Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity
ABSTRACTGlioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for drug screening.
[…] Individual tumor-bearing or mNSC-transplanted animals were anesthetized using tricaine (160 μg/ml) at 2, 5, 7 and 10 dpt and imaged in a FluoroDish (WPI, Inc., Sarasota, FL, USA) under the spinning disk confocal microscope (Andor). Using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) and Fiji (), images captured through the zebrafish tumors were reconstructed using a z-stack to analyze tumor volume. Using MetaMorph, a standard cell volume in fluorescent pixels was averaged from six cells. This ‘standard’ cell volume was then applied to the entire z-stack. These measurements were converted into volume (in micrometers cubed) using a MetaMorph software formula that converts a standardized pixel analysis of one ‘cell’ into a volumetric number. Images were analyzed using Fiji/ImageJ and Excel. The same animal was imaged on 2, 5, 7 and 10 dpt, and tumor burden was quantified and plotted for each of these days using Excel. […]