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 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 (dβh) 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.
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 (dβh) 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 dβh 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 dβh promoter (designated MYCN line) was developed by injecting the dβh-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, dβh-LMO1 and dβh-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.
All research methods using zebrafish and animal care/maintenance were performed in compliance with the institutional guidelines at Mayo Clinic.
1. Preparation and microinjection of transgene constructs for the development of LMO1 transgenic zebrafish line with overexpression in PSNS
2. Screen and verify LMO1 transgenic fish line for germline transmission of LMO1 and mCherry
3. Outcross of LMO1 and MYCN transgenic lines to create metastatic model
4. Visualizing tumor burden in transgenic zebrafish lines
5. Tissue processing and paraffin sectioning of tumor-bearing fish
NOTE: Perform this step to characterize the spontaneously developed primary and/or metastatic tumors in MYCN and MYCN;LMO1 transgenic fish.
6. Hematoxylin and eosin (H&E) staining of paraffin sections for pathology review
7. Immunohistochemical Analysis (IHC) with antibodies against NB marker and overexpressed transgenes to further confirm the spread of tumor and their sympathoadrenal lineage property
8. Picrosirius red staining of paraffin slides for collagen accumulation in tumors as mechanism study
To determine whether LMO1 synergizes with MYCN to affect NB pathogenesis, transgenic constructs that drive expression of either LMO1 (dβh:LMO1 and dβh:mCherry) or MYCN (dβh:EGFP-MYCN) in the PSNS cells under control of the dβh 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 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&E staining of the sagittal sections showed that the primary tumor arose from the interrenal gland region, the zebrafish 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 paraffin sections from MYCN-only and MYCN;LMO1 tumors were stained with Picrosirius red (PSR), a highly selective stain for collagen fibers, to assess collagen deposition and extracellular matrix (ECM) stiffness26,27. As demonstrated in Figure 3A-E, there were significantly amplified amounts and increased thickness of PSR-stained collagen fibers 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.
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 µm. 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. Please click here to view a larger version of this figure.
Figure 2: Co-overexpression of MYCN and LMO1 promotes distant metastases of neuroblastoma in transgenic zebrafish model. (A-G) H&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 µm (A) and 50 µm (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. Please click here to view a larger version of this figure.
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 µm (A and C) and 50 µm (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 ± 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. Please click here to view a larger version of this figure.
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'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.
The authors have nothing to disclose.
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.).
3,3’-Diaminobenzidine (DAB) Vector Kit | Vector | SK-4100 | |
Acetic Acid | Fisher Scientific / Acros Organic | 64-19-7 | |
Agarose GP2 | Midwest Scientific | 009012-36-6 | |
Anti-Tyrosine Hydroxylase (TH) Antibody | Pel-Freez | P40101 | |
Avidin/Biotin Blocking Kit | Vector | SP-2001 | |
BOND Intense R Detection | Leica Biosystems | DS9263 | |
BOND primary antibody diluent | Leica Biosystems Newcastle, Ltd. | AR9352 | |
BOND-MAX IHC instrument | Leica Biosystems Newcastle, Ltd. | N/A | fully automated IHC staining system |
CH211-270H11 BAC clone | BACPAC resources center (BRFC) | N/A | |
Compound microscope equipped with DP71 camera | Olympus | AX70 | |
Cytoseal XYL (xylene based mounting medium) | Richard-Allan Scientific | 8312-4 | |
Eosin | Leica | 3801601 | ready-to-use (no preparation needed) |
Ethanol | Carolina | 86-1263 | |
Expand Long Template PCR System | Roche Applied Science, IN | 11681834001 | |
Gateway BP Clonase II enzyme mix | Invitrogen, CA | 11789-020 | |
Gateway LR Clonase II enzyme mix | Invitrogen, CA | 11791-100 | |
Goat anti-Rb secondary antibody (Biotinylated) | Dako | E0432 | |
Hematoxylin Solution, Harris Modified | Sigma Aldrich Chemical Company Inc. / SAFC | HHS-32-1L | |
HRP Avidin D | Vector | A-2004 | |
Hydrochloric Acid | Aqua Solutions | 4360-1L | |
Hydrogen Peroxide, 3% | Fisher Scientific | H324-500 | |
I-SceI enzyme | New England Biolabs, MA | R0694L | |
Kanamycin sulfate | Teknova, Inc. | K2150 | |
Kimberly-Clark Professional Kimtech Science Kimwipes | Fisher Scientific | 34133 | |
Lithium Carbonate | Sigma Aldrich Chemical Company Inc. / SAFC | 554-13-2 | |
Microtome for sectioning | Leica Biosystems | RM2255 | |
One Shot TOP10 Chemically Competent E. coli | Invitrogen | C404006 | |
p3E-polyA | Dr. Chi-Bin Chien, Univ. of Utah | N/A | a generous gift (Please refer to webpage http://tol2kit.genetics.utah.edu/index.php/Main_Page to obtain material, which is freely distrubted as described.) |
Parafin wax | Surgipath Paraplast | 39603002 | Parrafin to parafin |
Paraformaldehyde | Alfa Aesar | A11313 | |
pDEST vector (modified destination vector containing I-SceI recognition sites) | Dr. C. Grabher, Karlsruhe Institute of Technology, Karlsruhe, Germany | N/A | a generous gift |
pDONR 221 gateway donor vector | Thermo Fisher Scientific | 12536-017 | |
pDONRP4-P1R donor vector | Dr. Chi-Bin Chien, Univ. of Utah | N/A | a generous gift |
Phenol red, 0.5% | Sigma Aldrich | P0290 | |
Phosphate Buffered Saline (PBS), 10X | BioRad | 1610780 | |
Picrosirrius red stain kit | Polysciences | 24901-250 | |
pME-mCherry | Addgene | 26028 | (DBH construct) |
Proteinase K, recombinant, PCR Grade | Roche | 21712520 | |
QIAprep Spin MiniPrep Kit | Qiagen | 27104 | |
RDO Rapid Decalcifier | Apex Enginerring | RDO04 | |
Sodium Azide (NaN3) | Sigma Aldrich | 26628-22-8 | |
Stereo fluorescence microscope | Leica | MZ10F | |
Stereoscopic fluorescence microscope equipped with a digital sight DS-U1 camera for imaging | Nikon | SMZ-1500 | |
Taq DNA Polymerase | New England Biolabs, MA | M0273L | |
Tissue-Tek VIP® 6 AI Vacuum Infiltration Processor | Sakura | N/A | Model #: VIP-6-A1 |
Tricaine-S | Western Chemical Incorporated | 20513 | |
Xylene | Thermo Fisher Scientific | X3P1GAL |