A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Abstract

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.

Developing a Novel Embryo–Larval Zebrafish Xenograft Assay to Prioritize Human Glioblastoma Therapeutics

“…18,25 Based on work by others, we maintained our xenografts at 33°C, which is lower than the standard 37°C for culturing glioblastoma cells, but higher than the typical 28°C for zebrafish maintenance. 6,15 This temperature caused no observable zebrafish toxicity, yet resulted in adequate human glioblastoma growth and migration/invasion over the 3-4 day assay period to detect exposure-dependent differences between treatment groups.…”

“…Measurements of migration and invasion in embryonic zebrafish xenograft models has typically involved qualitative scoring or simply counting the number of cells to move away from the initial cell mass with no information on distance. 5,15,16 Our method provides a quantitative method to measure changes in the cells spread outward over concentric distances from the center of the cancer mass. This approach is similar to Zhang et al who measured chemical inhibition of leukemia stem cell migration in a zebrafish xenograft model.…”

“…Another group studying glioblastoma invasion in an embryonic zebrafish xenograft model only observed invasion of U87 cells implanted in the zebrafish yolk sac after injecting a subpopulation enriched for stem cells. 15 In a more similar xenograft model to the one described in this article, Welker et al quantified GBM9 neurosphere migration in the zebrafish brain up to 1.2 mm from the injection site after 7 days posttransplantation or *8.5 DPF. 32 However, this value is difficult to compare with our results since the measurements were made in a more mature zebrafish after 3-4 day longer growth period.…”

“…14 Existing embryo-larval zebrafish xenograft models have already demonstrated that human cancer cells, including glioma, can grow, divide, metastasize, and induce angiogenesis in zebrafish similarly to rodent xenograft models. 5,6,8,9,[15][16][17] A particular advantage of the zebrafish xenograft model is that only a small number of cancer cells are required for xenotransplantation, which better simulates earlier stages of cancer progression. 8,9 Many embryo-larval zebrafish xenograft glioma models involve transplanting the cells into the yolk sac or perivitelline space.…”

“…8,9 Many embryo-larval zebrafish xenograft glioma models involve transplanting the cells into the yolk sac or perivitelline space. 5,6,9,15 These transplant locations lack a complex brain microenvironment, which is filled with glial cells, neuronal processes, extracellular matrix, and a functional blood-brain barrier important for studying brain cancer. 4 By transplanting glioblastoma cells into the 48-72 h postfertilization zebrafish brain, which possess a functioning blood-brain barrier similar to mammalian models, our laboratory has developed an efficient and relevant assay using high-content imaging to study glioblastoma progression and prioritize the development of new glioblastoma therapies.…”