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.     

Baohuoside i suppresses breast cancer metastasis by downregulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 pathway

BackgroundBreast cancer is the most common malignancy in women and metastasis is the leading cause of breast cancer-related deaths. Our previous studies have shown that XIAOPI formula, a newly approved drug by the State Food and Drug Administration of China (SFDA), can dramatically inhibit breast cancer metastasis by modulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 (TAMs/CXCL1) pathway. However, the bioactive compound accounting for the anti-metastatic effect of XIAOPI formula remains unclear.PurposeThis study was designed to separate the anti-metastatic bioactive compound from XIAOPI formula and to elucidate its action mechanisms.Study Design/MethodsTAMs/CXCL1 promoter activity-guided fractionation and multiple chemical structure identification approaches were conducted to screen the bioactive compound from XIAOPI formula. Breast cancer cells and TAMs were co-cultured in vitro or co-injected in vivo to simulate their coexistence. Multiple molecular biology experiments, zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts were applied to validate the anti-metastatic activity of the screened compound.ResultsBioactivity-guided fractionation identified baohuoside I (BHS) as the key bioactive compound of XIAOPI formula in inhibiting TAMs/CXCL1 promoter activity. Functional studies revealed that BHS could significantly inhibit the migration and invasion as well as the expression of metastasis-related proteins in both human and mouse breast cancer cells, along with decreasing the proportion of breast cancer stem cells (CSCs). Furthermore, BHS could suppress the M2 phenotype polarization of TAMs and therefore attenuate their CXCL1 expression and secretion. Notably, mechanistic investigations validated TAMs/CXCL1 as the crucial target of BHS in suppressing breast cancer metastasis as exogenous addition of CXCL1 significantly abrogated the anti-metastatic effect of BHS on breast cancer cells. Moreover, BHS was highly safe in vivo as it exhibited no observable embryotoxicity or teratogenic effect on zebrafish embryos. More importantly, BHS remarkably suppressed breast cancer metastasis and TAMs/CXCL1 activity in both zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts.ConclusionThis study not only provides novel insights into TAMs/CXCL1 as a reliable screening target for anti-metastatic drug discovery, but also suggests BHS as a promising candidate drug for metastatic breast cancer treatment.     

A new zebrafish model produced by TILLING of SOD1-related amyotrophic lateral sclerosis replicates key features of the disease and represents a tool for in vivo therapeutic screening

Mutations in the superoxide dismutase gene (SOD1) are one cause of familial amyotrophic lateral sclerosis [ALS; also known as motor neuron disease (MND)] in humans. ALS is a relentlessly progressive neurodegenerative disease and, to date, there are no neuroprotective therapies with significant impact on the disease course. Current transgenic murine models of the disease, which overexpress mutant SOD1, have so far been ineffective in the identification of new therapies beneficial in the human disease. Because the human and the zebrafish (Danio rerio) SOD1 protein share 76% identity, TILLING (‘targeting induced local lesions in genomes’) was carried out in collaboration with the Sanger Institute in order to identify mutations in the zebrafish sod1 gene. A T70I mutant zebrafish line was characterised using oxidative stress assays, neuromuscular junction (NMJ) analysis and motor function studies. The T70I sod1 zebrafish model offers the advantage over current murine models of expressing the mutant Sod1 protein at a physiological level, as occurs in humans with ALS. The T70I sod1 zebrafish demonstrates key features of ALS: an early NMJ phenotype, susceptibility to oxidative stress and an adult-onset motor neuron disease phenotype. We have demonstrated that the susceptibility of T70I sod1 embryos to oxidative stress can be used in a drug screening assay, to identify compounds that merit further investigation as potential therapies for ALS.KEY WORDS: MND, ALS, SOD1, Zebrafish     

Automated whole animal bio-imaging assay for human cancer dissemination

A quantitative bio-imaging platform is developed for analysis of human cancer dissemination in a short-term vertebrate xenotransplantation assay. Six days after implantation of cancer cells in zebrafish embryos, automated imaging in 96 well plates coupled to image analysis algorithms quantifies spreading throughout the host. Findings in this model correlate with behavior in long-term rodent xenograft models for panels of poorly- versus highly malignant cell lines derived from breast, colorectal, and prostate cancer. In addition, cancer cells with scattered mesenchymal characteristics show higher dissemination capacity than cell types with epithelial appearance. Moreover, RNA interference establishes the metastasis-suppressor role for E-cadherin in this model. This automated quantitative whole animal bio-imaging assay can serve as a first-line in vivo screening step in the anti-cancer drug target discovery pipeline.     

