From Trace: Transgenic Zebrafish Models of Pediatric Cancer as Tools for Defining Oncogenic Pathways

-Trace Jolly, University of Kentucky, Blackburn Lab

The Blackburn lab uses zebrafish to study a variety of pediatric cancers. Our lab does this through two main techniques: 1-Transplants and 2- Transgenic Models. Zebrafish transplants/engraftments are a valuable model for examining how cells derived from an external source behave when implanted into a living organism. This methodology can draw samples from a wide variety of sources, including other zebrafish (allograft), human cell lines (xenograft), and even human patient samples received from the clinic (patient-derived xenograft).

 

 

 

 

 

 

The Blackburn also uses a variety of transgenic zebrafish to study cancer progression. Transgenic zebrafish contain alterations in their genome to cause a specific effect in the adult organism (phenotype). Often, transgenic animals are created to label a specific organ or system in the fish with a fluorescent reporter to watch how the organ develops and changes over time. A transgenic animal will frequently rely on a tissue-specific promoter. An example of this is the fli:GFP zebrafish line. Fli is an active promoter in endothelial cells. By putting a green fluorescent protein (GFP) under this promoter’s control, the fish’s endothelial cells will glow green, and we can visualize the blood vessels in the living organism.

 

 

 

 

 

In addition to labeling the structure of live animals, transgenesis can create zebrafish that spontaneously develop cancers. This methodology allows us to determine the time of onset, aggressiveness, and overall cancer progression in real-time in the living organism. Mammalian models frequently rely on the sacrifice and single time point analysis of cancer progression, which is bypassed by optically clear animal strains such as the “Casper” zebrafish.

 

 

 

 

 

In the lab, we routinely utilize two trangenic models of pediatric cancer. These are a model of T-Cell Acute Lymphoblastic Leukemia (T-ALL) and Embryonal Rhabdomyosarcoma (ERMS), both devastating types of pediatric cancers with very different types of presentation and progression. Both models rely on introducing an oncogene under a tissue-specific promoter to drive the spontaneous formation of the cancers. The T-ALL model is driven by the c-Myc oncogene, and the ERMS model is by the KRAS.G12D oncogene. Both of these drivers are frequently observed in their human counterparts. These drivers are then paired with a fluorescent protein to mark the cancer cells in the animal (typically either red or green). After around 25 days post fertilization (DPF), the animals will begin to develop the respective cancer types as illustrated below.

 

 

 

 

 

 

 

 

 

 

This model’s power comes from pairing these oncogenic drivers with additional proteins of interest to determine if the new protein will enhance/inhibit cancer progression. One example is the Phosphatase of Regenerating Liver 3 (PRL-3). PRL-3 enhances cancer metastasis in humans, and high PRL-3 levels correlate with a poor patient prognosis. However, it is unclear if the oncogenic mechanism of PRL-3 is due to the protein phosphatase activity of the ability to bind cell magnesium transporters.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

To answer these questions, we paired wildtype (WT) PRL-3 and a phosphatase dead PRL-3 mutant (C104D) with the oncogenic KRAS.G12D model of Embryonal Rhabdomyo Sarcoma. As expected, the WT PRL-3 protein enhanced the progression of the ERMS tumors by increasing the tumor size and promoting an invasive phenotype. Surprisingly, the phosphatase dead PRL-3 mutant (C104D) could also enhance this model. These findings suggest that the phosphatase activity of the protein is not critical for its oncogenic activity.

 

 

 

 

 

 

 

This is an example of how transgenic zebrafish cancer models can characterize oncogenic pathways in a quick, effective, and cost-efficient manner. We are excited to continue using our transgenic models to study the progression and further treat pediatric cancers.

 

References:

Joshua M WeissDianne Lumaquin-YinEmily MontalShruthy SureshCarl S LeonhardtRichard M White (2022) Shifting the focus of zebrafish toward a model of the tumor microenvironment eLife 11:e69703.

Wei, M., Haney, M.G., Rivas, D.R. et al. Protein tyrosine phosphatase 4A3 (PTP4A3/PRL-3) drives migration and progression of T-cell acute lymphoblastic leukemia in vitro and in vivo. Oncogenesis 9, 6 (2020). https://doi.org/10.1038/s41389-020-0192-5