Canthin-6-One Inhibits Developmental and Tumour-Associated Angiogenesis in Zebrafish.

Tumour-associated angiogenesis play key roles in tumour growth and cancer metastasis. Consequently, several anti-angiogenic drugs such as sunitinib and axitinib have been approved for use as anti-cancer therapies. However, the majority of these drugs target the vascular endothelial growth factor A (VEGFA)/VEGF receptor 2 (VEGFR2) pathway and have shown mixed outcome, largely due to development of resistances and increased tumour aggressiveness. In this study, we used the zebrafish model to screen for novel anti-angiogenic molecules from a library of compounds derived from natural products. From this, we identified canthin-6-one, an indole alkaloid, which inhibited zebrafish intersegmental vessel (ISV) and sub-intestinal vessel development. Further characterisation revealed that treatment of canthin-6-one reduced ISV endothelial cell number and inhibited proliferation of human umbilical vein endothelial cells (HUVECs), suggesting that canthin-6-one inhibits endothelial cell proliferation. Of note, canthin-6-one did not inhibit VEGFA-induced phosphorylation of VEGFR2 in HUVECs and downstream phosphorylation of extracellular signal-regulated kinase (Erk) in leading ISV endothelial cells in zebrafish, suggesting that canthin-6-one inhibits angiogenesis independent of the VEGFA/VEGFR2 pathway. Importantly, we found that canthin-6-one impairs tumour-associated angiogenesis in a zebrafish B16F10 melanoma cell xenograft model and synergises with VEGFR inhibitor sunitinib malate to inhibit developmental angiogenesis. In summary, we showed that canthin-6-one exhibits anti-angiogenic properties in both developmental and pathological contexts in zebrafish, independent of the VEGFA/VEGFR2 pathway and demonstrate that canthin-6-one may hold value for further development as a novel anti-angiogenic drug.

Microinjection of ß-glucan Into Larval Zebrafish (Danio rerio) for the Assessment of a Trained-Like Immunity Phenotype.

The innate immune system can remember previous inflammatory insults, enabling long-term heightened responsiveness to secondary immune challenges in a process termed "trained immunity." Trained innate immune cells undergo metabolic and epigenetic remodelling and, upon a secondary challenge, provide enhanced protection with therapeutic potential. Trained immunity has largely been studied in innate immune cells in vitro or following ex vivo re-stimulation where the primary insult is typically injected into a mouse, adult zebrafish, or human. While highly informative, there is an opportunity to investigate trained immunity entirely in vivo within an unperturbed, intact whole organism. The exclusively innate immune response of larval zebrafish offers an attractive system to model trained immunity. Larval zebrafish have a functional innate immune system by 2 days post fertilisation (dpf) and are amenable to high-resolution, high-throughput analysis. This, combined with their optical transparency, conserved antibacterial responses, and availability of transgenic reporter lines, makes them an attractive alternative model to study trained immunity in vivo. We have devised a protocol where ß-glucan (one of the most widely used experimental triggers of trained immunity) is systemically delivered into larval zebrafish using microinjection to stimulate a trained-like phenotype. Following stimulation, larvae are assessed for changes in gene expression, which indicate the stimulatory effect of ß-glucan. This protocol describes a robust delivery method of one of the gold standard stimulators of trained immunity into a model organism that is highly amenable to several non-invasive downstream analyses. Key features • This protocol outlines the delivery of one of the most common experimental stimulators of trained immunity into larval zebrafish. • The protocol enables the assessment of a trained-like phenotype in vivo. • This protocol can be applied to transgenic or mutant zebrafish lines to investigate cells or genes of interest in response to ß-glucan stimulation.

In vitro and in vivo accumulation of the anticancer Ru complexes [RuII(cym)(HQ)Cl] and [RuII(cym)(PCA)Cl]Cl.

