ZeOncoTest: Refining and Automating the Zebrafish Xenograft Model for Drug Discovery in Cancer

Abstract

The xenograft of human cancer cells in model animals is a powerful tool for understanding tumor progression and metastatic potential. Mice represent a validated host, but their use is limited by the elevated experimental costs and low throughput. To overcome these restrictions, zebrafish larvae might represent a valuable alternative. Their small size and transparency allow the tracking of transplanted cells. Therefore, tumor growth and early steps of metastasis, which are difficult to evaluate in mice, can be addressed. In spite of its advantages, the use of this model has been hindered by lack of experimental homogeneity and validation. Considering these facts, the aim of our work was to standardize, automate, and validate a zebrafish larvae xenograft assay with increased translatability and higher drug screening throughput. The ZeOncoTest reliability is based on the optimization of different experimental parameters, such as cell labeling, injection site, automated individual sample image acquisition, and analysis. This workflow implementation finally allows a higher precision and experimental throughput increase, when compared to previous reports. The approach was validated with the breast cancer cell line MDA-MB-231, the colorectal cancer cells HCT116, and the prostate cancer cells PC3; and known drugs, respectively RKI-1447, Docetaxel, and Mitoxantrone. The results recapitulate growth and invasion for all tested tumor cells, along with expected efficacy of the compounds. Finally, the methodology has proven useful for understanding specific drugs mode of action. The insights gained bring a step further for zebrafish larvae xenografts to enter the regulated preclinical drug discovery path.

Aggressiveness and Metastatic Potential of Breast Cancer Cells Co-Cultured with Preadipocytes and Exposed to an Environmental Pollutant Dioxin: An in Vitro and in Vivo Zebrafish Study

“…Since the larvae are optically transparent, the implementation of fluorescent cancer cells (which are xenografts) and the follow-up of their growth or intra-/extravasation (early metastatic events) is facilitated. This model now is considered as a powerful tool for the study such events ( Cornet et al. 2019 ; Cabezas-Sáinz et al.…”

Ouabain and Digoxin Activate the Proteasome and the Degradation of the ERα in Cells Modeling Primary and Metastatic Breast Cancer

“…Zebrafish xenograft experiments were performed by ZeClinics (Barcelona, Spain) according to the procedure published in [ 31 ], with modifications in image analysis to allow measurement of the tumor area, rather than tumor volume. Briefly, Casper adult Zebrafish ( Danio Rerio ) were mated to obtain embryos.…”

Zebrafish Xenografts Unveil Sensitivity to Olaparib beyond BRCA Status

“…The zebrafish model presents numerous advantages and has been used in several experimental approaches, including drug screenings and the generation of patient-derived zebrafish Avatars, using embryonic-larval stages or even adult zebrafish [30,31,33,[88][89][90][91]. The optical transparency and single-cell resolution allow for the evaluation of different readouts apart from the classically used tumor size analysis [32,91].…”