Robotic injection of zebrafish embryos for high-throughput screening in disease models

Spaink, Herman P.; Cui, Chao; Wiweger, Małgorzata; Jansen, Hans J.; Veneman, Wouter J.; Marín-Juez, Rubén; Sonneville, Jan de; Ordas, Anita; Torraca, Vincenzo; Ent, Wietske van der; Leenders, William P. J.; Meijer, Annemarie H.; Snaar-Jagalska, B. Ewa; Dirks, Ron P.

doi:10.1016/j.ymeth.2013.06.002

Cited by 92 publications

(85 citation statements)

References 87 publications

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“…Finally, the ZF embryo is particularly suited for high-throughput screening, as shown recently for Mycobacterium marinum (36,43,44). Work is currently in progress in our laboratory to develop an in vivo platform for high-throughput screening of molecules against M. abscessus in order to speed up the process of identifying promising drug candidates, particularly warranted due to the extreme resistance of M. abscessus to most current antibiotics.…”

Section: Discussionmentioning

confidence: 99%

In Vivo Assessment of Drug Efficacy against Mycobacterium abscessus Using the Embryonic Zebrafish Test System

Bernut

Moigne

Lesne

et al. 2014

Antimicrob Agents Chemother

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c Mycobacterium abscessus is responsible for a wide spectrum of clinical syndromes and is one of the most intrinsically drug-resistant mycobacterial species. Recent evaluation of the in vivo therapeutic efficacy of the few potentially active antibiotics against M. abscessus was essentially performed using immunocompromised mice. Herein, we assessed the feasibility and sensitivity of fluorescence imaging for monitoring the in vivo activity of drugs against acute M. abscessus infection using zebrafish embryos. A protocol was developed where clarithromycin and imipenem were directly added to water containing fluorescent M. abscessusinfected embryos in a 96-well plate format. The status of the infection with increasing drug concentrations was visualized on a spatiotemporal level. Drug efficacy was assessed quantitatively by measuring the index of protection, the bacterial burden (CFU), and the number of abscesses through fluorescence measurements. Both drugs were active in infected embryos and were capable of significantly increasing embryo survival in a dose-dependent manner. Protection from bacterial killing correlated with restricted mycobacterial growth in the drug-treated larvae and with reduced pathophysiological symptoms, such as the number of abscesses within the brain. In conclusion, we present here a new and efficient method for testing and compare the in vivo activity of two clinically relevant drugs based on a fluorescent reporter strain in zebrafish embryos. This approach could be used for rapid determination of the in vivo drug susceptibility profile of clinical isolates and to assess the preclinical efficacy of new compounds against M. abscessus. The emerging pathogen Mycobacterium abscessus is the etiological agent of a wide spectrum of infections in humans, including severe chronic pulmonary and disseminated infections, mostly in immunosuppressed and cystic fibrosis (CF) patients (1), and cutaneous diseases, often posttraumatic and postsurgical. This neglected pathogen causes a higher fatality rate than other rapidly growing mycobacteria (RGM), and CF patient infections is becoming a major threat in most CF centers worldwide (2). M. abscessus infections occur in early childhood (3), are severe and sometimes fatal, especially following transplantation (4-6), and may lead to outbreaks of infection (6). It is also the main RGM responsible for nosocomial and iatrogenic infections in humans (postinjection abscesses, cardiac surgery, and plastic surgery) (7-9). It has been reported to cross the blood-brain barrier and cause important central nervous system (CNS) lesions. Although a rapid grower, M. abscessus possesses several important pathogenic traits, such as the ability to (i) persist silently for years and even decades in the human host (10) and to (ii) induce lung disease with caseous lesions and granuloma formation in the parenchyma (11, 12).The major issue with M. abscessus relies on its intrinsic resistance to the most available antibiotics. The American Thoracic Society has recommended different...

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Section: Discussionmentioning

confidence: 99%

In Vivo Assessment of Drug Efficacy against Mycobacterium abscessus Using the Embryonic Zebrafish Test System

Bernut

Moigne

Lesne

et al. 2014

Antimicrob Agents Chemother

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“…Although the pipeline is currently only partly automated, screening of small adenoviral RNAi sublibraries ($100 genes) is feasible. Integration of the established automated imaging and quantitative image analysis with recently described methods for automated injection and sorting of zebrafish embryos can widen applicability to larger scale screening (45).…”

Section: Discussionmentioning

confidence: 99%

SYK Is a Candidate Kinase Target for the Treatment of Advanced Prostate Cancer

Ghotra

Horst

et al. 2015

Cancer Research

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Improved targeted therapies are needed to combat metastatic prostate cancer. Here, we report the identification of the spleen kinase SYK as a mediator of metastatic dissemination in zebrafish and mouse xenograft models of human prostate cancer. Although SYK has not been implicated previously in this disease, we found that its expression is upregulated in human prostate cancers and associated with malignant progression. RNAi-mediated silencing prevented invasive outgrowth in vitro and bone colonization in vivo, effects that were reversed by wild-type but not kinase-dead SYK expression. In the absence of SYK expression, cell surface levels of the progression-associated adhesion receptors integrin a2b1 and CD44 were diminished. RNAimediated silencing of a2b1 phenocopied SYK depletion in vitro and in vivo, suggesting an effector role for a2b1 in this setting. Notably, pharmacologic inhibitors of SYK kinase currently in phase I-II trials for other indications interfered similarly with the invasive growth and dissemination of prostate cancer cells. Our findings offer a mechanistic rationale to reposition SYK kinase inhibitors for evaluation in patients with metastatic prostate cancer. Cancer Res; 75(1); 230-40. Ó2014 AACR.

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“…To assess the quantity of drugs present in zebrafish, larvae were exposed to rifampin, a first-line anti-TB drug used as a positive control in our drug-screening experiments (10,24), as well as to moxifloxacin, a gyrase inhibitor currently under exploration for novel antitubercular regimen development to shorten the duration of TB treatment (37).…”

Section: Resultsmentioning

confidence: 99%

“…The easy availability of large numbers of larvae and their small size makes this model particularly suitable for high-throughput in vivo screening of drugs added to the medium (1,2,10,(24)(25)(26), significantly reducing material requirements for testing. However, caution must be taken when interpreting the data in the absence of knowledge on drug absorption into the tissues of zebrafish larvae (27).…”

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confidence: 99%

Testing Tuberculosis Drug Efficacy in a Zebrafish High-Throughput Translational Medicine Screen

Ordas

Raterink

Cunningham

et al. 2015

Antimicrob Agents Chemother

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dThe translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

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Robotic injection of zebrafish embryos for high-throughput screening in disease models

Cited by 92 publications

References 87 publications

In Vivo Assessment of Drug Efficacy against Mycobacterium abscessus Using the Embryonic Zebrafish Test System

In Vivo Assessment of Drug Efficacy against Mycobacterium abscessus Using the Embryonic Zebrafish Test System

SYK Is a Candidate Kinase Target for the Treatment of Advanced Prostate Cancer

Testing Tuberculosis Drug Efficacy in a Zebrafish High-Throughput Translational Medicine Screen

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