Development of a Novel Embryonic Zebrafish Xenograft Model for Early-Stage Glioblastoma Tumor Cell Invasion

Background:Glioblastoma multiforme is the most common primary brain tumor in adults ages 45-70, with over 22,000 Americans diagnosed in 2010 alone. Despite decades of research efforts, glioblastoma is still one of the most difficult cancers to treat because the tumor cells have the ability to invade into the surrounding tissue with finger-like projections, making it difficult to completely remove all of the tumor tissue without resulting in more damage. But by slowing down the invasion of the tumor, a patient's survival rate can be increased. Still, the 5 year survival rate remains low -- less than 5%, and without treatment, median survival time is only 4.5 months after diagnosis. To study this, we have developed a new embryonic zebrafish xenograft model to better understand key factors in tumor cell invasion and decision-making. These include the usage of specific attachment sites, links between tumor invasiveness and distance from blood vessels, and the velocity and directionality of the tumor cells. Zebrafish are ideal model organisms for this process due to a variety of characteristics including, but not limited to their transparent nature, presence of blood circulation and vertebrate anatomy, as well as a dense microenvironment comparable to that of a human brain's. Specifically, transgenic fli fish were selected. These fli fish express Green Fluorescent Protein (GFP) in the endothelial layer of their blood vessels and were used to help monitor the interaction of tumor cells with blood vessels within the brain.

Methods: Our current imaging protocol involves an efficient seven day process. Transgenic fli fish 0 days post fertilization (dpf) were acquired from the Sinnhuber Research Laboratory. Starting 1 dpf, the fli fish were continuously treated with phenylthiourea to further inhibit melanogenesis, enhancing image clarity and reducing background. At 3 dpf, the fish were sedated and microinjected with fluorescently dyed cell cultured glioblastoma cells (U251 & U87). From 4-7 dpf, 18 hours of live, overnight, multidimentional images (both GFP and red fluorescent CM-Dil) of the fli fish were captured using a High Content Imager. Finally, after imaging, the GFP and CM-Dil images were overlayed and analyzed. Specifically, this new model will be useful for the quantification tumor cell velocity and colocalization of blood vessels and glioblastoma cells over time, and the investigation of whether certain morphological characteristics determine directionality and affect tumor cell decision-making.