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

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.

Catch of the day: zebrafish as a human cancer model

“…We have also observed in preliminary studies that human cancer cells, when injected directly into the vasculature of 2-day-old zebrafish embryos, lodge in small vessels in various organs and extravasate into the surrounding tissue (Stoletov et al, 2007; Figure 3). Haldi et al (2005) using human melanoma cells reported similar results. While these initial results are encouraging, additional work needs to be done to confirm that fish and human cancer cells metastasize using the typical vascular and lymphatic routes, and that similar mechanisms known to drive these processes in mammals also operate in zebrafish.…”

“…Transplantation of human cancer cells into early-stage (6-48 h post-fertilization) zebrafish embryos has also revealed important information about cancer biology as reported by several groups (Haldi et al, 2005;Lee et al, 2005;Topczewska et al, 2006;Nicoli et al, 2007). The major advantage of this model is that the immune system is still immature that permits human tumor cell engraftment.…”

“…Cancer cells derived from various species (mouse and human) and tissues (adenocarcinoma, fibrosarcoma, melanoma) have been introduced into both young embryos (2-5-days old post fertilization; Haldi et al, 2005;Lee et al, 2005;Topczewska et al, 2006;Nicoli et al, 2007) and juvenile 30-day-old zebrafish (Stoletov et al, 2007). Both transplantation models have their specific benefits and limitations when it comes to understanding cancer cell behavior in a living organism (Table 1).…”

“…Early embryo model: human tumor cell-induced angiogenesis and the role of VE-cadherin Haldi et al (2005) injected several human cell lines (WM-266-4 metastatic melanoma, FG pancreatic carcinoma and SW620 colon carcinoma) into the yolk sac of 2-day-old zebrafish embryos. Upon injection into the yolk sac these tumor cell lines survived, proliferated and migrated for up to a week, while normal human foreskin fibroblasts did not.…”

“…Cancer progression in these animals recapitulates many aspects of human disease and opens the door for studies to identify genetic and chemical modifiers of cancer (Amatruda et al, 2002;Smolowitz et al, 2002;Stern and Zon, 2003;Berghmans et al, 2005a;Goessling et al, 2007a;Kari et al, 2007). The attention has been further fueled by the development of xenograft models that allow the propagation and visualization of human cancer cells engrafted in optically transparent zebrafish (Haldi et al, 2005;Lee et al, 2005;Topczewska et al, 2006;Nicoli et al, 2007;Stoletov et al, 2007). The integration of zebrafish genetics with the large tool chest of reagents available to study human cancer cells provides a powerful new vertebrate model to visualize and dissect the mechanisms that drive cancer formation, angiogenesis and metastasis.…”