An orthotopic glioblastoma animal model suitable for high-throughput screenings

An orthotopic glioblastoma animal model suitable for high-throughput screenings 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. animal model, brain cancer, glioblastoma, orthotopic transplantation, zebrafish Importance of the study The development of novel drugs fighting GBM is challenging, as no orthotopic animal model exists which can readily be implemented in large-scale drug discovery screens. The zebrafish is a clinically relevant model for human malignancies, and orthotopic transplantation of GBM material was found to faithfully recapitulate the human disease. However, none of the currently available transplantation procedures in zebrafish can be used in high-throughput screens, as the injection process is time-consuming and technically challenging. We have refined existing orthotopic zebrafish models and developed an approach which allows for transplantation of thousands of embryos per hour. This model can facilitate automated drug discovery and profiling as well as help to identify novel candidate drugs against GBM. Glioblastoma (GBM) is one of the most common malignancies of the central nervous system (CNS), and despite aggressive combinatory treatment, including maximal surgical resection, radiotherapy, and adjuvant chemotherapy, prognosis remains dismal. GBM in particular is characterized by extensive tumor heterogeneity as well as cancer cell plasticity and the presence of cancer stem cells.1,2 These multifaceted factors contribute to the ability of GBM to evade almost any treatment strategy.1,2 Understanding the complex biology of GBM in its physiological environment is a key for the development of new therapeutic options, and improvement of currently available animal models is a crucial prerequisite.3 Xenograft models, which are based on the orthotopic transplantation of either patient-derived glioblastoma cells or fresh brain tumor biopsy spheroids into immune-deficient rodents, resemble closely the genetic and phenotypic heterogeneity of the original patient tumor. Due to their clinical relevance, xenograft models remain the gold standard for understanding the biology of GBM.3 However, while recapitulating human gliomablastoma sufficiently well, orthotopic xenografts in rodents are technically challenging. In addition, ethical and financial constraints make them unsuitable for use in drug screening and profiling projects. During the last decade, the zebrafish (Danio rerio) has been characterized as a powerful and clinically relevant model for human diseases, including cancer.4–6 Besides its ex utero development, small size, high fecundity, and cost-efficient husbandry, the absence of a functional adaptive immune system until embryonic day 21 makes it an ideal animal model for xenotransplantation. Furthermore, the zebrafish is highly suitable for large-scale drug discovery projects, and its predictive capacity has already been exploited in various high-throughput screens.7 Orthotopic xenograft models of GBM in zebrafish have been reported by several groups8,9 and recent studies highlighted their extraordinary recapitulative potential and clinical relevance.10,11 However, since intracranial transplantation of GBM material into single zebrafish embryos is technically challenging and time-consuming, the use of zebrafish to understand the complex biology of GBM and to facilitate high-throughput screens for candidate therapies is currently limited. A novel method allowing for easy, fast, and automatable orthotopic injection of GBM material into zebrafish embryos would considerably help not only to understand GBM biology but also to develop, characterize, and verify novel treatment options. Materials and Methods Zebrafish Maintenance and Transgenic Strains The zebrafish tupfel longfin, fli1a:EGFP,12 HuC (elavl3):GFP,13 mpeg1:mCherry,14 and the Zebrabow15 strains were used throughout this study and raised and staged according to standard conditions.16 Transplanted zebrafish embryos were kept at 33°C. As described before,17,18 neither embryo growth/viability nor GBM proliferation (Supplementary Fig. S1) was affected.17,18 All experimental protocols were performed in accordance with the national ethical guidelines and regulations (N207/14). Cryosections and Immunocytochemistry Transplanted zebrafish embryos were prepared for cryosections as described previously19 and sections were stained with anti-Ki67 (#P6834, Sigma) and anti‒green fluorescent protein (GFP) antibodies (#SC8334, Santa Cruz) together with an anti-mouse/Alexa Fluor 488 secondary antibody using routine protocols. Images were taken with a Zeiss LSM 700 confocal microscope. Cell Culture All cells have been growing at 37°C in a humidified atmosphere (95% humidity) with 5% CO2. The GBM cell line U343-MGA-GFP20 as well as the primary GBM culture #1821 have been propagated in Minimum Essential Medium (Gibco) containing 2 mM glutamine (Gibco). Primary GBM cultures #310122 and #320422 have been propagated as stated in the