Changes in tumor cell heterogeneity after chemotherapy treatment in a xenograft model of glioblastoma.

Glioblastoma (GBM) is a highly aggressive brain cancer with limited treatments and poor patient survival. GBM tumors are heterogeneous containing a complex mixture of dividing cells, differentiated cells, and cancer stem cells. It is unclear, however, how these different cell populations contribute to tumor growth or whether they exhibit differential responses to chemotherapy. Here we set out to address these questions using a zebrafish xenograft transplant model (Welker et al., 2016). We found that a small population of differentiated vimentin-positive tumor cells, but a majority of Sox2-positive putative cancer stem cells, were dividing during tumor growth. We also observed co-expression of Sox2 and GFAP, another suggested marker of glioma cancer stem cells, indicating that the putative cancer stem cells in GBM9 tumors expressed both of these markers. To determine how these different tumor cell populations responded to chemotherapy, we treated animals with temozolomide (TMZ) and assessed these cell populations immediately after treatment and 5 and 10days after treatment cessation. As expected we found a significant decrease in dividing cells after treatment. We also found a significant decrease in vimentin-positive cells, but not in Sox2 or GFAP-positive cells. However, the Sox2-positive cells significantly increased 5days after TMZ treatment. These data support that putative glioma cancer stem cells are more resistant to TMZ treatment and may contribute to tumor regrowth after chemotherapy.

Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity.

Glioblastoma (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.