure 1: Illustration of the tumor screening assay on offspring from the breeding of MYCN and LMO1 transgenic zebrafish lines. (A) Schematic illustration of sorting and tumor screening of MYCN and/or LMO1-overexpressing stable transgenic fish for neuroblastoma study. Upper panels: Representative pictures of EGFP-MYCN+ embryos at 1 day postfertilization (dpf) (dorsal view on the left and lateral view on the right). Lower panels: Representative pictures of mCherry-LMO1+ embryos at 3 dpf (lateral view on the left and ventral view on the right). AAC, arch-associated catecholaminergic neurons; CA, cranial ganglia; and MO, medulla oblongata. Scale bar, 100 &#181;m. Created with BioRender.com (B) Coexpression of LMO1 and MYCN promotes neuroblastoma metastasis. Left: Transgenic fish overexpressing MYCN alone (MYCN) with EGFP-expressing tumor (white arrow) at 36 months of age. Right: Transgenic fish overexpressing both MYCN and LMO1 (MYCN;LMO1) with a mCherry-positive tumor mass at the primary site (IRG, white arrow) and multiple metastatic sites (solid arrowheads) at 36 months of age. Scale bar, 1 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62416/62416fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62416/62416fig02.jpg" /> 
Figure 2: Co-overexpression of MYCN and LMO1 promotes distant metastases of neuroblastoma in transgenic zebrafish model.&#160;(A-G) H&#38;E-stained sagittal sections of MYCN;LMO1 transgenic fish while at 6 months of age. (H-M) Magnified views of immunohistochemical analyses of MYCN;LMO1 transgenic fish in sagittal tissue sections, using tyrosine hydroxylase (TH) antibody. White box outlines the interrenal gland (b, h), with magnified views in panels B and H. Disseminated tumor cells were found in kidney marrow (c, i, C, and I with solid black arrowheads), the sclera of the eye (d, j, D, and J with black outlined and white filled open arrows), the gill (e, k, E, and K with black outlined and white filled open arrowheads), the spleen (f, l, F, and L with solid black arrows), and the heart chamber (G and M with black outlined and white filled double arrowheads). Scale bars, 100 &#181;m (A) and 50 &#181;m (B-M). This figure has been modified from Zhu, S. et al. LMO1 Synergizes with MYCN to Promote Neuroblastoma Initiation and Metastasis. Cancer Cell. 32, 310-323 (2017)13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62416/62416fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62416/62416fig03.jpg" /> 
Figure 3: Increased LMO1 expression promotes collagen deposition and ECM stiffness leading to facilitated tumor cell dissemination in zebrafish models. (A-D) Representative light microscopy images of collagen fibers stained by Picrosirius red (PSR) in MYCN only (A,B) or MYCN;LMO1 (C,D) transgenic zebrafish. (B) and (D) are magnified from the boxed areas of (A) and (C), using arrows (A and B) and arrowheads (C and D) to indicate the PSR-positive collagen fibers, respectively. Scale bars, 100 &#181;m (A and C) and 50 &#181;m (B and D). (E) Quantification of PSR-stained areas on tumor sections of MYCN only or MYCN;LMO1 transgenic fish. Results were normalized to the mean of PSR-stained areas in MYCN-only tumors. The statistics present as the mean &#177; SD of three MYCN-only or three MYCN;LMO1 tumors; p = 0.02 by two-tailed t-test. This figure has been modified from Zhu, S. et al. LMO1 Synergizes with MYCN to Promote Neuroblastoma Initiation and Metastasis. Cancer Cell. 32, 310-323 (2017)13. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62416/62416fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Zebrafish Model of Neuroblastoma Metastasis at JoVE.com

Zebrafish Model of Neuroblastoma Metastasis

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 ...

zebrafish-model-of-neuroblastoma-metastasis

Nanyang Technological University

Research

JoVE Journal

Cancer Research

2.7K Views.  Mayo Clinic College of Medicine, Mayo Clinic Cancer Center. Deze methoden werden gebruikt om het eerste zebravismodel van neuroblastoommetastase te ontwikkelen. En om aan te tonen dat dit model veel kenmerken van metastase bij patiënten met een hoog risico op neuroblastoom getrouw kan samenvatten. Het belangrijkste voordeel van dit gemetastaseerde zebravismodel is de real-time in vivo beeldvorming van tumorverspreiding om beter te begrijpen wanneer en mogelijk hoe metastase optreedt.Deze techniek is niet specifiek voor neuroblastoom. Het kan w...

Video: Zebrafish Model of Neuroblastoma Metastasis

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

This method utilizes zebrafish embryos to efficiently test the vascular invasive ability of cancer cells. Fluorescent cancer cells are injected into the precardiac sinus or yolk sac of developing embryos. Cancer cell vascular invasion and extravasation is assessed via fluorescence microscopy of the tail region 24 to 96 hr later.

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

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

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

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

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

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

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

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

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

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

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 ...

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 ...

Live Imaging of Microtubule Dynamics in Glioblastoma Cells Invading the Zebrafish Brain

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment failure is mainly due to the invasiveness of GBM cells, which speaks for the need for a better understanding of GBM motile properties. To investigate the molecular mechanism supporting GBM invasion, new physiological models enabling in-depth characterization of protein dynamics during invasion are required. These observations would pave the way to the discovery of novel targets to block tumor infiltration and improve patient outcomes. This paper reports how an orthotopic xenograft of GBM cells in the zebrafish brain permits subcellular intravital live imaging. Focusing on microtubules (MTs), we describe a procedure for MT labeling in GBM cells, microinjecting GBM cells in the transparent brain of 3 days post fertilization (dpf) zebrafish larvae, intravital imaging of MTs in the disseminating xenografts, altering MT dynamics to assess their role during GBM invasion, and analyzing the acquired data.

We report a technique permitting live imaging of microtubule dynamics in glioblastoma (GBM) cells invading a vertebrate brain tissue. Coupling the orthotopic injection of fluorescently tagged GBM cells into a transparent zebrafish brain with high-resolution intravital imaging allows the measurement of cytoskeleton dynamics during in situ cancer invasion.

With a dismal median survival time in real populations-between 6 to 15 months-glioblastoma (GBM) is the most devastating malignant brain tumor. Treatment ...

Zebrafish Model of Neuroblastoma Metastasis

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