reference. SW620 cells have been propagated following the suggestions of the American Type Culture Collection. All cells have been dissociated with Accutase (Sigma) at subconfluency. For details on generation and authentication of patient-derived GBM cell lines used in this study, see the respective references. Generation of GBM#18 cells expressing an octamer-binding transcription factor 4 (Oct4)/sex determining region Y–box 2 (Sox2) reporter has been described before.23 GBM cells stably expressing tdTomato or luciferase were generated with a lentivirus-based approach using the vector #32904 or #17477 (Addgene). Regular mycoplasma tests have been performed in all cell cultures using Mycoalert (#LT07, Lonza). Transplantation of Glioblastoma Cultures and Treatment Glioblastoma cultures were maintained as stated above and transferred to complete neural stem cell medium 7 days before transplantation. All cell cultures were prepared for injection as previously described.25 Briefly, cells were washed with phosphate buffered saline (PBS) and labeled with DiI, a fluorescent dye (1:250 in PBS, #V22885, Molecular Probes) for 15 min, followed by several washing steps using PBS to remove residual dye particles. Subsequently, cells were harvested, passed over a 20 µM nylon mesh, and resuspended in medium containing 2% polyvinylpyrrolidone (Sigma) to avoid clogging of the micro-injection capillary. Directly before transplantation, cells were spun down and medium was almost entirely removed. The cell suspension was loaded in nonfilament microcapillaries (World Precision Instruments) and approximately 100 tumor cells were injected into blastula-stage zebrafish embryos which have been immobilized in an agarose mold. After injection, embryos were screened for successful transplantation, transferred into a 10 cm dish, and incubated at 33°C for 24 hours before analysis or subsequent treatment. For treatment, embryos were distributed to 6-well plates (20 embryos/well) in a total volume of 3 mL E3 medium containing 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid). Tyrosine kinase inhibitors (TKIs; dissolved in dimethyl sulfoxide [DMSO] for 10 mM) were added directly to the medium to a final concentration of 20 µM. DMSO was used as control. For documentation, embryos were mounted in 1% methyl cellulose (Sigma) and pictures were taken with a Leica MZ16 microscope equipped with a Leica DCF3000FX camera. Time-Lapse Confocal Imaging Transplanted embryos were anesthetized with MS222 (Sigma), mounted in low-melting agarose (Thermo Scientific), and covered in E3 medium. Imaging was performed using an upright Leica TCS SP8 MP microscope, equipped with a 10x/0.3 numerical aperture (NA) long working distance apochromat immersion lens (Leica, 506142). The fluorophores were excited with 488-nm and 552-nm lasers, and the images were registered in bidirectional nonresonant scanning mode. Stacks were captured at 12 min intervals with a step size of 10 µm, for 24 hours, using the minimal necessary laser power (optical power meter, Thorlabs, PM100D). Image processing (denoising, enhancing signal intensity) and visualization were performed with Imaris Software (Bitplane) and ImageJ (NIH). Luminescence Measurement Single zebrafish embryos were lysed for 15 min in 96-well plates (10% glycerol, 1% Triton-X100, 1 mM dithiothreitol [DTT], pH 7.8) followed by incubation with an equal amount of substrate solution (1 mM DTT, 1 mM ATP, 0.3 mg/mL D-luciferin, pH 7.8) for 5 min and subsequent luminescence measurement (Hidex Sense). Light-Sheet Microscopy Twenty-four hours post fertilization (hpf), embryos were mounted in a glass capillary containing 1% low melt agarose and, following polymerization, were extruded into an imaging chamber containing E3 medium supplemented with MS222. Time-lapse images were acquired on a Zeiss Lightsheet Z.1 using a 10x detection objective (W-Plan-Apochromat-0.5NA) and 10x illumination objectives (light-sheet fluorescence microscopy, 0.2NA). Samples were illuminated from one or both directions and Z-stacks were acquired every 15 min using a 1x optical zoom, 6.4 µm light-sheet thickness, and 1.8 µm Z-interval. The Contour Surface tool in Imaris 8.4.1 (Bitplane) was used to measure tumor volume at each time point. Max intensity projections were generated and sample drift was corrected for using a rigid body transformation in the StackReg plugin of ImageJ. Statistical Analysis All experiments were performed at minimum in triplicates. The results are presented as mean ± SD. Statistical significance was determined using the 2-tailed Student’s t-test (GraphPad Prism v6). The following P-values were considered significant: **P < 0.01; ***P < 0.001; ****P < 0.0001. Results Based on fate-map analysis and the observation that early zebrafish embryos could potentially provide lineage-specific trophic support to human cells,24 we reasoned that human brain cancer cells injected into zebrafish blastula stages might migrate into CNS structures of the developing zebrafish embryo (Fig. 1A). To test