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Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish

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Abstract

In this research, we optimized parameters for xenotransplanting WM-266-4, a metastatic melanoma cell line, including zebrafish site and stage for transplantation, number of cells, injection method, and zebrafish incubation temperature. Melanoma cells proliferated, migrated and formed masses in vivo. We transplanted two additional cancer cell lines, SW620, a colorectal cancer cell line, and FG CAS/Crk, a pancreatic cancer cell line and these human cancers also formed masses in zebrafish. We also transplanted CCD-1092Sk, a human fibroblast cell line established from normal foreskin and this cell line migrated, but did not proliferate or form masses. We quantified the number of proliferating melanoma and normal skin fibroblasts by dissociating xenotransplant zebrafish, dispensing an aliquot of CM-DiI labeled human cells from each zebrafish onto a hemocytometer slide and then visually counting the number of fluorescently labeled cancer cells. Since zebrafish are transparent until approximately 30 dpf, the interaction of labeled melanoma cells and zebrafish endothelial cells (EC) can be visualized by whole-mount immunochemical staining. After staining with Phy-V, a mouse anti-zebrafish monoclonal antibody (mAb) that specifically labels activated EC and angioblasts, using immunohistology and 2-photon microscopy, we observed activated zebrafish EC embedded in human melanoma cell masses. The zebrafish model offers a rapid efficient approach for assessing human cancer cells at various stages of tumorigenesis.

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Abbreviations

Alexa 488-Phy-V:

Alexa 488 conjugated directly with Phy-V monoclonal antibody

Alexa 488-2nd Ab:

Alexa 488 conjugated secondary antibody

BSA:

Bovine serum albumin

DMSO:

Dimethyl sulfoxide

dpf:

Days post fertilization

dpi:

Days post injection

EC:

Vascular endothelial cell

hpf:

Hours post fertilization

hpi:

Hours post injection

HBSS:

Hanks’ balanced salt solution

mAb:

Monoclonal antibody

Phy-V:

A mouse, anti-zebrafish monoclonal antibody that specifically labels EC and angioblasts

msec:

Millisecond

PFA:

Paraformaldehyde in PBST

PBS:

Phosphate buffered saline, pH 7.0

PBST:

PBS containing 0.1% Tween-20

psi:

Pounds per square inch

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Acknowledgments

We acknowledge the expert assistance of Dr. Thomas Diefenbach, DDRC Imaging Core (Children’s Hospital, Boston, MA) with 2-photon microscopy, Poh Kheng Loi of the University of Oregon for preparation of histological samples and Susie Tang for expert technical assistance. We also acknowledge insightful collaborative discussions with Dr. Richard Klemke (UCSD, San Diego, CA). A portion of this research was supported by a Small Business Innovation Research grant from the National Institute for Diabetes and Digestive and Kidney Disease: 1 R43DK074169.

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  1. Phylonix Pharmaceuticals, Inc., 100 Inman Street, suite 300, Cambridge, MA, 02139, USA

    Maryann Haldi, Christopher Ton, Wen Lin Seng & Patricia McGrath

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  1. Maryann Haldi
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  2. Christopher Ton
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  3. Wen Lin Seng
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  4. Patricia McGrath
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Correspondence to Maryann Haldi.

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Haldi, M., Ton, C., Seng, W.L. et al. Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish. Angiogenesis 9, 139–151 (2006). https://doi.org/10.1007/s10456-006-9040-2

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  • DOI: https://doi.org/10.1007/s10456-006-9040-2

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