Zebrafish embryo, a tool to study tumor angiogenesis

Zebrafish (Danio rerio) represents a powerful model system in cancer research. Recent observations have shown the possibility to exploit zebrafish to investigate tumor angiogenesis, a pivotal step in cancer progression and target for anti-tumor therapies. Experimental models have been established in zebrafish adults, juveniles, and embryos, each one with its own advantages and disadvantages. Novel genetic tools and high resolution in vivo imaging techniques are also becoming available in zebrafish. It is anticipated that zebrafish will represent an important tool for chemical discovery and gene targeting in tumor angiogenesis. This review focuses on the recently developed tumor angiogenesis models in zebrafish, with particular emphasis to tumor engrafting in zebrafish embryos.     

Zebrafish Models of p53 Functions

Zebrafish models have significantly contributed to our understanding of vertebrate development and, more recently, human disease. The growing number of genetic tools available in zebrafish research has resulted in the identification of many genes involved in developmental and disease processes. In particular, studies in the zebrafish have clarified roles of the p53 tumor suppressor in the formation of specific tumor types, as well as roles of p53 family members during embryonic development. The zebrafish has also been instrumental in identifying novel mechanisms of p53 regulation and highlighting the importance of these mechanisms in vivo. This article will summarize how zebrafish models have been used to reveal numerous, important aspects of p53 function.The zebrafish, Danio rerio, is a small model organism that has long been used to study vertebrate development. Zebrafish embryos are optically clear and develop externally to the mother, facilitating the study of early developmental processes. In addition, zebrafish have increasingly been used in modeling human diseases, including a number of cancers. The availability of forward and reverse genetic tools in the zebrafish has resulted in the identification and characterization of many genes involved in development and disease. One gene that has been extensively studied is the p53 tumor suppressor gene, which is structurally and functionally conserved in the zebrafish. This article will discuss how studies in the zebrafish have increased our understanding of how p53 contributes to the formation of specific tumor types, resulted in the identification of novel mechanisms of p53 regulation, and showed how p53 and p53 family members are involved in embryonic development.     

Zebrafish as a cancer model

The zebrafish has developed into an important model organism for biomedical research over the last decades. Although the main focus of zebrafish research has traditionally been on developmental biology, keeping and observing zebrafish in the lab led to the identification of diseases similar to humans, such as cancer, which subsequently became a subject for study. As a result, about 50 articles have been published since 2000 in which zebrafish were used as a cancer model. Strategies used include carcinogenic treatments, transplantation of mammalian cancer cells, forward genetic screens for proliferation or genomic instability, reverse genetic target-selected mutagenesis to inactivate known tumor suppressor genes, and the generation of transgenics to express human oncogenes. Zebrafish have been found to develop almost any tumor type known from human, with similar morphology and, according to gene expression array studies, comparable signaling pathways. However, tumor incidences are relatively low, albeit highly comparable between different mutants, and tumors develop late in life. In addition, tumor spectra are sometimes different when compared with mice and humans. Nevertheless, the zebrafish model has created its own niche in cancer research, complementing existing models with its specific experimental advantages and characteristics. Examples of these are imaging of tumor progression in living fish by fluorescence, treatment with chemical compounds, and screening possibilities not only for chemical modifiers but also for genetic enhancers and suppressors. This review aims to provide a comprehensive overview of the state of the art of zebrafish as a model in cancer research. (Mol Cancer Res 2008;6(5):685-94).     