Zebrafish embryos and larvae: a new generation of disease models and drug screens

Technological innovation has helped the zebrafish embryo gain ground as a disease model and an assay system for drug screening. Here, we review the use of zebrafish embryos and early larvae in applied biomedical research, using selected cases. We look at the use of zebrafish embryos as disease models, taking fetal alcohol syndrome and tuberculosis as examples. We discuss advances in imaging, in culture techniques (including microfluidics), and in drug delivery (including new techniques for the robotic injection of compounds into the egg). The use of zebrafish embryos in early stages of drug safety-screening is discussed. So too are the new behavioral assays that are being adapted from rodent research for use in zebrafish embryos, and which may become relevant in validating the effects of neuroactive compounds such as anxiolytics and antidepressants. Readouts, such as morphological screening and cardiac function, are examined. There are several drawbacks in the zebrafish model. One is its very rapid development, which means that screening with zebrafish is analogous to "screening on a run-away train." Therefore, we argue that zebrafish embryos need to be precisely staged when used in acute assays, so as to ensure a consistent window of developmental exposure. We believe that zebrafish embryo screens can be used in the pre-regulatory phases of drug development, although more validation studies are needed to overcome industry scepticism. Finally, the zebrafish poses no challenge to the position of rodent models: it is complementary to them, especially in early stages of drug research.     

Aiduqing formula suppresses breast cancer metastasis via inhibiting CXCL1-mediated autophagy

BackgroundMetastasis is the most common lethal cause of breast cancer-related death. Recent studies have implied that autophagy is closely implicated in cancer metastasis. Therefore, it is of great significance to explore autophagy-related molecular targets involved in breast cancer metastasis and to develop therapeutic drugs.PurposeThis study was designed to investigate the anti-metastatic effects and autophagy regulatory mechanisms of Aiduqing (ADQ) formula on breast cancer.Study Design/MethodsMultiple cellular and molecular experiments were conducted to investigate the inhibitory effects of ADQ formula on autophagy and metastasis of breast cancer cells in vitro. Meanwhile, autophagic activator/inhibitor as well as CXCL1 overexpression or interference plasmids were used to investigate the underlying mechanisms of ADQ formula in modulating autophagy-mediated metastasis. Furthermore, the zebrafish xenotransplantation model and mouse xenografts were applied to validate the inhibitory effect of ADQ formula on autophagy-mediated metastasis in breast cancer in vivo.ResultsADQ formula significantly inhibited the proliferation, migration, invasion and autophagy but induced apoptosis of high-metastatic breast cancer cells in vitro. Similar results were also observed in starvation-induced breast cancer cells which exhibited elevated metastatic ability and autophagy activity. Mechanism investigations further approved that either CXCL1 overexpression or autophagic activator rapamycin can significantly abrogated the anti-metastatic effects of ADQ formula, suggesting that CXCL1-mediated autophagy may be the crucial pathway of ADQ formula in suppressing breast cancer metastasis. More importantly, ADQ formula suppressed breast cancer growth, autophagy, and metastasis in both the zebrafish xenotransplantation model and the mouse xenografts.ConclusionOur study not only revealed the novel function of CXCL1 in mediating autophagy-mediated metastasis but also suggested ADQ formula as a candidate drug for the treatment of metastatic breast cancer.     

Small molecule screening in the zebrafish

The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have successfully been used in chemical genetic screens to elucidate biological pathways and find chemical suppressors. Small molecules discovered by screening zebrafish disease models may also be useful as lead compounds for drug development as there appears to be a high level of conservation of drug activity between mammals and zebrafish. Here we provide the technical aspects of treating embryos with small molecules and performing chemical screens with zebrafish.     

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.     

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.     

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.     

Significance of the zebrafish model in the discovery of bioactive molecules from nature

Natural products have immense therapeutic potential not only due to their structural variation and complexity but also due to their range of biological activities. Research based on natural products has led to the discovery of molecules with biomedical and pharmaceutical applications in different therapeutic areas like cancer, inflammation responses, diabetes, and infectious diseases. There are still several challenges to be overcome in natural product drug discovery research programs and the challenge of high throughput screening of natural substances is one of them. Bioactivity screening is an integral part of the drug discovery process and several in vitro and in vivo biological models are now available for this purpose. Among other well-reported biological models, the zebrafish (Danio rerio) is emerging as an important in vivo model for preclinical studies of synthetic molecules in different therapeutic areas. Zebrafish embryos have a short reproductive cycle, show ease of maintenance at high densities in the laboratory and administration of drugs is a straightforward procedure. The embryos are optically transparent, allowing for the visualization of drug effects on internal organs during the embryogenesis process. In this review, we illustrate the importance of using zebrafish as an important biological model in the discovery of bioactive drugs from natural sources.     