The cellular accumulation and the underlying mechanisms for the two ruthenium-based anticancer complexes [RuII(cym)(HQ)Cl] 1 (cym = η6-p-cymene, HQ = 8-hydroxyquinoline) and [RuII(cym)(PCA)Cl]Cl 2 (PCA = N-fluorophenyl-2-pyridinecarbothioamide) were investigated in HCT116 human colorectal carcinoma cells. The results showed that the cellular accumulation of both complexes increased over time and with higher concentrations, and that 2 accumulates in greater quantities in cells than 1. Inhibition studies of selected cellular accumulation mechanisms indicated that both 1 and 2 may be transported into the cells by both passive diffusion and active transporters, similar to cisplatin. Efflux experiments indicated that 1 and 2 are subjected to efflux through a mechanism that does not involve p-glycoprotein, as addition of verapamil did not make any difference. Exploring the influence of the Cu transporter by addition of CuCl2 resulted in a higher accumulation of 1 and 2 whilst the amount of Pt detected was slightly reduced when cells were treated with cisplatin. Complexes 1 and 2 were further explored in zebrafish where accumulation and distribution were determined with ICP-MS and LA-ICP-MS. The results correlated with the in vitro observations and zebrafish treated with 2 showed higher Ru contents than those treated with 1. The distribution studies suggested that both complexes mainly accumulated in the intestines of the zebrafish.

Infection-experienced HSPCs protect against infections by generating neutrophils with enhanced mitochondrial bactericidal activity.

Hematopoietic stem and progenitor cells (HSPCs) respond to infection by proliferating and generating in-demand neutrophils through a process called emergency granulopoiesis (EG). Recently, infection-induced changes in HSPCs have also been shown to underpin the longevity of trained immunity, where they generate innate immune cells with enhanced responses to subsequent microbial threats. Using larval zebrafish to live image neutrophils and HSPCs, we show that infection-experienced HSPCs generate neutrophils with enhanced bactericidal functions. Transcriptomic analysis of EG neutrophils uncovered a previously unknown function for mitochondrial reactive oxygen species in elevating neutrophil bactericidal activity. We also reveal that driving expression of zebrafish C/EBPß within infection-naïve HSPCs is sufficient to generate neutrophils with similarly enhanced bactericidal capacity. Our work suggests that this demand-adapted source of neutrophils contributes to trained immunity by providing enhanced protection toward subsequent infections. Manipulating demand-driven granulopoiesis may provide a therapeutic strategy to boost neutrophil function and treat infectious disease.

Platinum(terpyridine) complexes with N-heterocyclic carbene co-ligands: high antiproliferative activity and low toxicity in vivo.

Pt(terpyridine) complexes are well-known DNA intercalators. The introduction of an NHC co-ligand rendered such a complex highly antiproliferative in cancer cells compared to its chlorido derivative. Despite the high potency, zebrafish embryos tolerated the compound well, especially compared to cisplatin. DNA interaction studies support a mode of action related to intercalation.

Uricase-Deficient Larval Zebrafish with Elevated Urate Levels Demonstrate Suppressed Acute Inflammatory Response to Monosodium Urate Crystals and Prolonged Crystal Persistence.

Gout is caused by elevated serum urate leading to the deposition of monosodium urate (MSU) crystals that can trigger episodes of acute inflammation. Humans are sensitive to developing gout because they lack a functional urate-metabolizing enzyme called uricase/urate oxidase (encoded by the UOX gene). A hallmark of long-standing disease is tophaceous gout, characterized by the formation of tissue-damaging granuloma-like structures ('tophi') composed of densely packed MSU crystals and immune cells. Little is known about how tophi form, largely due to the lack of suitable animal models in which the host response to MSU crystals can be studied in vivo long-term. We have previously described a larval zebrafish model of acute gouty inflammation where the host response to microinjected MSU crystals can be live imaged within an intact animal. Although useful for modeling acute inflammation, crystals are rapidly cleared following a robust innate immune response, precluding analysis at later stages. Here we describe a zebrafish uox null mutant that possesses elevated urate levels at larval stages. Uricase-deficient 'hyperuricemic' larvae exhibit a suppressed acute inflammatory response to MSU crystals and prolonged in vivo crystal persistence. Imaging of crystals at later stages reveals that they form granuloma-like structures dominated by macrophages. We believe that uox-/- larvae will provide a useful tool to explore the transition from acute gouty inflammation to tophus formation, one of the remaining mysteries of gout pathogenesis.

Plexin D1 negatively regulates zebrafish lymphatic development.