A proteoliposome containing apolipoprotein A-I mutant (V156K) enhances rapid tumor regression activity of human origin oncolytic adenovirus in tumor-bearing zebrafish and mice

We recently reported that the efficiency of adenoviral gene delivery and virus stability are significantly enhanced when a proteoliposome (PL) containing apolipoprotein (apo) A-I is used in an animal model. In the current study, we tested tumor removal activity of oncolytic adenovirus (Ad) using PL-containing wildtype (WT) or V156K. Oncolytic Ad with or without PL was injected into tumors of zebrafish and nude mice as a Hep3B tumor xenograft model. The V156K-PL-Ad-injected zebrafish, group showed the lowest tumor tissue volume and nucleic acids in the tumor area, whereas injection of Ad alone did not result in adequate removal of tumor activity. Reactive oxygen species (ROS) contents increased two-fold in tumor-bearing zebrafish; however, the V156K-PL-Ad injected group showed a 40% decrease in ROS levels compared to that in normal zebrafish. After reducing the tumor volume with the V156K-PL-Ad injection, the swimming pattern of the zebrafish changed to be more active and energetic. The oncolytic effect of PL-Ad containing either V156K or WT was about two-fold more enhanced in mice than that of Ad alone 34 days after the injection. Immunohistochemical analysis revealed that the PL-Ad-injected groups showed enhanced efficiency of viral delivery with elevated Ad-E1A staining and a diminished number of proliferating tumor cells. Thus, the antitumor effect of oncolytic Ad was strongly enhanced by a PL-containing apoA-I and its mutant (V156K) without causing side effects in mice and zebrafish models.     

Quantifying the frequency of tumor-propagating cells using limiting dilution cell transplantation in syngeneic zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as tumor-propagating cells, or tumor-initiating cells. It is thought that targeting these self-renewing cells for destruction will block tumor progression and stop relapse, greatly improving patient prognosis. The most common way to determine the frequency of self-renewing cells within a tumor is a limiting dilution cell transplantation assay, in which tumor cells are transplanted into recipient animals at increasing doses; the proportion of animals that develop tumors is used the calculate the number of self-renewing cells within the original tumor sample. Ideally, a large number of animals would be used in each limiting dilution experiment to accurately determine the frequency of tumor-propagating cells. However, large scale experiments involving mice are costly, and most limiting dilution assays use only 10-15 mice per experiment. Zebrafish have gained prominence as a cancer model, in large part due to their ease of genetic manipulation and the economy by which large scale experiments can be performed. Additionally, the cancer types modeled in zebrafish have been found to closely mimic their counterpart human disease. While it is possible to transplant tumor cells from one fish to another by sub-lethal irradiation of recipient animals, the regeneration of the immune system after 21 days often causes tumor regression. The recent creation of syngeneic zebrafish has greatly facilitated tumor transplantation studies. Because these animals are genetically identical, transplanted tumor cells engraft robustly into recipient fish, and tumor growth can be monitored over long periods of time. Syngeneic zebrafish are ideal for limiting dilution transplantation assays in that tumor cells do not have to adapt to growth in a foreign microenvironment, which may underestimate self-renewing cell frequency. Additionally, one-cell transplants have been successfully completed using syngeneic zebrafish and several hundred animals can be easily and economically transplanted at one time, both of which serve to provide a more accurate estimate of self-renewing cell frequency. Here, a method is presented for creating primary, fluorescently-labeled T-cell acute lymphoblastic leukemia (T-ALL) in syngeneic zebrafish, and transplanting these tumors at limiting dilution into adult fish to determine self-renewing cell frequency. While leukemia is provided as an example, this protocol is suitable to determine the frequency of tumor-propagating cells using any cancer model in the zebrafish.     

Different Regulation of Physiological and Tumor Angiogenesis in Zebrafish by Protein Kinase D1 (PKD1)

