Zebrafish Tumor Imaging and Analysis

Imaging of embryos was performed 24 hpi and 72 hpi at 32–34 °C using the ImageXpress Micro Confocal High-Content Microscope (Molecular Devices, San José, CA, USA). Embryos were anesthetized using 1x tricaine and were put into Hashimoto (Funakoshi Co., Ltd., Tokyo, Japan) zebrafish 96-well plates with one embryo/well. Images were taken with 4x magnification in multiple z-stacks (35 z-stacks per image; step size 20 µM) and two sites per well covering the whole yolk sac, using brightfield and Cy3 settings, respectively. The tumor volume was calculated with an automated analysis pipeline (in-house macro for ImageJ), carefully discriminating specific signals from yolk sac-caused blurry background signal, as described previously [26 (link)]. To determine the difference (e.g., increase due to growth) of tumor volume from baseline until end of treatment, the percentage of volume difference was calculated between these two imaging time points.
The zebrafish-adapted Response Evaluation Criteria in Solid Tumors (RECIST) as described previously [26 (link)] was used to evaluate and visualize the drug response. To be classified as progressive disease (PD) the tumor volume must have increased at least 20%, and to be classified as partial response (PR) the tumor volume must have decreased by more than 30%.