Lymphangiogenesis is a dynamic process that involves the directed migration of lymphatic endothelial cells (LECs) to form lymphatic vessels. The molecular mechanisms that underpin lymphatic vessel patterning are not fully elucidated and, to date, no global regulator of lymphatic vessel guidance is known. In this study, we identify the transmembrane cell signalling receptor Plexin D1 (Plxnd1) as a negative regulator of both lymphatic vessel guidance and lymphangiogenesis in zebrafish. plxnd1 is expressed in developing lymphatics and is required for the guidance of both the trunk and facial lymphatic networks. Loss of plxnd1 is associated with misguided intersegmental lymphatic vessel growth and aberrant facial lymphatic branches. Lymphatic guidance in the trunk is mediated, at least in part, by the Plxnd1 ligands, Semaphorin 3AA and Semaphorin 3C. Finally, we show that Plxnd1 normally antagonises Vegfr/Erk signalling to ensure the correct number of facial LECs and that loss of plxnd1 results in facial lymphatic hyperplasia. As a global negative regulator of lymphatic vessel development, the Sema/Plxnd1 signalling pathway is a potential therapeutic target for treating diseases associated with dysregulated lymphatic growth.

A New Transgenic Line for Rapid and Complete Neutrophil Ablation.

Zebrafish lines expressing nitroreductase (NTR) in specific cell compartments, which sensitizes those cells to metronidazole (MTZ)-mediated ablation, have proven extremely useful for studying tissue regeneration and investigating cell function. In contrast to many cells, neutrophils are comparatively resistant to the NTR/MTZ targeted ablation strategy. Recently, a rationally engineered variant of NTR (NTR 2.0) has been described that exhibits greatly improved MTZ-mediated ablation efficacy in zebrafish. We show that a transgenic line with neutrophil-restricted expression of NTR 2.0 demonstrates complete neutrophil ablation, with an MTZ dose 100-fold less than current treatment regimens, and with treatment durations as short as 5 h.

Towards a new model of trained immunity: Exposure to bacteria and ß-glucan protects larval zebrafish against subsequent infections.

Once thought to be a feature exclusive to lymphocyte-driven adaptive immunity, immune memory has also been shown to operate as part of the innate immune system following infection to provide an elevated host response to subsequent pathogenic challenge. This evolutionarily conserved process, termed 'trained immunity', enables cells of the innate immune system to 'remember' previous pathogen encounters and mount stronger responses to the same, or different, pathogens after returning to a non-activated state. Here we show that challenging larval zebrafish, that exclusively rely on innate immunity, with live or heat-killed Salmonella typhimurium provides protection to subsequent infection with either Salmonella typhimurium or Streptococcus iniae, that lasts for at least 12 days. We also show that larvae injected with ß-glucan, the well-known trigger of trained immunity, demonstrate enhanced survival to similar live bacterial infections, a phenotype supported by increased cxcl8 expression and neutrophil recruitment to the infection site. These results support the conservation of a trained immunity-like phenotype in larval zebrafish and provide a foundation to exploit the experimental attributes of larval zebrafish to further understand this form of immunological memory.

3,4-Difluorobenzocurcumin Inhibits Vegfc-Vegfr3-Erk Signalling to Block Developmental Lymphangiogenesis in Zebrafish.

Lymphangiogenesis, the formation of new lymphatic vessels from pre-existing vasculature, plays critical roles in disease, including in cancer metastasis and chronic inflammation. Preclinical and recent clinical studies have now demonstrated therapeutic utility for several anti-lymphangiogenic agents, but optimal agents and efficacy in different settings remain to be determined. We tested the anti-lymphangiogenic property of 3,4-Difluorobenzocurcumin (CDF), which has previously been implicated as an anti-cancer agent, using zebrafish embryos and cultured vascular endothelial cells. We used transgenic zebrafish labelling the lymphatic system and found that CDF potently inhibits lymphangiogenesis during embryonic development. We also found that the parent compound, Curcumin, does not inhibit lymphangiogenesis. CDF blocked lymphatic and venous sprouting, and lymphatic migration in the head and trunk of the embryo. Mechanistically, CDF impaired VEGFC-VEGFR3-ERK signalling in vitro and in vivo. In an in vivo pathological model of Vegfc-overexpression, treatment with CDF rescued endothelial cell hyperplasia. CDF did not inhibit the kinase activity of VEGFR3 yet displayed more prolonged activity in vivo than previously reported kinase inhibitors. These findings warrant further assessment of CDF and its mode of action as a candidate for use in metastasis and diseases of aberrant lymphangiogenesis.