our hypothesis, we transplanted a patient-derived GBM culture (GBM#18)21 into embryos at the blastula stage (3.5 hpf) and found that within 24 hours post transplantation (hpt) 70 ± 7.9% of 99 injected embryos developed a congregated tumor in the CNS (Fig. 1C). Intrigued, we transplanted those human GBM cells into transgenic zebrafish embryos expressing a pan-neuronal marker (elavl3:GFP) and followed their migration by time-lapse confocal microscopy. This real-time analysis revealed that the transplanted cells coalesce in the anterior part of the developing zebrafish embryo before the specification of neurons and that the human GBM cells remained in the neurogenic region of the developing zebrafish embryo during brain formation (see Fig. 1B, Supplementary movie S1). In order to elucidate if this migratory behavior might be a general phenomenon of GBM cultures, we continued transplanting an established GBM culture, U343-MGA-GFP, as well as primary GBM material, GBM#310115 and #3024,15 into blastula-stage zebrafish embryos. Our analysis showed that at 24 hpf, 67.2 ± 5.9% of embryos injected with U343-MGA-GFP (n = 444), 88.3 ± 1.2 of embryos transplanted with GBM#3101 (n = 178), and 90.5 ± 0.7% of embryos transplanted with GBM#3024 (n = 154) suffered from an orthotopic intracranial tumor (Fig. 2A, B). We continued by investigating if the transplantation site within the blastoderm of zebrafish embryos could influence the migratory behavior of GBM cells and transplanted GBM#18 cells either apically or basically into the blastoderm. At 24 hours after apical injection into blastula embryos, 74 ± 10% of tumors were located in the fore/midbrain, whereas no tumor was found in the hindbrain and 25 ± 10% of tumors were located in the tail (n = 26). One day after basal injection into blastula embryos, 73 ± 2.5% of tumors were established in the fore/midbrain, 3 ± 2.5% were found in the hindbrain, and 25 ± 10% were located in the tail (n = 40) (Fig. 2C). Besides, we transplanted SW620 colon cancer cells into blastula-stage embryos, with only 7 ± 3% of intracranial tumors being established until 24 hpf (n = 123) (Fig. 2A, B). Transplantation at blastula stage was tolerated by the embryos and at 24 hpf, we found 90 ± 3.0% of U343-MGA-GFP, 74 ± 8.7% of GBM#3101, 65.5 ± 0.7% of GBM#3024, and 81 ± 23% of SW620 transplanted embryos alive (Fig. 2D). Fig. 1 View largeDownload slide (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. 1 View largeDownload slide (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 View largeDownload slide (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. 2 View largeDownload slide (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. After confirming the robustness of our novel transplantation procedure, we analyzed the xenotransplants by light-sheet microscopy and observed that U343-MGA cells extended tumor microtubules into the host CNS 2 days after transplantation, reminiscent of the human disease (Fig. 3A, Supplementary movie S2). Real-time imaging revealed that this network of protrusions starts to form as early as 24 hpt (Fig. 3B, C, Supplementary movies S3, S4). Intriguingly, the tumor microtubule network continued to grow in complexity until the humane endpoint of the experiment was reached at 6 days post fertilization (Fig. 3E, F and Supplementary movie S5). Simultaneously with sending out protrusions, the volume of U343-MGA transplants increased by ~20% within 24 hours (Fig. 3D). Besides, orthotopically transplanted GBM#18 as well as primary GBM material #3024 also actively proliferated in zebrafish embryos, as shown with anti-Ki67 staining (demarking proliferating cells) on cryosections (Fig. 4A, B). Moreover, transplantation into fli:EGFP embryos with a fluorescent blood vessel system followed by cryosectioning revealed ongoing tumor vascularization (Fig. 4A, B). Active tumor vascularization was also confirmed by light-sheet real-time imaging of fli:EGFP embryos transplanted with GBM#18 cells expressing a red fluorescent protein (Fig. 4F, Supplementary movie S6). Besides active tumor angiogenesis, which is a prerequisite for a clinically relevant GBM animal model, the interaction of GBM with host macrophages/microglia plays a crucial role for tumor aggressiveness and infiltration.25 In order to detect a possible interaction, we orthotopically transplanted U343-MGA into mpeg1:mCherry transgenic zebrafish that express a red fluorescent protein in macrophages/microglia.14 Applying light-sheet microscopy, we could confirm a tight interaction of host macrophages/microglia with the U343-MGA transplant; single transplanted GBM cells were also engulfed and resorbed (Fig. 4C, Supplementary movie S7). Besides the interaction with macrophages/microglia, the aggressiveness of GBM is also driven by the presence of cancer initiating cells which can be identified by Oct4/Sox2 expression.26 Transplantation of GBM#18 cells expressing GFP under the control of the Oct4/Sox2 recognition site revealed that cancer initiating cells persist in the zebrafish embryo over time (Fig. 4E). Fig. 3 View largeDownload slide (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. 