title: Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion.
abstract: In the last decade, high-throughput chemical screening has become the dominant approach for discovering novel compounds with therapeutic properties. Automated screening using in vitro or cultured cell assays have yielded thousands of candidate drugs for a variety of biological targets, but these approaches have not resulted in an increase in drug discovery despite major increases in expenditures. In contrast, phenotype-driven screens have shown a much stronger success rate, which is why we developed an in vivo assay using transgenic zebrafish with a GFP-marked migrating posterior lateral line primordium (PLLp) to identify compounds that influence collective cell migration. We then conducted a high-throughput screen using a compound library of 2160 annotated bioactive synthetic compounds and 800 natural products to identify molecules that block normal PLLp migration. We identified 165 compounds that interfere with primordium migration without overt toxicity in vivo. Selected compounds were confirmed in their migration-blocking activity by using additional assays for cell migration. We then proved the screen to be successful in identifying anti-metastatic compounds active in vivo by performing orthotopic tumor implantation assays in mice. We demonstrated that the Src inhibitor SU6656, identified in our screen, can be used to suppress the metastatic capacity of a highly aggressive mammary tumor cell line. Finally, we used CRISPR/Cas9-targeted mutagenesis in zebrafish to genetically validate predicted targets of compounds. This approach demonstrates that the migrating PLLp in zebrafish can be used for large-scale, high-throughput screening for compounds that inhibit collective cell migration and, potentially, anti-metastatic compounds.
authors: Developmental Genomics Section, Genome Technology Branch National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Viviana E
authors: VE
authors: Gallardo
authors: Developmental Genomics Section, Genome Technology Branch National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Gaurav K
authors: GK
authors: Varshney
authors: Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Minnkyong
authors: M
authors: Lee
authors: Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Sujata
authors: S
authors: Bupp
authors: Developmental Genomics Section, Genome Technology Branch National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Lisha
authors: L
authors: Xu
authors: Department of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
authors: Paul
authors: P
authors: Shinn
authors: Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Nigel P
authors: NP
authors: Crawford
authors: Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA Department of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
authors: James
authors: J
authors: Inglese
authors: Developmental Genomics Section, Genome Technology Branch National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA burgess@mail.nih.gov.
authors: Shawn M
authors: SM
authors: Burgess
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D004353:Drug Evaluation, Preclinical; D004625:Embryo, Nonmammalian / Q000187:drug effects; D005260:Female; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D053403:Lateral Line System / Q000166:cytology / Q000378:metabolism; D008807:Mice, Inbred BALB C; D008954:Models, Biological; D008969:Molecular Sequence Data; D009362:Neoplasm Metastasis; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology*; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D015027:Zebrafish / Q000196:embryology / Q000378:metabolism*; D029961:Zebrafish Proteins / Q000737:chemistry / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D007211:Indoles; D012333:RNA, Messenger; C416927:SU 6656; D054852:Small Molecule Libraries; D013449:Sulfonamides; D029961:Zebrafish Proteins; D019061:src-Family Kinases
keywords: Drug screening; Metastasis; Orthotopic implantation; Src; Tks5; Zebrafish