A Multitargeted Approach: Organorhodium Anticancer Agent Based on Vorinostat as a Potent Histone Deacetylase Inhibitor.

The combination of more than one bioactive moiety in a multitargeted anticancer agent may result in synergistic activity of its components. Using this concept, bioorganometallic compounds were designed to feature a metal center, a 2-pyridinecarbothioamide (PCA), and a hydroxamic acid, which is found in the anticancer drug vorinostat (SAHA). The organometallics showed inhibitory activity in the nanomolar range against histone deacetylases (HDACs) as the key target for SAHA. In particular, the Rh complex was a potent inhibitor of HDAC6 over HDAC1 and HDAC8. Whereas this complex was highly cytotoxic in human cancer cells, it showed low toxicity in hemolysis studies and zebrafish, demonstrating the role of the metal center. For this complex a slightly reduced expression of vascular endothelial growth factor receptor 2 (VEGFR2) was established, which was upregulated by SAHA. This finding indicates that the new organometallics display different modes of action than their bioactive components.

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes.

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Isolation of Neutrophils from Larval Zebrafish and Their Transplantation into Recipient Larvae for Functional Studies.

Live imaging of neutrophils within optically transparent larval zebrafish has proved a powerful technique to investigate how specific gene products control neutrophil function. To resolve whether a gene contributes to neutrophil function in a cell-autonomous manner necessitates a way to examine gene-deficient neutrophils in an otherwise wild type background. To this end, here we describe methods to harvest fluorescent neutrophils from larval donor zebrafish and transplant them into age-matched recipients. We show that transplanted neutrophils can survive in recipient larvae for at least 3 days providing ample opportunity for functional studies. Focusing on bactericidal activity, we show that transplanted neutrophils phagocytose and kill live bacteria with similar kinetics to nontransplanted neutrophils, indicating that the transplantation process does not influence these neutrophil effector functions. Following the methods described here to transplant neutrophils between gene-deficient and wild type larval zebrafish will enable investigations into whether a gene's contribution to neutrophil function is cell-autonomous.

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

Anticancer organorhodium and -iridium complexes with low toxicity in vivo but high potency in vitro: DNA damage, reactive oxygen species formation, and haemolytic activity.

Redox-modulating anticancer drugs allow the exploitation of altered redox biology observed in many cancer cells. We discovered dinuclear RhIII(Cp*) and IrIII(Cp*) complexes that have in vitro anticancer activity superior to cisplatin and the investigational drug IT-139, while being less toxic in haemolysis and in vivo zebrafish models. The mode of action appears to be related to DNA damage and ROS-mediated stress pathways.

Glycogen synthase kinase 3-ß inhibition induces lymphangiogenesis through ß-catenin-dependent and mTOR-independent pathways.

Lymphatic vessels play an important role in health and in disease. In this study, we evaluated the effects of GSK3-ß inhibition on lung lymphatic endothelial cells in vitro. Pharmacological inhibition and silencing of GSK3-ß resulted in increased lymphangiogenesis of lung lymphatic endothelial cells. To investigate mechanisms of GSK3-ß-mediated lymphangiogenesis, we interrogated the mammalian/mechanistic target of rapamycin pathway and found that inhibition of GSK3-ß resulted in PTEN activation and subsequent decreased activation of AKT, leading to decreased p-P70S6kinase levels, indicating inhibition of the mTOR pathway. In addition, consistent with a negative role of GSK3-ß in ß-catenin stability through protein phosphorylation, we found that GSK3-ß inhibition resulted in an increase in ß-catenin levels. Simultaneous silencing of ß-catenin and inhibition of GSK3-ß demonstrated that ß-catenin is required for GSK3-ß-induced lymphangiogenesis.

Zebrafish facial lymphatics develop through sequential addition of venous and non-venous progenitors.

