Project no. 037220
Project acronym ZF-TOOLS
Project title: High-throughput Tools for Biomedical Screens in Zebrafish
Instrument SPECIFIC TARGETED RESEARCH PROJECT
Thematic Priority Life Sciences, Genomics and Biotechnology for Health
ZF-TOOLS
Publishable final activity report
Period covered:
Date of preparation:
from 1st of January 2007 to 30st June2010
September 28, 2010
Start date of project:
1st of January 2007
Project coordinator name:
Project coordinator organisation name:
Duration: 42 months
Dr. Annemarie H. Meijer
Institute of Biology, Leiden University (LEI)
Publishable final activity report
Project Title:
High-throughput Tools for Biomedical Screens in Zebrafish
Project Acronym: ZF-TOOLS
Contract Number: LSHG-CT-2006-037220
EC Contribution: 1,739,000 €
Duration in months: 42
Starting date: 01-01-2007
Instrument: STREP
Project web page:
http://www.science.leidenuniv.nl/index.php/ibl/mcb/research_themes/zf_tools
Coordinator
Dr. Annemarie H. Meijer
Institute of Biology, Leiden University
Einsteinweg 55
2333 CC, Leiden, The Netherlands
Phone: +31-71-5274927
Fax: +31-71-5274999
E-mail: a.h.meijer@biology.leidenuniv.nl
http://www.science.leidenuniv.nl/index.php/ibl/meijer
Other contractors
ZF-screens BV, Niels Bohrweg 11, 2333 CA, Leiden, The Netherlands, www.zfscreens.com
BaseClear B.V., Einsteinweg 5, 2302 BH, Leiden, The Netherlands, www.baseclear.com
Zenon Bio Ltd., Maros St. 40., H-6721, Szeged, Hungary, www.zenonbio.hu
Szeged University, Dr. Mátyás Mink, Dept. of Genetics, Kozepfasor 52, H-6726, Szeged,
Hungary, www.u-szeged.hu
Karlsruhe Institut für Technologie (KIT), Prof. Dr. Nicholas Foulkes, Hermann-vonHelmholtz-Platz 1, Eggenstein-Leopoldshafen, 76344, Germany, www.kit.edu
1. Project execution
Summary
The zebrafish holds much promise as a high-throughput drug screening model for
immune-related diseases, including inflammatory and infectious diseases and cancer.
This is due not only to the remarkable similarity of the zebrafish and human immune
systems, but also to the excellent possibilities for in vivo imaging in combination with
advanced tools for genomic and large scale mutant analysis. Furthermore, the millimeter
size of the zebrafish embryos and larvae makes them highly suited for screening chemical
libraries, since compounds can be administered simply to the embryo medium and only
minute quantities are needed. In the ZF-TOOLS project we developed zebrafish-based
screening systems for anti-tumor and anti-microbial drugs. We demonstrated that a novel
robotic system can be used to implant fluorescently labeled tumor cells or microbes into
zebrafish embryos at an unprecedented high-throughput level. The growth and metastatic
behavior of implanted tumor cells or the proliferation of bacteria and their interaction
with the zebrafish immune cells can be excellently visualized in the optically transparent
zebrafish embryos, and quantified by coupling the robotic injector to an automated
system for fluorescence sorting and image acquisition. This set-up was benchmarked for
anti-tumor and anti-tuberculosis drug screening in the ZF-TOOLS project. Using a
multidisciplinary functional genomics approach, where we integrated results from
microarray analysis and next-generation deep sequencing, marker genes and microRNAs
were identified with a predictive value for tumor progression or for immune defense
responses of the zebrafish embryo. A subset of these markers was used to develop highthroughput assays for expression analysis. In addition, several oncogenic cell lines as
well as luciferase reporter lines driven by oncogene or immune gene promoters were
generated that present useful tools for biomedical research. Three biotech enterprises will
commercially exploit the expertise on bioinformatics, high-throughput genomics analysis,
and high-throughput drug screening that was gained in the ZF-TOOLS project.
Objectives
The ZF-TOOLS project was a coordinated effort
of three research laboratories and three biotech
enterprises (SMEs) aimed at the following
objectives:
 Genomic-based marker discovery for
biomedical screens in zebrafish.
 Use of high-throughput marker analysis and
tumor cell implants for the identification of
tumor growth and metastasis factors and
organismal defense factors.