Protein kinase D isoenzymes (PKDs, Prkds) are serine threonine kinases that belong to the CAMK superfamily. PKD1 is expressed in endothelial cells and is a major mediator of biological responses downstream of the VEGFRs that are relevant for angiogenesis such as endothelial cell migration, proliferation and tubulogenesis in vitro. PKDs also play a critical role in tumor development and progression, including tumor angiogenesis. However, given the plethora of signaling modules that drive angiogenesis, the precise role of PKD1 in both physiological and tumor angiogenesis in vivo has not been worked out so far. This study aimed at dissecting the contribution of PKD1 to physiological blood vessel formation, PKD1 was found to be widely expressed during zebrafish development. As far as physiological angiogenesis was concerned, morpholino-based silencing of PKD1 expression moderately reduced the formation of the intersomitic vessels and the dorsal longitudinal anastomotic vessel in tg(fli1:EGFP) zebrafish. In addition, silencing of PKD1 resulted in reduced formation of the parachordal lymphangioblasts that serves as a precursor for the developing thoracic duct. Interestingly, tumor angiogenesis was completely abolished in PKD1 morphants using the zebrafish/tumor xenograft angiogenesis assay. Our data in zebrafish demonstrate that PKD1 contributes to the regulation of physiological angiogenesis and lymphangiogenesis during zebrafish development and is essential for tumor angiogenesis.     

The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages

Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.     

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis.     

Hypoxia-induced metastasis model in embryonic zebrafish

Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average experimental time for this model is 7 d. Our protocol offers a remarkable opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis.     

Zebrafish models to study hypoxia-induced pathological angiogenesis in malignant and nonmalignant diseases

Most in vivo preclinical disease models are based on mouse and other mammalian systems. However, these rodent-based model systems have considerable limitations to recapitulate clinical situations in human patients. Zebrafish have been widely used to study embryonic development, behavior, tissue regeneration, and genetic defects. Additionally, zebrafish also provides an opportunity to screen chemical compounds that target a specific cell population for drug development. Owing to the availability of various genetically manipulated strains of zebrafish, immune privilege during early embryonic development, transparency of the embryos, and easy and precise setup of hypoxia equipment, we have developed several disease models in both embryonic and adult zebrafish, focusing on studying the role of angiogenesis in pathological settings. These zebrafish disease models are complementary to the existing mouse models, allowing us to study clinically relevant processes in cancer and nonmalignant diseases, which otherwise would be difficult to study in mice. For example, dissemination and invasion of single human or mouse tumor cells from the primary site in association with tumor angiogenesis can be studied under normoxia or hypoxia in zebrafish embryos. Hypoxia-induced retinopathy in the adult zebrafish recapitulates the clinical situation of retinopathy development in diabetic patients or age-related macular degeneration. These zebrafish disease models offer exciting opportunities to understand the mechanisms of disease development, progression, and development of more effective drugs for therapeutic intervention.     

The zebrafish/tumor xenograft angiogenesis assay

Here we describe a method to study tumor angiogenesis in zebrafish (Danio rerio) based on the injection of proangiogenic mammalian tumor cells into the perivitelline space of zebrafish embryos at 48 h post-fertilization. Within 24-48 h, proangiogenic tumor grafts induce a neovascular response originating from the developing subintestinal vessels. This can be observed at macroscopic and microscopic levels after whole-mount alkaline phosphatase staining of wild-type zebrafish embryos, or by fluorescence microscopy in transgenic VEGFR2:G-RCFP embryos in which endothelial cells express the green fluorescent protein under the control of the VEGFR2/KDR promoter. Angiogenesis inhibitors added to the injected cell suspension or to the fish water prevent tumor-induced neovascularization. The assay is rapid and inexpensive, representing a novel tool for investigating tumor angiogenesis and for antiangiogenic drug discovery. Also, gene inactivation by antisense morpholino oligonucleotides injection in zebrafish embryos may allow the identification of genes involved in tumor angiogenesis.     

Zebrafish embryo as a tool to study tumor/endothelial cell cross-talk

Tumor/endothelial cell cross-talk plays a pivotal role in the growth, neovascularization and metastatic dissemination of human cancer. Recent observations have shown that the teleost zebrafish (Danio rerio) may represent a powerful experimental platform in cancer research. Various tumor models have been established in zebrafish adults, juveniles, and embryos and novel genetic tools and high resolution in vivo imaging techniques have been exploited. In particular, grafting of mammalian tumor cells in zebrafish embryo body may simulate early stages of tumor development, neovascularization, and local invasion whereas the injection of cancer cells in the bloodstream of zebrafish embryo may allow the study of metastatic homing and colonization. This review focuses on the recent advances in tumor xenotransplantation in zebrafish embryo for the in vivo study of the cancer neovascularization, invasion and metastatic processes. This article is part of a Special Issue entitled: Animal Models of Disease.     

