Zebrafish modelling of apoptosis and inflammation in aggressive B-cell lymphoma

Apoptosis is a well-orchestrated programmed cell death. In cancer biology the evasion of apoptosis has been considered as one of the key events for tumour development and paradoxically, studies also show that apoptosis has detrimental effects that may even promote cancer. High rates of apoptosis have been observed in many cancers and aggressive B cell lymphoma (prototypically Burkitt’s lymphoma (BL)) present characteristic ‘starry sky’ appearance due to extensive apoptotic tumour cells engulfed by infiltrating tumour-associated macrophages (TAM). Previous work using a murine BL cell model has shown that constitutive apoptosis promotes both angiogenesis and the accumulation of pro-tumour TAMs. However, the detailed cellular and molecular mechanisms underlying how apoptosis fosters this pro-tumour growth microenvironment are still not fully understood, especially the role of apoptosis during early steps of tumour initiation. The aim of this project is to establish an in vivo model system to dissect the mechanisms as to how apoptotic B lymphoma cells enhance angiogenesis and how apoptotic B lymphoma cells interact with cells within the host microenvironment to promote tumour progression. Zebrafish (Danio Rerio), a small tropical fresh water fish, has become increasingly popular as a biomedical research model organism. Not only is it amenable for genetic manipulation, but also due to its transparency in the larval stages, is one of the most important models for in vivo live imaging studies. Therefore efforts were made to establish a novel transgenic B lymphoma model in zebrafish. Complementary to the transgenic model, I also established a Xenograft model using a B lymphoma cell line BL2 and its apoptosis resistant derivative BL2-bcl2 cell lines. Using a Tol2 based transgenesis system and zebrafish promoter for IgM heavy chain, I have generated transgenic zebrafish with either constitutive B cells expressing oncogenic cmyc, Tg (IgM1::cmyc-eGFP), or a tamoxifen inducible version, Tg(IgM1::CreERT2/IgM::lox-H2BmCherry-lox-cmyc-eGFP) which allows induction of oncogenic cmyc expression in B-cells with precise temporal control. Unfortunately, neither of these models developed B cell lymphoma, and fish appear to be generally healthy. Although flow cytometic analysis showed normal expression of the transgene in Tg(IgM::lox- H2BmCherry-lox-cmyc-eGFP), further analysis of the constitutive model failed to detect any CMYC expressing eGFP positive B-cells in the head-kidney. Therefore, unexpectedly IgM driven cmyc expression in zebrafish might drive B-cell death instead of B-cell malignancy. This is in contrast to the mouse B lymphoma model. More work is needed in choosing a suitable promoter and/or oncogene combination to generate a transgenic zebrafish B lymphoma model. Xenograft models using zebrafish larvae provided an additional opportunity to study interaction between tumour cell and cells within the tumour microenvironment. Available transgenic reporter zebrafish strains labelling various cell lineages facilitate in vivo imaging of host cellular responses to B lymphoma cells. In order to identify putative roles that apoptotic tumour cells might play in the tumour microenvironment, I have established a reliable and consistent xenograft protocol to graft tumour cells into yolk sack of 2 days post fertilization (dpf) zebrafish larvae. Consistent with previous observations in mice, BL2 (an apoptotic prone lymphoma cell line) cells survive better than their apoptotic resistant derivative BL2-bcl2 (over-expressing bcl2 in BL2 cells). However, the overall longest survival time is no more than 4 days post grafting even with BL2 cells. A possible explanation for this could be lack of some key B cell survival factors in zebrafish larvae, as normally mature B cells do not develop until two weeks post fertilization. The mouse models of BL indicate that TAMs have been attracted by apoptotic BL cells and accumulate at the BL microenvironment. To evaluate whether macrophages modulated by apoptotic cells promote BL survival in the zebrafish model, human monocyte-derived macrophages were activated by either apoptotic BL2 cells or IFN-γ / lipopolysaccharide (LPS) and co-injected with BL2 cells into zebrafish. Results showed that macrophages activated by apoptotic BL2 cells, but not IFN-γ /LPS, enhanced the survival of BL2 cells. In the next part I further investigated how apoptotic BL2 cells might modulate macrophages and the tumour microenvironment. Extracellular vesicles (EVs) are small membrane-bounded vesicles whose molecular profile is regulated by their cellular origin and the types of stimuli. EVs have been shown to be critical messengers in tumor progression and metastasis. The study of apoptosis-induced EVs (Apo-EVs) is sparse. I hypothesised that EVs released by apoptotic cells might mediate their pro-tumourigenesis properties. I used a recently developed novel protocol in my laboratory to isolate Apo-EVs from BL2 cells. EVs from apoptosis resistant BL2-bcl2 cells (non-Apo-EVs) were used as a control. I show here for the first time that Apo- EVs are pro-angiogenic in vivo. Further analysis of the secretome from apoptotic BL2 cells as well as their Apo-EVs indicates that soluble protein component(s) mediate the pro-angiogenic function. Combining macrophage reporter fish Tg(mpeg1::mCherry) with TNFα reporter Tg(tnfα::eGFP), I show that Apo-EVs promote macrophage activation but not TNFα in vivo. In conclusion, this project suggests that apoptotic tumour cells execute their pro-oncogenic functions by modulating macrophage activation, enhancing tumour angiogenesis, possibly through releasing Apo-EVs. Apo-EVs are recognized as a key player in fostering a pro-tumour growth microenvironment. Thus a further understanding of how apoptotic cells exert their tumour promoting roles will help us to optimise cancer therapy by maximizing tumour cell death while minimizing unwanted pro-tumorigenic effects. The models established during this project may be used to identify factors that are key to the survival and growth of B lymphoma