3 View largeDownload slide (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 View largeDownload slide (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. 4 View largeDownload slide (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. After a thorough characterization of our improved orthotopic GBM animal model, we sought to assess the antitumor activity of small molecules using our platform. Since neither U343-MGA nor GBM#18 is sensitive to today’s standard-care temozolomide,23 we tested several TKIs which are in clinical trials to treat astrocytoma, including glioblastoma. Zebrafish embryos were transplanted with luciferase expressing U343-MGA or GBM#18 cells and from 24 hpt on exposed to the TKI erlotinib, R-crizotinib, gefitinib, or afatinib for 2 days. Because the tumor cells were engineered to express luciferase, single embryos could be transferred to 96-well plates for quantification of the bioluminescence of remaining tumor. We found erlotinib to reduce the tumor burden of U343-MGA cells most significantly—all other tested TKIs had only limited albeit significant effect (Fig. 4G). We could also measure a significant reduction of tumor burden, despite being generally less sensitive to the TKIs, in all groups of the more aggressive primary GBM culture #18 (Fig. 4G). Discussion Glioblastoma is one of the most intractable forms of cancer. Its complex biology as well as the lack of predictive animal models which can be used in high-throughput screens severely hamper the development of new treatments.3 Although many orthotopic and clinically relevant rodent models have provided invaluable insights, they remain technically challenging, ethically problematic, and expensive. Therefore, orthotopic rodent models can neither be easily implemented in research laboratories to address the complex biology of GBM nor be used in high-throughput drug discovery screens, leaving academic and commercial drug exploratory research without any clinically relevant, orthotopic GBM model for large-scale screening and high-throughput profiling of small molecules. The zebrafish is a well-established and clinically relevant model for cancer4,5,27 and suitable for automated high-throughput screens, since embryos can be raised, treated, and screened in 96-well plate formats, and robotics for automated zebrafish embryo handling are commercially available.7,28,29 Orthotopic transplantation procedures of GBM material into embryonic zebrafish have been developed during the last years and their extraordinary disease recapitulating potential described in detail.10,11,30–32 However, all currently published orthotopic zebrafish models depend on direct intracranial injection, which requires sedation and precise positioning of embryonic zebrafish, and the transplantation process remains slow and technically challenging. Therefore, those models, although clinically relevant, cannot readily be implemented in high-throughput screens to profile novel candidate compounds. Here, we present a substantial improvement of available orthotopic GBM animal models. Instead of transplanting directly into the cranium of embryonic zebrafish, we injected GBM cells at 3.5 hpf into blastula-stage embryos. At this stage, hundreds of embryos can easily be lined up in agarose molds and transplanted quickly; neither sedation nor precise orientation is required compared with all previously published orthotopic animal models. Intriguingly all our analyzed GBM cultures, both established cell lines as well as primary patient-derived material, migrated from their injection site within the blastoderm into the CNS and established a congregated, orthotopic tumor within 24 hpt, indicating a general migration capacity of GBM material. We did not observe that the transplantation site within the blastoderm influenced their migration into the CNS, likely because ectoderm precursor cells are found at all latitudes in the blastoderm fate map.33 Homing to the natural microenvironment of human cells transplanted into blastula embryos has been observed before, and it was postulated that the early zebrafish embryo contains homing cues and trophic support which can be interpreted by human cells.24 Interestingly, during preparation of our tumor cells for transplantation we observed that dissociating GBM cultures using trypsin, an enzyme which degrades surface molecules, significantly reduced their CNS homing capacity compared with dissociation with Accutase, which is a much milder dissociation reagent (Supplementary Figure. S2), indicating that indeed surface molecules of the transplanted cells are involved in the perception of homing cues. Both tumor volume measurements and immunohistochemical analysis confirmed that GBM cell lines and primary GBM cultures proliferate in the zebrafish. Furthermore, actively ongoing tumor vascularization as well as the tight interaction of host macrophages/microglia with the xenotransplants imply