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. 2018 Oct 9;20(11):1475-1484.
doi: 10.1093/neuonc/noy071.

An Orthotopic Glioblastoma Animal Model Suitable for High-Throughput Screenings

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Free PMC article

An Orthotopic Glioblastoma Animal Model Suitable for High-Throughput Screenings

Linda Pudelko et al. Neuro Oncol. .
Free PMC article

Abstract

Background: Glioblastoma (GBM) is an aggressive form of brain cancer with poor prognosis. Although murine animal models have given valuable insights into the GBM disease biology, they cannot be used in high-throughput screens to identify and profile novel therapies. The only vertebrate model suitable for large-scale screens, the zebrafish, has proven to faithfully recapitulate biology and pathology of human malignancies, and clinically relevant orthotopic zebrafish models have been developed. However, currently available GBM orthotopic zebrafish models do not support high-throughput drug discovery screens.

Methods: We transplanted both GBM cell lines as well as patient-derived material into zebrafish blastulas. We followed the behavior of the transplants with time-lapse microscopy and real-time in vivo light-sheet microscopy.

Results: We found that GBM material transplanted into zebrafish blastomeres robustly migrated into the developing nervous system, establishing an orthotopic intracranial tumor already 24 hours after transplantation. Detailed analysis revealed that our model faithfully recapitulates the human disease.

Conclusion: We have developed a robust, fast, and automatable transplantation assay to establish orthotopic GBM tumors in zebrafish. In contrast to currently available orthotopic zebrafish models, our approach does not require technically challenging intracranial transplantation of single embryos. Our improved zebrafish model enables transplantation of thousands of embryos per hour, thus providing an orthotopic vertebrate GBM model for direct application in drug discovery screens.

Figures

Fig. 1
Fig. 1
(A) Transplantation of GBM material into the blastula stage of zebrafish embryos leads to the development of orthotopic tumors within 24 hours. (B) Still images of time-lapse confocal microscopy of elavl3:GFP blastula embryos transplanted with DiI-labeled GBM#3101 (see also Supplementary movie S1). (C) Confocal image of 24 hpf fli1a:EGFP embryo transplanted with DiI-labeled GBM#18 cells at the blastula stage. Elavl3 is a pan-neuronal marker; fli1a is a marker for endothelial cells.
Fig. 2
Fig. 2
(A) Embryos transplanted 24 hpf with a DiI-labeled GBM cell line and primary GBM material as well as SW620 colon carcinoma cells at the blastula stage. (B) Intracranial location of tumors is independent of injection site (apical or basal) within the blastula. (C) Frequency of intra-CNS tumor location after transplantation at blastula stage. (D) Viability of embryos transplanted at the blastula stage.
Fig. 3
Fig. 3
(A) Light-sheet images of orthotopic U343-MGA tumors 48 hpt (see also Supplementary movie S2). (B) Still images of time-lapse light-sheet microscopy on U343-MGA-GFP intracranial tumors transplanted at blastula stage with growing tumor microtubules (see also Supplementary movies S3, S4). (C) Magnification of tumor microtubules sent out by orthotopically transplanted U343-MGA tumors. (D) Increase of U343-MGA tumor volume based on surface calculation between 24 and 48 hpt. (E) Light-sheet images taken on orthotopic U343-MGA tumors between 5 and 6 days post transplantation with tumor microtubules encircling the otholit of the embryo (see also Supplementary movie S5). (F) Epifluorescent images of living 6-day-old embryos with orthotopically transplanted U343-MGA tumors; tumor protrusions marked with arrows.
Fig. 4
Fig. 4
(A, B) Immunohistochemical staining of cryosections of orthotopically transplanted fli:EGFP zebrafish embryos at 72 hpt. Ki67 demarks proliferative cells, fli:EGFP stains host blood vessels. (C) Still images of light-sheet real-time imaging on orthotopically transplanted U343-MGA tumors into mpeg1:mCherry zebrafish embryos that express a red fluorescent protein in macrophages/microglia. Engulfed tumor cell is encircled (see also Supplementary movie S6). (D) Light-sheet images of orthotopically transplanted GBM#18 cells expressing GFP under the control of the Sox2/Oct4 promoter demarking glioblastoma stem cells. (E) Still image of real-time light-sheet imaging on orthotopically transplanted GBM#18 cells expressing a red fluorescent protein into fli:EGFP embryos. Host vasculature is stained in green, arrows demark ongoing tumor vascularization (see also Supplementary movie S7). (F) Orthotopically transplanted embryos were treated at 1 day post transplantation with 20 µM of the respective TKI for 48 hours followed by tumor size determination via bioluminescence measurements. Experiments have been performed at least in quadruplicates using >20 embryos/replicate.
Fig. 5
Fig. 5
Potential setup for a large-scale drug screen. (A) Hundreds of blastula-stage embryos can be lined up in agarose molds and transplanted with robotic systems. (B) At 24 hours after injection, transplanted embryos are screened for tumor size by a commercially available automated imaging system and (C) automatically distributed into 96-well plates preloaded with candidate compounds. (D) After 2 days incubation time, embryos are automatically sampled, imaged, and analyzed with commercially available robotics.

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