Innervation of the developing kidney in vivo and in vitro

Within the adult kidney, renal neurites can be observed alongside the arteries where they play a role in regulating blood flow. However, their role and localization during development has so far not been described in detail. In other tissues, such as the skin of developing limb buds, neurons play an important role during arterial differentiation. Here, we aim to investigate whether renal nerves could potentially carry out a similar role during arterial development in the mouse kidney. In order to do so, we used whole-mount immunofluorescence staining to identify whether the timing of neuronal innervation correlates with the recruitment of arterial smooth muscle cells. Our results show that neurites innervate the kidney between day 13.5 and 14.5 of development, arriving after the recruitment of smooth muscle actin-positive cells to the renal arteries

Selective Inhibitors of Human mPGES-1 from Structure-Based Computational Screening

Human mPGES-1 is recognized as a promising target for next generation of anti-inflammatory drugs. Although various mPGES-1 inhibitors have been reported in literature, few have entered clinical trials and none has been proven clinically useful so far. It is highly desired for developing the next generation of therapeutics for inflammation-related diseases to design and discover novel inhibitors of mPGES-1 with new scaffolds. Here, we report the identification of a series of new, potent and selective inhibitors of human mPGES-1 with diverse scaffolds through combined computational and experimental studies. The computationally modeled binding structures of these new inhibitors with mPGES-1 provide some interesting clues for rational design of modified structures of the inhibitors to more favorably bind with mPGES-1

A Quantitative LC-MS/MS Method for Simultaneous Determination of Cocaine and Its Metabolites in Whole Blood

As new metabolic pathways of cocaine were recently identified, a high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed to simultaneously determine cocaine and nine cocaine-related metabolites in whole blood samples. One-step solid phase extraction was used to extract all of the ten compounds and corresponding internal standards from blood samples. All compounds and internal standards extracted were separated on an Atlantis T3 (100 Å, 3 μm, 2.1 mm × 150 mm I.D) column and detected in positive ion and high sensitivity mode with multiple reaction monitoring. This method was validated for its sensitivity, linearity, specificity, accuracy, precision, recovery, and stability. All of the ten compounds were quantifiable ranging from the lower limit of quantification (LLOQs) of ∼10 nM (1.9–3.2 ng/ml) to ∼1000 nM (190–320 ng/ml) without any interfering substance. Accuracy and precision were determined, and both of them were within the acceptance criteria of the United States (US) Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines. The recovery was above 66.7% for all compounds. Stability tests demonstrated the stability of compounds under different storage conditions in whole blood samples. The method was successfully applied to a pharmacokinetic study with co-administration of cocaine and alcohol in rats

