Ortho- and Ectopic Zebrafish Xeno-Engraftment of Ocular Melanoma to Recapitulate Primary Tumor and Experimental Metastasis Development

Abstract

There are currently no animal models for metastatic ocular melanoma. The lack of metastatic disease models has greatly hampered the research and development of novel strategies for the treatment of metastatic ocular melanoma. In this protocol we delineate a quick and efficient way to generate embryonic zebrafish models for both the primary and disseminated stage of ocular melanoma, using retro-orbital orthotopic and intravascular ectopic cell engraftment, respectively. Combining these two different engraftment strategies we can recapitulate the etiology of cancer in its totality, progressing from primary, localized tumor growth under the eye to a perivascular metastasis formation in the tail. These models allow us to quickly and easily modify the cancer cells prior to implantation with specific labeling, genetic or chemical interference; and to treat the engrafted hosts with (small molecular) inhibitors to attenuate tumor development.Here, we describe the generation and quantification of both orthotopic and ectopic engraftment of ocular melanomas (conjunctival and uveal melanoma) using fluorescently labelled stable cell lines. This protocol is also applicable for engraftment of primary cells derived from patient biopsy and patient/PDX derived material (manuscript in preparation). Within hours post engraftment cell migration and proliferation can be visualized and quantified. Both tumor foci are readily available for imaging with both epifluorescence microscopy and confocal microscopy. Using these models, we can confirm or refute the activity of either chemical or genetic inhibition strategies within as little as 8 days after the onset of the experiment, allowing not only highly efficient screening on stable cell lines, but also enables patient directed screening for precision medicine approaches.

Patient-derived zebrafish xenograft models reveal ferroptosis as a fatal and druggable weakness in metastatic uveal melanoma

“…After 5 days of treatment, we imaged 20-40 whole zebrafish larvae per condition using a fluorescent stereo microscope, and quantified the red fluorescent integrated density, as a measure of cancer cell survival, normalizing to DMSO control (as described by Groenewoud et al 2021) 29 .…”

Patient-derived zebrafish xenografts of uveal melanoma reveal ferroptosis as a drug target

“…Following centrifugation (200 x g, 5 min, room temperature), the red fluorescent lipid dye CM-DiL (Sigma) was used to stain the cells to visualize cancer cell proliferation and metastatic initiation as reported previously [49].…”

“…Either Tg(fli:GFPx casper) [45,51] [52] fish were bred prior to the start of an experiment, and Petri dishes containing larvae were cleaned every day, removing dead and malformed larvae, after harvesting up to 2 days post fertilization. Approximately 300 to 400 cells were injected into the Duct of Cuvier (doC, the embryonic common cardinal vein) of 2 dpf zebrafish larvae as described [49].…”

XePhIR: the zebrafish xenograft phenotype interactive repository

“…To ensure a high quality of uploaded data, we will curate all final submissions based on proper quality and experimental design. All permissible data will be generated using either fluorescent proteins as cell tracers or will have to be generated using transient chemical tracers ( 12–19 ). In the case of chemical tracers, these have to be preferentially followed by anti-human specific immunostaining or in a minimal case with a nuclear dye to show the presence of intact nuclei within the established tumor ( 18 , 19 ).…”

“…Perivitelline space injection, originally described as a model for the generation and assessment of angiogenesis, more recently developed as a model for the generation of primary-like tumors ( 6 ). Retro-orbital engraftment, used for the generation of orthotopic primary-like tumors derived from eye tumors, allows for the development of distant metastases ( 12 ). Hindbrain cavity injection models are used for the generation of orthotopic brain cancer models and for the generation of brain metastasis models ( 13 ).…”