ABSTRACT
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.
ABSTRACT
A simple and completely all-fiber Yb chirped pulse amplifier that uses a dispersion matched fiber stretcher and a spliced-on hollow core photonic bandgap fiber compressor is applied in nonlinear optical microscopy. This stretching-compression approach improves compressibility and helps to maximize the fluorescence signal in two-photon laser scanning microscopy as compared with approaches that use standard single mode fibers as stretcher. We also show that in femtosecond all-fiber systems, compensation of higher order dispersion terms is relevant even for pulses with relatively narrow bandwidths for applications relying on nonlinear optical effects. The completely all-fiber system was applied to image green fluorescent beads, a stained lily-of-the-valley root and rat-tail tendon. We also demonstrated in vivo imaging in zebrafish larvae, where we simultaneously measure second harmonic and fluorescence from two-photon excited red-fluorescent protein. Since the pulses are compressed in a fiber, this source is especially suited for upgrading existing laser scanning (confocal) microscopes with multiphoton imaging capabilities in space restricted settings or for incorporation in endoscope-based microscopy.
ABSTRACT
The orphan receptor CLEC-1 is part of a subfamily of C-type lectin-like receptors, which is encoded in the human natural killer gene complex and comprises several pattern recognition receptors important for innate immune functions. As information on human CLEC-1 is still very limited, we aimed to further characterize this receptor. Similar to another subfamily member, LOX-1, expression of CLEC-1 mRNA was detected in myeloid cells as well as in endothelial cells. CLEC-1 protein displayed N-linked glycosylation and formed dimers. However, in contrast to other members of the subfamily, expression levels were upregulated by transforming growth factor (TGF)-ß, but not significantly affected by proinflammatory stimuli. It is intriguing that human CLEC-1 could only be detected intracellularly with a staining pattern resembling endoplasmic reticulum proteins. Neither TGF-ß nor inflammatory stimuli could promote significant translocation to the cell surface. These findings are in accordance with a primarily intracellular localization and function of human CLEC-1.