Clinical Potential of an Enzyme-Based Novel Therapy for Cocaine Overdose

It is a grand challenge to develop a truly effective medication for treatment of cocaine overdose. The current available, practical emergence treatment for cocaine overdose includes administration of a benzodiazepine anticonvulsant agent (e.g. diazepam) and/or physical cooling with an aim to relieve the symptoms. The inherent difficulties of antagonizing physiological effects of drugs in the central nervous system have led to exploring protein-based pharmacokinetic approaches using biologics like vaccines, monoclonal antibodies, and enzymes. However, none of the pharmacokinetic agents has demonstrated convincing preclinical evidence of clinical potential for drug overdose treatment without a question mark on the timing used in the animal models. Here we report the use of animal models, including locomotor activity, protection, and rescue experiments in rats, of drug toxicity treatment with clinically relevant timing for the first time. It has been demonstrated that an efficient cocaine-metabolizing enzyme developed in our previous studies can rapidly reverse the cocaine toxicity whenever the enzyme is given to a living rat, demonstrating promising clinical potential of an enzyme-based novel therapy for cocaine overdose as a successful example in comparison with the commonly used diazepam

Structure-Based Discovery of mPGES-1 Inhibitors Suitable for Preclinical Testing in Wild-Type Mice as a New Generation of Anti-Inflammatory Drugs

Human mPGES-1 is recognized as a promising target for next generation of anti-inflammatory drugs without the side effects of currently available anti-inflammatory drugs, and various inhibitors have been reported in the literature. However, none of the reported potent inhibitors of human mPGES-1 has shown to be also a potent inhibitor of mouse or rat mPGES-1, which prevents using the well-established mouse/rat models of inflammation-related diseases for preclinical studies. Hence, despite of extensive efforts to design and discover various human mPGES-1 inhibitors, the promise of mPGES-1 as a target for the next generation of anti-inflammatory drugs has never been demonstrated in any wild-type mouse/rat model using an mPGES-1 inhibitor. Here we report discovery of a novel type of selective mPGES-1 inhibitors potent for both human and mouse mPGES-1 enzymes through structure-based rational design. Based on in vivo studies using wild-type mice, the lead compound is indeed non-toxic, orally bioavailable, and more potent in decreasing the PGE2 (an inflammatory marker) levels compared to the currently available drug celecoxib. This is the first demonstration in wild-type mice that mPGES-1 is truly a promising target for the next generation of anti-inflammatory drugs

SCOTfluors: Small, Conjugatable, Orthogonal and Tunable Fluorophores for in vivo Imaging of Cell Metabolism

The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small-sized multi-colored fluorophores for real-time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin. © 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.The authors acknowledge funding from Medical Research Scotland (S.B.: 879-2015), MSCA Individual Fellowship (A.F.: 704912), OPTIMA (N.D.B.: EP/L016559/1), Wellcome Trust Sir Henry Dale Fellowship (Y.F.: 100104/Z/12/Z) and the Spanish Ministry of Science, Innovation and Universities (J.L.A, A.D.: CTQ2017-85378-R). M.V. acknowledges funds from ERC Consolidator Grant (771443), Biotechnology and Biological Sciences Research Council (BB/M025160/1) and the Royal Society (IEC\R3\170132). The authors thank the technical support from the Flow Cytometry and the Confocal Advanced Light Microscopy units at the University of Edinburgh.Peer reviewe

Potential of Core-Collapse Supernova Neutrino Detection at JUNO

JUNO is an underground neutrino observatory under construction in Jiangmen, China. It uses 20kton liquid scintillator as target, which enables it to detect supernova burst neutrinos of a large statistics for the next galactic core-collapse supernova (CCSN) and also pre-supernova neutrinos from the nearby CCSN progenitors. All flavors of supernova burst neutrinos can be detected by JUNO via several interaction channels, including inverse beta decay, elastic scattering on electron and proton, interactions on C12 nuclei, etc. This retains the possibility for JUNO to reconstruct the energy spectra of supernova burst neutrinos of all flavors. The real time monitoring systems based on FPGA and DAQ are under development in JUNO, which allow prompt alert and trigger-less data acquisition of CCSN events. The alert performances of both monitoring systems have been thoroughly studied using simulations. Moreover, once a CCSN is tagged, the system can give fast characterizations, such as directionality and light curve