Lymphatic vessels are known to be derived from veins; however, recent lineage-tracing experiments propose that specific lymphatic networks may originate from both venous and non-venous sources. Despite this, direct evidence of a non-venous lymphatic progenitor is missing. Here, we show that the zebrafish facial lymphatic network is derived from three distinct progenitor populations that add sequentially to the developing facial lymphatic through a relay-like mechanism. We show that while two facial lymphatic progenitor populations are venous in origin, the third population, termed the ventral aorta lymphangioblast (VA-L), does not sprout from a vessel; instead, it arises from a migratory angioblast cell near the ventral aorta that initially lacks both venous and lymphatic markers, and contributes to the facial lymphatics and the hypobranchial artery. We propose that sequential addition of venous and non-venous progenitors allows the facial lymphatics to form in an area that is relatively devoid of veins. Overall, this study provides conclusive, live imaging-based evidence of a non-venous lymphatic progenitor and demonstrates that the origin and development of lymphatic vessels is context-dependent.

Liposome-Mediated Drug Delivery in Larval Zebrafish to Manipulate Macrophage Function.

Chemical interventions are regularly used to examine and manipulate macrophage function in larval zebrafish. Given chemicals are typically administered by simple immersion or injection, it is not possible to resolve whether their impact on macrophage function is direct or indirect. Liposomes provide an attractive strategy to target drugs to specific cellular compartments, including macrophages. As an example, injecting liposomal clodronate into animal models, including zebrafish, is routinely used to deliver toxic levels of clodronate specifically to macrophages for targeted cell ablation. Here we show that liposomes can also target the delivery of drugs to zebrafish macrophages to selectively manipulate their function. We utilized the drugs etomoxir (a fatty acid oxidation inhibitor) and MitoTEMPO (a scavenger of mitochondrial reactive oxygen species [mROS]), that we have previously shown, through free drug delivery, suppress monosodium urate (MSU) crystal-driven macrophage activation. We generated poloxamer 188 modified liposomes that were readily phagocytosed by macrophages, but not by neutrophils. Loading these liposomes with etomoxir or MitoTEMPO and injecting into larvae suppressed macrophage activation in response to MSU crystals, as evidenced by proinflammatory cytokine expression and macrophage-driven neutrophil recruitment. This work reveals the utility of packaging drugs into liposomes as a strategy to selectively manipulate macrophage function.

Macrophages enhance Vegfa-driven angiogenesis in an embryonic zebrafish tumour xenograft model.

Tumour angiogenesis has long been a focus of anti-cancer therapy; however, anti-angiogenic cancer treatment strategies have had limited clinical success. Tumour-associated myeloid cells are believed to play a role in the resistance of cancer towards anti-angiogenesis therapy, but the mechanisms by which they do this are unclear. An embryonic zebrafish xenograft model has been developed to investigate the mechanisms of tumour angiogenesis and as an assay to screen anti-angiogenic compounds. In this study, we used cell ablation techniques to remove either macrophages or neutrophils and assessed their contribution towards zebrafish xenograft angiogenesis by quantitating levels of graft vascularisation. The ablation of macrophages, but not neutrophils, caused a strong reduction in tumour xenograft vascularisation and time-lapse imaging demonstrated that tumour xenograft macrophages directly associated with the migrating tip of developing tumour blood vessels. Finally, we found that, although macrophages are required for vascularisation in xenografts that either secrete VEGFA or overexpress zebrafish vegfaa, they are not required for the vascularisation of grafts with low levels of VEGFA, suggesting that zebrafish macrophages can enhance Vegfa-driven tumour angiogenesis. The importance of macrophages to this angiogenic response suggests that this model could be used to further investigate the interplay between myeloid cells and tumour vascularisation.

Characterization of Zebrafish Facial Lymphatics.

Zebrafish are well-established as a model of vascular development. The genetic tractability, external development, permeability to small molecules and optical transparency of zebrafish larvae are all attributes that make this model attractive to the vascular biologist. There are an increasing number of lymphatic reporter lines that enable the visualization of zebrafish lymphatic vessel growth in vivo; these tools, coupled with either forward or reverse genetics, have provided new insights into the process of lymphatic specification and development. Zebrafish larvae have three main lymphatic networks: the trunk lymphatics, the intestinal lymphatics, and the facial lymphatics and it is therefore possible to use zebrafish to determine network-specific roles for molecules implicated in lymphatic development. This chapter provides protocols for visualization and analysis of facial lymphatic development in the zebrafish and may be applied in developmental or drug discovery studies.