斑马鱼模型在药物筛选中的应用

斑马鱼具有子代数量多、体外受精、胚胎透明、可以做大规模遗传突变筛选等生物学特性, 因此成为一种良好的脊椎动物模式生物。随着研究的深入, 斑马鱼不仅应用于遗传学和发育生物学研究, 而且拓展和延伸到疾病模型和药物筛选领域。作为一种整体动物模型, 斑马鱼能够全面地检测评估化合物的活性和副作用, 实现高内涵筛选。近年来, 科学家们不断地发展出新的斑马鱼疾病模型和新的筛选技术, 并找到了一批活性化合物。这些化合物大多数在哺乳动物模型中也有相似的效果, 其中前列腺素E2(dmPGE2)和来氟米特(Leflunomide)已经进入临床实验, 分别用来促进脐带血细胞移植后的增殖和治疗黑素瘤。这些成果显示了斑马鱼模型很适合用于药物筛选。文章概括介绍了斑马鱼模型的特点和近年来在疾病模型和药物筛选方面的进展, 希望能够帮助人们了解斑马鱼在新药研发中的应用, 并开展基于斑马鱼模型的药物筛选。     

Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation

Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs.     

Zebrafish xenografts as a tool for in vivo studies on human cancer

The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish. This model provides a unique opportunity to monitor tumor-induced angiogenesis, invasiveness, and response to a range of treatments in vivo and in real time. Despite the high conservation of gene function between fish and humans, concern remains that potential differences in zebrafish tissue niches and/or missing microenvironmental cues could limit the relevance and translational utility of data obtained from zebrafish human cancer cell xenograft models. Here, we summarize current data on xenotransplantation of human cells into zebrafish, highlighting the advantages and limitations of this model in comparison to classical murine models of xenotransplantation.     

Zebrafish as a model system for biomedical studies

Zebrafish (Danio rerio) is now firmly recognized as a powerful research model for many areas of biology and medicine. Here, we review some achievements of zebrafish-based assays for modeling human diseases and for drug discovery and development. For drug discovery, zebrafish is especially valuable during the earlier stages of research as its represents a model organism to demonstrate a new treatment’s efficacy and toxicity before more costly mammalian models are used. This review considers some examples of known compounds which exhibit both physiological activity and toxicity in humans and zebrafish. The major advantages of zebrafish embryos consist in their permeability to small molecules added to their incubation medium and chorion transparency that enables the easy observation of the development. Assay of acute toxicity (LC₅₀ estimation) in embryos can also include the screening for developmental disorders as an indicator of teratogenic effects. We have used the zebrafish model for toxicity testing of new drugs based on phospholipid nanoparticles (e.g. doxorubicin). Genome organization and the pathways involved into control of signal transduction appear to be highly conserved between zebrafish and humans and therefore zebrafish may be used for modeling of human diseases. The review provides some examples of zebrafish application in this field.     

异源器官移植研究进展

本文针对异源器官移植中面临的免疫排斥及病原微生物的感染等主要问题,综述了近年来国内外相关方面的最新研究进展,同时对解决这些难题的方法和技术路线进行了总结,并展望了异源器官移植领域的发展道路。     

Generation and Characterization of a genetic zebrafish model of SMA carrying the human SMN2 gene

Background

Animal models of human diseases are essential as they allow analysis of the disease process at the cellular level and can advance therapeutics by serving as a tool for drug screening and target validation. Here we report the development of a complete genetic model of spinal muscular atrophy (SMA) in the vertebrate zebrafish to complement existing zebrafish, mouse, and invertebrate models and show its utility for testing compounds that alter SMN2 splicing.

Results

The human motoneuron disease SMA is caused by low levels, as opposed to a complete absence, of the survival motor neuron protein (SMN). To generate a true model of SMA in zebrafish, we have generated a transgenic zebrafish expressing the human SMN2 gene (hSMN2), which produces only a low amount of full-length SMN, and crossed this onto the smn ^-/- background. We show that human SMN2 is spliced in zebrafish as it is in humans and makes low levels of SMN protein. Moreover, we show that an antisense oligonucleotide that enhances correct hSMN2 splicing increases full-length hSMN RNA in this model. When we placed this transgene on the smn mutant background it rescued the neuromuscular presynaptic SV2 defect that occurs in smn mutants and increased their survival.

Conclusions

We have generated a transgenic fish carrying the human hSMN2 gene. This gene is spliced in fish as it is in humans and mice suggesting a conserved splicing mechanism in these vertebrates. Moreover, antisense targeting of an intronic splicing silencer site increased the amount of full length SMN generated from this transgene. Having this transgene on the smn mutant fish rescued the presynaptic defect and increased survival. This model of zebrafish SMA has all of the components of human SMA and can thus be used to understand motoneuron dysfunction in SMA, can be used as an vivo test for drugs or antisense approaches that increase full-length SMN, and can be developed for drug screening.     

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.