RESUMO
Zebrafish xenotransplantation models are increasingly applied for phenotypic drug screening to identify small compounds for precision oncology. Larval zebrafish xenografts offer the opportunity to perform drug screens at high-throughput in a complex in vivo environment. However, the full potential of the larval zebrafish xenograft model has not yet been realized and several steps of the drug screening workflow still await automation to increase throughput. Here, we present a robust workflow for drug screening in zebrafish xenografts using high-content imaging. We established embedding methods for high-content imaging of xenografts in 96-well format over consecutive days. In addition, we provide strategies for automated imaging and analysis of zebrafish xenografts including automated tumor cell detection and tumor size analysis over time. We also compared commonly used injection sites and cell labeling dyes and show specific site requirements for tumor cells from different entities. We demonstrate that our setup allows us to investigate proliferation and response to small compounds in several zebrafish xenografts ranging from pediatric sarcomas and neuroblastoma to glioblastoma and leukemia. This fast and cost-efficient assay enables the quantification of anti-tumor efficacy of small compounds in large cohorts of a vertebrate model system in vivo. Our assay may aid in prioritizing compounds or compound combinations for further preclinical and clinical investigations.
RESUMO
The WD40 repeat containing angio-associated migratory cell protein (AAMP) was identified as a new binding partner of the human nucleotide-binding domain, leucine rich repeat containing (NLR) family member Nod2 in a yeast two-hybrid screen. Co-immunoprecipitations from human cells verified this interaction and revealed that an internal peptide of AAMP spanning three WD40 domains was sufficient for this interaction. AAMP was found to be ubiquitously expressed in different human cell-lines and exhibited a predominant cytosolic localization in epithelial cells. Functionally, using overexpression and siRNA knock-down, we showed that AAMP modulates Nod2- and Nod1-mediated NF-kappaB activation in HEK293T cells. Taken together, our data support a new function of AAMP in regulating innate immune responses initiated by the NLR protein Nod2.