The project aimed to develop a case study for an
anti-tumor drug screening system, based on the
implantation of fluorescently labeled tumor cells
into zebrafish embryos. In this system, growth
and metastasis properties of implanted tumor cells can be efficiently monitored by
fluorescence microscopy during development of the transparent zebrafish embryos. We
envisage that a powerful screening system can arise from the combination of highthroughput marker analysis with the possibility to visualize tumor growth and metastasis
in an optically transparent vertebrate model organism. However, for realization of such a
screening system, the identification of relevant disease marker genes in zebrafish formed
a crucial step. In the ZF-TOOLS project different genomics approaches were used to
discover novel markers, which will be suitable for application in the ZF-TOOLS tumor
screening system and also will have a broader utility for disease research in the zebrafish
model. The experimental design of our genomics approach was aimed to result in the
identification of two classes of markers: 1) markers correlating with growth and
metastasis of tumor cells and 2) markers correlating with the immune or other defense
responses of the organism towards tumor cells. The reason for concentrating on both
classes of markers is that interactions between developing tumors and the tumor
microenvironment are decisive for tumor survival or rejection. Strategies to boost antitumor immunity have been explored for many years. Therefore, knowledge of tumor
markers as well as defense response markers will increase the versatility of the anti-tumor
drug screening system developed in the project.
Approach
Zebrafish cell lines carrying constitutive or inducible expression constructs of various
oncogenes and cell lines derived from human tumors were fluorescently labeled and
tested by implantation into zebrafish embryos. Several of these cell lines, most notably
those carrying inducible oncogene constructs and those derived from human tumors,
showed reproducible proliferation and migration behavior inside the zebrafish larval body
and also elicited angiogenic responses. The best performing zebrafish cell lines were
selected for transcriptome profiling using microarray analysis and deep sequencing. The
data were compared with expression data of tumor tissues. In addition, extensive
transcriptome profiling studies were performed to characterize the zebrafish host defense
response toward tumor implants and these data were compared with responses to
infectious agents. Immuno-deficient zebrafish were characterized to gain insight into the
specificity of the defense response markers. For transcriptome sequencing we have used
Illumina deep sequencing technology with which we could obtain up to 10 million
sequence reads per sample. A dedicated data warehouse, the ZF-TOOLS database, was
developed for storage and bioinformatic analysis of transcriptome sequencing data.
Furthermore, a data processing pipeline for functional annotation of zebrafish genes was
developed. Based on the genomic profiling studies, we have developed high-throughput
expression assays. For this purpose we applied a multiplex PCR technique, RT-MLPA,
which allows the analysis of 40-50 markers in a single one-tube assay. In addition,
microarray data of tumor-and defense related small regulatory RNAs (microRNAs) were
used to set up quantitative PCR assays, and the promoters of several tumor- or defense
specific genes identified were used to generate luciferase reporter lines for cell-based
assays. Finally, a novel robotics system was applied to obtain a proof-of principle for
high-throughput screening of anti-microbial and anti-tumor drugs. To demonstrate the
versatility of this system we have tested it using fluorescent tumor cell implants as well
as mycobacteria that induce granuloma formation in zebrafish embryos similar as in
human tuberculosis. Drugs used for cancer and tuberculosis treatment in human were
tested in the zebrafish system.
End results
We have developed a high-throughput anti-tumor and anti-tuberculosis drug screening
system, based on implantation of fluorescently labeled tumor cells or mycobacteria into
zebrafish embryos. This screening system has the following unique features:
 It is based on novel robotics technology for injecting bacterial pathogens or tumor
cells which has been benchmarked in the ZF-TOOLS project.
 It combines robotic injection of fluorescently labeled tumor cells or mycobacteria
with automated image analysis for in-vivo monitoring of respectively tumor
progression or mycobacterial granuloma formation in transparent zebrafish embryos.
 Visual monitoring of disease progression in transparent larvae can be combined with
high-throughput molecular screening tools for tumor and defense response markers
developed within the project.
A prototype of the injection robot was benchmarked for anti-tuberculosis and anti-tumor
drug screens and the screening procedures were fully standardized for case study screens
with drugs used in human cancer and tuberculosis therapy.