Cross-Species Array Comparative Genomic Hybridization Identifies Novel Oncogenic Events in Zebrafish and Human Embryonal Rhabdomyosarcoma

Human cancer genomes are highly complex, making it challenging to identify specific drivers of cancer growth, progression, and tumor maintenance. To bypass this obstacle, we have applied array comparative genomic hybridization (array CGH) to zebrafish embryonal rhabdomyosaroma (ERMS) and utilized cross-species comparison to rapidly identify genomic copy number aberrations and novel candidate oncogenes in human disease. Zebrafish ERMS contain small, focal regions of low-copy amplification. These same regions were commonly amplified in human disease. For example, 16 of 19 chromosomal gains identified in zebrafish ERMS also exhibited focal, low-copy gains in human disease. Genes found in amplified genomic regions were assessed for functional roles in promoting continued tumor growth in human and zebrafish ERMS – identifying critical genes associated with tumor maintenance. Knockdown studies identified important roles for Cyclin D2 (CCND2), Homeobox Protein C6 (HOXC6) and PlexinA1 (PLXNA1) in human ERMS cell proliferation. PLXNA1 knockdown also enhanced differentiation, reduced migration, and altered anchorage-independent growth. By contrast, chemical inhibition of vascular endothelial growth factor (VEGF) signaling reduced angiogenesis and tumor size in ERMS-bearing zebrafish. Importantly, VEGFA expression correlated with poor clinical outcome in patients with ERMS, implicating inhibitors of the VEGF pathway as a promising therapy for improving patient survival. Our results demonstrate the utility of array CGH and cross-species comparisons to identify candidate oncogenes essential for the pathogenesis of human cancer.     

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer.KEY WORDS: HRASV12, Cancer, Chordoma, Drug treatment, Rapamycin, Zebrafish     

斑马鱼胚胎和肿瘤细胞评价细胞毒药物活性比较研究*

目的:比较斑马鱼胚胎和肿瘤细胞作为药物筛选模型的优缺点.方法:采用MTT法检测顺铂、紫杉醇、阿霉素、5-氟尿嘧啶四种药物对HL-60和Hela细胞的增殖影响;同时,观察药物对斑马鱼胚胎发育的影响.结果:阿霉素、顺铂及紫杉醇作用于HL-60及Hela细胞的IC50均显著高于作用于斑马鱼胚胎的LD50;而5-FU作用于肿瘤细胞和斑马鱼胚胎的结果与其它药物相反;四种抗肿瘤药物对斑马鱼胚胎的生长发育均有致畸作用.结论:斑马鱼胚胎作为细胞毒类药物筛选模型,对于抗微管类药物较为敏感,但对于抗代谢药敏感性较肿瘤细胞差.     

A Novel Xenograft Model in Zebrafish for High-Resolution Investigating Dynamics of Neovascularization in Tumors

Tumor neovascularization is a highly complex process including multiple steps. Understanding this process, especially the initial stage, has been limited by the difficulties of real-time visualizing the neovascularization embedded in tumor tissues in living animal models. In the present study, we have established a xenograft model in zebrafish by implanting mammalian tumor cells into the perivitelline space of 48 hours old Tg(Flk1:EGFP) transgenic zebrafish embryos. With this model, we dynamically visualized the process of tumor neovascularization, with unprecedented high-resolution, including new sprouts from the host vessels and the origination from VEGFR2⁺ individual endothelial cells. Moreover, we quantified their contributions during the formation of vascular network in tumor. Real-time observations revealed that angiogenic sprouts in tumors preferred to connect each other to form endothelial loops, and more and more endothelial loops accumulated into the irregular and chaotic vascular network. The over-expression of VEGF165 in tumor cells significantly affected the vascularization in xenografts, not only the number and size of neo-vessels but the abnormalities of tumor vascular architecture. The specific inhibitor of VEGFR2, SU5416, significantly inhibited the vascularization and the growth of melanoma xenografts, but had little affects to normal vessels in zebrafish. Thus, this zebrafish/tumor xenograft model not only provides a unique window to investigate the earliest events of tumoral neoangiogenesis, but is sensitive to be used as an experimental platform to rapidly and visually evaluate functions of angiogenic-related genes. Finally, it also offers an efficient and cost-effective means for the rapid evaluation of anti-angiogenic chemicals.     

