Embryonic zebrafish xenograft assay of human cancer metastasis

Abstract

Cancer metastasis is the most important prognostic factor determining patient survival, but currently there are very few drugs or therapies that specifically inhibit the invasion and metastasis of cancer cells. Currently, human cancer metastasis is largely studied using transgenic and immunocompromised mouse xenograft models, which are useful for analysing end-point tumour growth but are unable to accurately and reliably monitor invasion, intravasation, extravasation or secondary tumour in vivo formation of human cancer cells. Furthermore, limits in our ability to accurately monitor early stages of tumour growth and detect micro-metastases likely results in pain and suffering to the mice used for cancer xenograft experiments. Zebrafish ( ) embryos, however, Danio rerio offer many advantages as a model system for studying the complex, multi-step processes involved during cancer metastasis. This article describes a detailed method for the analysis of human cancer cell invasion and metastasis in zebrafish embryos before they reach protected status at 5 days post fertilisation. Results demonstrate that human cancer cells actively invade within a zebrafish microenvironment, and form metastatic tumours at secondary tissue sites, suggesting that the mechanisms involved during the different stages of metastasis are conserved between humans and zebrafish, supporting the use of zebrafish embryos as a viable model of human cancer metastasis. We suggest that the embryonic zebrafish xenograft model of human cancer is a tractable laboratory model that can be used to understand cancer biology, and as a direct replacement of mice for the analysis of drugs that target cancer invasion and metastasis. Potential applications:• Studying the heterogeneity of tumours and cooperation of different cancer cells from patient-derived tumours.• Studying the remodelling of the extracellular matrix during tumour invasion.• As a screening assay to identify new agents/drugs that reduce the metastatic behaviour of cancer cells. Supplementary material Supplementary File 1. Complete step-by-step protocol for Zebrafish xenograft of human cancer cells.Click here to access the data Supplementary File 2. Completed ARRIVE checklist.Click here to access the data Supplementary Movie 1. Representative time lapse confocal movie showing active invasion of a DiI-labelled A375 melanoma cell through the yolk sac and into a kdrl-GFP labelled blood vessel of a Casper zebrafish embryo.Images were taken every 15 minutes for 5 hours. Click here to access the data Supplementary Movie 2. 3D rendering of confocal movie showing active invasion of a DiI-labelled A375 melanoma cell through the yolk sac and into a kdrl-GFP labelled blood vessel of a Casper zebrafish embryo.Transverse field of view through the blood vessel demonstrates that the red melanoma is within the green blood vessel and actively moving against the flow of blood. Click here to access the data Supplementary Movie 3. Representative time lapse confocal movie showing movement of a DiI-labelled A375 melano...

Benefits of Zebrafish Xenograft Models in Cancer Research

“…Currently, metastasis of human cancers is largely studied via transgenic and immunocompromised mPDX models, which are useful for measuring the end-point tumor volume but are unable to quantify or recapitulate in vivo invasion, intravasation, extravasation, or the secondary tumor growth of patient-derived cancer cells in real time (Morton and Houghton, 2007 ; Konantz et al, 2012 ; van der Ent et al, 2014b ; Barriuso et al, 2015 ; Hill et al, 2018 ; Yan et al, 2019 ). The methods used to establish an mPDX metastasis model include cell injection into the tail vein, spleen, or left ventricle, but the key step of metastasis, cancer cell intravasation, cannot be simulated (Goddard et al, 2016 ; Lizardo and Sorensen, 2018 ).…”

“…The methods used to establish an mPDX metastasis model include cell injection into the tail vein, spleen, or left ventricle, but the key step of metastasis, cancer cell intravasation, cannot be simulated (Goddard et al, 2016 ; Lizardo and Sorensen, 2018 ). Zebrafish, however, can offer many advantages as a model system for studying the complex, multistep processes involved during spontaneous cancer metastasis (Fior et al, 2017 ; Follain et al, 2018 ; Hill et al, 2018 ; Wong et al, 2019 ). Implantation into the yolk sac, perivitelline space (PVS) and subcutaneous tissues allow the direct injection of cancer cells into the zebrafish's body (Abercrombie and Ambrose, 1958 ; Pruvot et al, 2011 ; Canella et al, 2017 ; Letrado et al, 2018 ).…”

“…The same report also suggested that the key to successfully treating metastatic tumors is interfering with endothelial remodeling (Follain et al, 2018 ). It is worth noting that individual cancer cells can be dynamically visualized in the zPDX model, especially during metastasis (Hill et al, 2018 ; Astell and Sieger, 2019 ; Yan et al, 2019 ).…”

Unveiling Tumor Microenvironment Interactions Using Zebrafish Models

“…The utility of zebrafish to probe metastatic events is poised largely on its optical clarity, which permits both close monitoring of individual cells and organismal structures in real time (Teng et al, 2013 ; Heilmann et al, 2015 ; Hill et al, 2018 ; Asokan et al, 2020 ). Combined with tools for genetic manipulation and fluorescent reporting (Lawson and Weinstein, 2002 ), every step in a malignant cell's journey, from invasion to colonization, can be interrogated in vivo ( Figure 2 ) (Astell and Sieger, 2019 ; Osmani and Goetz, 2019 ).…”

Reporting animal research: Explanation and Elaboration for the ARRIVE guidelines 2019

“…Each measurement outcome will be analysed by 2-way ANOVA to determine the effect of drug treatment on growth, survival and invasion of each cancer cell line." [20] 1b. The experimental unit (e.g.…”