In addition, the ZF-TOOLS project generated extensive microarray and deep sequencing
data sets of zebrafish oncogenic cells and tumor samples together with data on defense
responses of the zebrafish host to tumor implants or infectious agents, resulting in:
 Knowledge on disease markers in zebrafish, more specifically tumor- and host
defense-specific genes and microRNAs
 Assays for quantitative analysis of disease marker sets
 A collection of constitutive and inducible, oncogenic and non-oncogenic reporter cell
lines useful for basic disease research and for application in screening systems
Fundamental knowledge gained from the use of zebrafish cancer and infection models
has been presented at scientific meetings and has now resulted in several publications in
journals such as Blood, Journal of Immunology, and Molecular Immunology.
Furthermore, with the knowledge of disease markers and expertise gained on
bioinformatics, high-throughput genomics analysis, and high-throughput drug screening
biotech enterprises involved in the project have strengthened their IP positions and
expanded their service activities.
Impact on the industry and research sector
The ZF-TOOLS objectives were focused on the incorporation of the zebrafish embryo
model into the preclinical drug screening pipelines. The use of mice for in vivo
monitoring of disease processes such as tumorigenesis, metastasis and immune response
to tumors or infectious diseases like tuberculosis is limited by costs and throughput level.
Introducing a high-throughput zebrafish embryo model will potentially contribute to costeffective and more efficient methods in the anti-tumor drug discovery process.
Acceleration of drug lead time benefits economy as well as quality of life of patients.
With the development of a robotic system for injection of tumor cells and bacterial
pathogens into zebrafish embryos the ZF-TOOLS project has provided a solution to
enable high-throughput cancer and tuberculosis drug screens in the zebrafish model. In
addition, the lack of basic knowledge of disease marker genes has been a bottleneck for
biomedical research in zebrafish and for genomics-based compound screens in this model
organism. The ZF-TOOLS project has used multidisciplinary functional genomics
approaches to discover novel disease markers. The identification of factors important for
tumorigenic properties and organismal defense responses has contributed fundamental
knowledge relevant to human health and will open the door to the establishment of
zebrafish-based biomedical research and screening tools.
2. Dissemination and use
Zebrafish-based anti-tuberculosis and anti-tumor drug screening
• Result description: Robotic system for injection of bacterial pathogens and tumor cells
into zebrafish embryos.
• Possible market applications: High throughput screening of small molecule libraries for
anti-microbial and anti-tumor drugs
• Stage of development: prototype benchmarked for anti-tuberculosis and anti-tumor drug
screens, standardizations performed, validation with human cancer drugs in progress
• Collaborator details: ZF-screens B.V., Leiden University, Institute of Biology
• Intellectual property rights: WO 2006/080841 A1, US # 61/233,805, Europe #
09164956.6
• Contact details: Dr. Ron Dirks, ZF-screens BV, Niels Bohrweg 11, 2333 CA, Leiden,
The Netherlands, www.zfscreens.com
Tumor markers in zebrafish
• Result description: Knowledge of tumor-specific markers in zebrafish
• Possible market applications: anti-tumor drug screening
• Stage of development: expression assays tested up to medium throughput level, high
throughput applications under development
• Intellectual property rights: US # 61/233,805, Europe # 09164956.6
• Collaborator details: ZF-screens B.V., Leiden University, Institute of Biology
• Contact details: Dr. Ron Dirks, ZF-screens BV, Niels Bohrweg 11, 2333 CA, Leiden,
The Netherlands, www.zfscreens.com
Macrophage and immune response markers in zebrafish
• Result description: Knowledge of infection-induced and macrophage-specific markers
in zebrafish (Hegedus et al., 2009, Stockhammer et al., 2010, Zakrzewska et al., 2010,
Ordas et al., 2010, Stockhammer et al., 2010)
• Possible market applications: application in anti-microbial drug screening
• Stage of development: Research will be continued in the context of the EU-funded ZFHEALTH consortium, the Smartmix consortium funded by the Dutch government, and
with a horizon grant from the Netherlands Organization of Scientific Research.
Transgenic reporter lines for immune-response and macrophage markers are currently
under construction.
• Collaborator details: Leiden University, Institute of Biology; Szeged University,
Department of genetics, ZF-screens B.V.
