Development of a systematic roadmap for cancer drug screening using zebrafish embryo xenograft models

Abstract

Zebrafish embryo tumor transplant models are widely utilized in cancer research. Compared with traditional murine models, the small size and transparency of zebrafish embryos combined with large clutch sizes that increase statistical power and cheap husbandry make them a cost-effective and versatile tool for in vivo drug discovery. However, the lack of a comprehensive analysis of key factors impacting the successful use of these models impedes the establishment of basic guidelines for systematic screening campaigns. Thus, we explored the following crucial factors: 1) user-independent inclusion criteria, focusing on sample homogeneity; 2) metric defi-nition for data analysis; 3) tumor engraftment criteria; 4) image analysis versus quantification of human cancer cells using qPCR (RNA and gDNA); 5) staining strategies evaluation for cancer cells; 6) temperature of incubation for xenografted zebrafish embryos; 7) number of cancer cells to implant; 8) tumor implantation sites; 9) compound distribution (intratumoral administration versus alternative inoculation sites); and 10) efficacy (intratumoral microinjection versus compound solution in media). Based on these analyses and corresponding assessments, we propose the first roadmap for systematic drug discovery screening in zebrafish xenograft cancer models. This study aims to help the wider cancer research community to consider the adoption of this versatile model for cancer drug screening projects.