Systematic Roadmap for Cancer Drug Screening Using Zebrafish Embryo Xenograft Cancer Models: Melanoma Cell Line as a Case Study

SIMPLE SUMMARY: Currently, there is no consensus in the scientific literature regarding the zebrafish embryo xenotransplantation procedure for drug screening. Thus, this study sets systematic guidelines for maximizing the reproducibility of drug screening in zebrafish-embryo cancer xenograft models based on evaluating every step of the procedure in a real case scenario in which the chemical properties of the compounds are unknown or not optimal. It aims to be a stepping stone to bring the versatility of zebrafish embryos to drug screening for cancer. The present work helps our group to pursue the objective of establishing zebrafish embryos as a valuable alternative to mice models; and hopefully, will help other groups in this field to progress in the same direction. 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: (i) user-independent inclusion criteria, focusing on sample homogeneity; (ii) metric definition for data analysis; (iii) tumor engraftment criteria; (iv) image analysis versus quantification of human cancer cells using qPCR (RNA and gDNA); (v) tumor implantation sites; (vi) compound distribution (intratumoral administration versus alternative inoculation sites); and (vii) 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 using a melanoma cell line as a case study. This study aims to help the wider cancer research community to consider the adoption of this versatile model for cancer drug screening projects.