• Contact details: Dr. Annemarie H. Meijer, Institute of Biology, Leiden University,
Einsteinweg 55, 2333 CC, Leiden, The Netherlands, Phone: +31-71-5274927, Fax: +3171-5274999, E-mail: a.h.meijer@biology.leidenuniv.nl
Mycobacterium-induced host genes as potential diagnostic tools for tuberculosis
• Result description: gene expression profiles specific to mycobacterium infection
• Possible market applications: clinical screening system for diagnosing tuberculosis
• Stage of development: The Hungarian Development Agency has granted a project
proposal aiming to develop the prototype of a clinical screening system for diagnosing
tuberculosis. This project is based on the hypotheses that the gene expressional
alterations observed in Mycobacterium marinum-infected zebrafish (Hegedus et al.,
2009) may occur in human patients and the expressional alterations are followed by
translational changes in the same directions. We plan to analyse serum proteins by
Western of about 25-30 tuberculosis patients and matching controls, provided by the
pulmonologists. The number of the candidate markers will be ~25. Markers with
statistically significant changes will be selected for an ELISA-based screening test. We
expect to develop a prototype, which is cheap (USD 5/test), high throughput (processing
of 50 blood samples a day) system that we will offer to tuberculosis-endemic countries
(Russia, China, India). These expected properties of the screening system may make
superior over the time-consuming, laborious, complicated and expensive screening
systems.
• Collaborator details: Szeged University, Department of Genetics; Szeged University,
Pulmonology Clinics; Creative Ltd. (Szeged, Hungary)
• Contact details: Dr. Mátyás Mink, Szeged University, Dept. of Genetics and Molecular
Biology
Kozepfasor 52, H-6726, Szeged, Hungary, Phone: +36 62 544269, Fax: +36 62 544651,
e-mail: mink@bio.u-szeged.hu.
High-throughput sequencing services
• Result description: Expertise on Digital Gene Expression library preparation and highthroughput sequencing using the Illumina Genome analyzer II system.
• Possible market applications: Illumina sequencing technology has a range of possible
applications in the area of biological research and analysis, including genome
sequencing, transcriptome sequencing and Chip-Seq.
• Stage of development: A Genome Analyzer II system was installed in December 2008
and commercial services are now being offered to customers with more than 18 months
of experience.
Collaborator details: BaseClear B.V and ZF-screens B.V.
• Contact details: Dr. Derek Butler, BaseClear B.V., P.O. Box 1336, 2302 BH, Leiden,
The Netherlands, www.baseclear.com
DeepSAGE sequencing bioinformatics service
• Result description: special knowledge about the large scale bioinformatics analysis of
data derived from high throughput SAGE based transcriptome profiling studies.
• Possible market applications: ZenonBio Ltd. offers their expertise on bioinformatics of
deep sequencing data for customers and potential collaborating partners requiring a
related custom bioinformatics data analysis service.
• Stage of development: The offered service covers the entire data processing workflow
from the first conversion steps of the raw sequencing data up to the final data annotation
and data interpretation subtasks. The bioinformatics pipeline we use contains modules
assessing the sequencing quality, mapping sequence tags to reference genome or
reference transcript databases and performing statistical comparison of different
experimental samples. The custom data analysis service ZenonBio Ltd. offers might be
primarily useful for research laboratories and biotech companies specialized for
genomics, transcriptomics and system biology.
• Contact details: Dr. Tamás Forrai, Zenon Bio Ltd., Maros St. 40., H-6721, Szeged,
Hungary, Phone: +36 62 424290, Fax: +36 62 476558, e-mail: tforrai@zenonbio.hu
MLPA technology custom service
• Result description: High-throughput expression assays based on multiplex PCR
technology (RT-MLPA). Developed protocol, and gained knowledge and experience on
the development of RT-MLPA probe sets and additionally in the area of analysis and
interpretation of MLPA data and results.
• Possible market applications: Possibility to offer a commercial RT-MLPA probe
development service based on the RT-MLPA probe sets developed. Additionally
BaseClear is already offering commercial MLPA analysis services and is currently
working on a number of projects in this area.
• Stage of development: A number of RT-MLPA probe sets are complete. The MLPA
assay services are currently being offered to customers and at least one project is
currently being performed.
• Contact details: Dr. Derek Butler, BaseClear B.V., P.O. Box 1336, 2302 BH, Leiden,
The Netherlands, www.baseclear.com