Zebrafish keratin 8 is expressed at high levels in the epidermis of regenerating caudal fin

Cytokeratins are structural proteins of the intermediate filament family and are mainly expressed in epithelial cells. In several vertebrates it has been shown that keratin 8 is expressed in simple epithelial tissues, some non-epithelial tissue and in hyper-proliferative tissues during development and tumor transformation. We previously cloned and characterised the zebrafish (Danio rerio) homologous cytokeratin 8 cDNA (zfk8) which was described as an epidermal marker during zebrafish development. It has been found that the zfk8 gene is normally expressed in simple epithelia in embryonic and mature zebrafish. Using whole-mount in situ hybridisation, we show in this report that expression of zfk8 is tightly linked to the regeneration of caudal fin and exclusively observed in epidermal cells. It is strongly expressed in the epidermis overlaying the inter-rays zone of regenerating caudal fin. Our results indicate that in zebrafish, cytokeratin 8 is a suitable epidermal marker during regeneration.     

Zebrafish model of KRAS-initiated pancreatic endocrine tumor

Pancreatic cancer constitutes a genetic disease in which somatic mutations in the KRAS proto-oncogene are detected in 95% of cases. Activation of the KRAS proto-oncogene represents an initiating event in pancreatic tumorigenesis. Here, we established a zebrafish pancreatic neoplasia model that recapitulates human pancreatic tumors. Toward this end, we generated a stable CRE/Lox-based zebrafish model system to express oncogenic KRAS^G12D  in the elastase3I domain of the zebrafish pancreas. Lineage tracing experiments showed that early KRAS^G12D -responsive pancreatic progenitors contribute to endocrine in addition to exocrine cells. In this system, 10% and 40% of zebrafish developed pancreatic tumors by 6 and 12 months, respectively. The histological profiles of these experimental tumors bore a striking resemblance to those of pancreatic endocrine tumors. Immunohistochemical analysis including the endocrine cell-specific marker confirmed the pancreatic tumor region as a characteristic endocrine tumor. Taken together, our zebrafish model data revealed that pancreatic endocrine tumors originate from early KRAS^G12D -responsive pancreatic progenitor cells. These findings demonstrated that this zebrafish model may be suitable as an experimental and preclinical system to evaluate different strategies for targeting pancreatic endocrine tumors and ultimately improve the outcome for patients with pancreatic endocrine tumors.     

Many ribosomal protein genes are cancer genes in zebrafish

We have generated several hundred lines of zebrafish (Danio rerio), each heterozygous for a recessive embryonic lethal mutation. Since many tumor suppressor genes are recessive lethals, we screened our colony for lines that display early mortality and/or gross evidence of tumors. We identified 12 lines with elevated cancer incidence. Fish from these lines develop malignant peripheral nerve sheath tumors, and in some cases also other tumor types, with moderate to very high frequencies. Surprisingly, 11 of the 12 lines were each heterozygous for a mutation in a different ribosomal protein (RP) gene, while one line was heterozygous for a mutation in a zebrafish paralog of the human and mouse tumor suppressor gene, neurofibromatosis type 2. Our findings suggest that many RP genes may act as haploinsufficient tumor suppressors in fish. Many RP genes might also be cancer genes in humans, where their role in tumorigenesis could easily have escaped detection up to now.