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

Abstract

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.

Keywords: GBM9 neurospheres; Glial fibrillary acidic protein; Glioblastoma; Sox2; Temozolomide; X12 cells.

Fig. 1.

Survival of zebrafish xenotransplants. (A) Control animals were transplanted with ∼50 mouse neural stem cells (mNSCs; blue line) or sham injected with 1-2 nl of HBSS (black line), with 87.5% survival in both groups. Animals transplanted with 51-90 GBM9 cells (dark green line) had a median survival of 5±1.0 dpt, compared with 2 dpt for animals transplanted with 91-140 GBM9 cells (light green line). Animals transplanted with 51-90 X12 cells (red line) engrafted and formed tumors only 50% of the time, with a median survival of 18 dpt, whereas animals transplanted with 91-140 X12 cells (orange line) had a median survival of 10±0.5 dpt. n=100 animals per group for GBM9 and X12 groups and n=24 for mNSCs and sham-injected groups. P<0.0001 for all tumor xenotransplants compared with mNSC or sham. (B) GBM9 xenotransplants were scored at 1 dpt for the number of engrafted tumor cells; 10-25 cells (blue line), 26-50 cells (green line) with a median survival of 10±0.7 dpt, 51-90 cells (red line) with a median survival of 5±1.0 dpt, or 91-140 cells (black line) with a median survival of 2 days. n=24 animals per group. P<0.0001 for GBM9 transplants of 25 cells or greater compared with those receiving <20 cells.

Fig. 2.

Analysis of tumor burden in live animals over time. Confocal images superimposed on bright field (anterior to the left) of two representative casper zebrafish transplanted with 50-75 GBM9 cells (A-A‴,B-B‴) and a casper animal transplanted with control mNSC cells (C-C‴) imaged at 2 (A,B,C), 5 (A′,B′,C′), 7 (A″,B″,C″) and 10 (A‴,B‴,C‴) dpt. Examples of a compact (A-A‴) and diffuse tumor (B-B‴) are shown. (D) Tumor burden were quantified using volume measurements of florescence in micrometers cubed. Approximately 50-75 GBM9 cells (green lines) and ∼50 mNSC cells (blue lines) were transplanted and followed over time in the same animal. n=8 animals per group. Scale bar: 50 μm.

Fig. 3.

GBM9 tumor cells grow throughout the brain tissue. (A) Representative area sectioned (red lines) in a 7 dpt zebrafish. (B-F) Transverse 20-μm-thick cryosections of a GBM9 compact tumor at the level of the forebrain (B), midbrain (C,D) and hindbrain (E,F). (G-K) Transverse cryosections of a diffuse tumor at the level of the forebrain (G,H) midbrain (I) and hindbrain (J,K). (L) Based on morphology, tumors were scored as compact (light green bar) or diffuse (dark green bar) then measured by Sholl analysis at 7 dpt to quantify cell spread. Largest radius (in micrometers) is the measure of the farthest radius intersecting a cell from the injection site. n=10 per group; 20 animals total. *P<0.001. Scale bar: 40 μm for B-K.

Fig. 4.

GBM9 histology staining for Hematoxalin and Eosin. (A-D) Paraffin-embedded GBM9 xenotransplanted animals at 7 dpt; 40× (A,B) and 100× (C,D) magnification of two separate animals (A,C and B,D) with tumors. Yellow dashed lines in A,B denote the tumor mass. Green arrows in C,D denote hyperchromatic and abnormal nuclei. Scale bar: 50 μm in A,B and 20 μm in C,D.

Fig. 5.

GBM9 and X12 xenotransplants contain a high number of dividing cells. (A-F) Confocal images of GBM9 and X12 on 2 (A,D), 5 (B,E) and 10 (C,F) dpt transverse cryosections. (A-C) GBM9 (green), DAPI (blue) and Ki67 (red) at 100×. (D-F) X12 (green), DAPI (blue) and Ki67 (red) at 100×. White boxes denote magnified area to the right of the image. n=5 animals per group; 30 animals total. Scale bar: 20 μm for main panels and 5 μm for insets. (E) Quantification of the total number of dividing cells per animal at each time point for GBM9 (green line) and X12 (gray line) transplants.

Fig. 6.

GBM9 and X12 tumors contain a combination of differentiated cells and stem cells. Confocal images of GBM9 and X12 on 2 (A,D,G,J,M,P), 5 (B,E,H,K,N,Q) and 10 (C,F,I,L,O,R) dpt transverse cryosections. (A-C) GBM9 (green), DAPI (blue) and vimentin (red) at 100×. (D-F) X12 (green), DAPI (blue) and vimentin (red) at 100×. (G-I) GBM9 (green), DAPI (blue) and GFAP (red) at 100×. (J-L) X12 (green), DAPI (blue) and GFAP (red) at 100×. (M-O) GBM9 (green), DAPI (blue) and Sox2 (red) at 100×. (P-R) X12 (green), DAPI (blue) and Sox2 (red) at 100×. White boxes denote area magnified to the right of the image. White arrow in R points to a cell with a migratory morphology. n=5 animals per group; 90 total animals. Scale bar: 20 μm for main panels and 5 μm for insets.

Fig. 7.

Chemotherapeutic agents decrease GBM9 xenotransplant tumor burden. GBM9 xenotransplants were treated with 50 μM drug continuously between 5 and 10 dpt. (A-D′) Confocal images superimposed on bright field (anterior to the left) of two GBM9 animals at 5 dpt (A,B) and at 10 dpt after 5 days of temozolomide (TMZ) treatment (A′,B′). (C,D) Confocal images superimposed on bright field (anterior to the left) of two GBM9 animals at 5 dpt (C,D) and at 10 dpt after 5 days of bortezomib (Bort) treatment (C′,D′). (E) Quantification of tumor burden (in micrometers cubed) before treatment (5 dpt) and after 5 days of treatment (10 dpt). n=10 animals per group. *P<0.001. (F) Kaplan–Meier survival curve of animals during drug treatment (5-10 dpt) with temozolomide (dark blue line) and bortezomib (light blue line). Control DMSO-treated GBM9 animals (green line) have a median survival of 8±0.6 days. Of the animals treated with TMZ, 70.8% lived until 25 days compared with 50.0% treated with bortezomib. Of the wild-type animals treated with 50 μm TMZ (dashed dark blue line) or bortezomib (dashed light blue line), 83.3 and 88.0%, respectively, survived. n=48 animals for all groups. P<0.0001 for GBM9 DMSO versus both GBM9 TMZ and GBM9 Bort. P=0.0672 for GBM9 TMZ versus GBM9 Bort. Scale bar: 50 μm.