a high clinical relevance of our animal model.25,34 During recent years it has been described that astrocytomas, including GBM, establish tumor microtubule networks which facilitate tumor invasion and proliferation and that those interconnections might be a new target for GBM therapy.35 Intriguingly, we observed similar microtubule structures as early as 24 hpt which were growing in length and complexity until the humane endpoint was reached, confirming again the clinical relevance of our improved orthotopic animal model. The fast orthotopic transplantation process described here allows the model to be used in compound screens. As neither U343-MGA nor GBM#18 responds to standard-care temozolomide,23 we tested different TKIs which are in clinical trials. After only 2 days of treatment we found erlotinib to have superior antitumor effects compared with the other tested TKIs on U343-MGA cells. In line with our observations, it has been published that erlotinib has the best blood–brain barrier passage and lowest brain efflux rate of our tested TKIs36–39 in humans and that it exhibits the highest response rate in patients,40 indicating that our model can faithfully recapitulate its pharmacoactivity in vivo. Besides, it has been published before that orthotopically transplanted mouse brain tumors into zebrafish retain their histological and pharmacological profile for erlotinib.10 In contrast to U343-MGA, erlotinib and the other TKIs had only limited but significant effect on the primary GBM#18 transplants. Although it has been published that GBM#18 is highly resistant to imatinib, another TKI,21 further sequencing of the cells to identify potential resistance mechanisms would be needed to fully understand the pharmacological profile. In summary, we provide evidence that our improved orthotopic animal model not only faithfully recapitulates the clinical characteristics of the tumor but could also be easily implemented in drug screens. Importantly, a robust and predictive drug screen can be done in as few as 3 days (Fig. 5). Additionally, as the whole experimental pipeline, including transplantation, drug exposure, and evaluation, can be performed in zebrafish embryos younger than 5 days, no ethical permit is required in most countries and therefore our model complies with the 3R guidelines (replace/reduce/refine). Lastly, it is tempting to speculate that our improved in vivo model could, due to its robustness, be used to transplant freshly excised tumor specimen from patients in order to rapidly evaluate the best option for GBM therapy. Fig. 5 View largeDownload slide 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. Fig. 5 View largeDownload slide 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. Supplementary Material Supplementary material is available at Neuro-Oncology online. Funding This work was supported by the Karolinska Institutes KID funding (L.P.), the Knut and Alice Wallenberg Foundation (KAW2014.273, T.H.), the Swedish Foundation for Strategic Research (RB13-0224, T.H.), the Swedish Cancer Society (T.H., D.N.), the Swedish Research Council (2015-00162, T.H., 2013–03950, D.N.), the Göran Gustafsson Foundation (T.H.), the Swedish Children’s Cancer Foundation (to T.H.), the Swedish Pain Relief Foundation (PR20140048, T.H.), and the Torsten and Ragnar Söderberg Foundation (T.H.) Acknowledgments We would like to thank the zebrafish core facility at Karolinska Institute for their excellent service, K. Edfeldt for administrative support, the KTH node of the National Microscopy Infrastructure, NMI (VR-RFI 2016-00968), as well as S. Eriksson and F. Pineiro for superior laboratory assistance. Primary glioblastoma cultures were provided from M. Nister and D. Hägerstrand (Karolinska Institutet) as well as the Uppsala Biobank. We further would like to acknowledge C. Berndt (Heinrich-Heine University of Düsseldorf) for artwork. Authorship: L.P., S.E., M.B., J.A-S., J.D., I.A., and L.B. performed experiments. L.P., D.N., T.H., and L.B. conceived the study. L.P. and L.B. wrote the manuscript. All authors have read and approved the final version. Conflict of interest statement. No conflicts of interest. References 1. Osuka S , Van Meir EG . Overcoming therapeutic resistance in glioblastoma: the way forward . J Clin Invest . 2017 ; 127 ( 2 ): 415 – 426 . 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One for all—a highly efficient and versatile method for fluorescent immunostaining in fish embryos . PLoS One . 2011 ; 6 ( 5 ): e19713 . Google Scholar Crossref Search ADS PubMed 20. Nistér M , Heldin CH , Westermark B . Clonal variation in the production of a platelet-derived growth factor-like protein and expression of corresponding receptors in a human malignant glioma . Cancer Res . 1986 ; 46 ( 1 ): 332 – 340 . Google Scholar PubMed 21. Hägerstrand D , Hesselager G , Achterberg S , et al. Characterization of an imatinib-sensitive subset of high-grade human glioma cultures . Oncogene . 2006 ; 25 ( 35 ): 4913 – 4922 . Google Scholar Crossref Search ADS PubMed 22. Xie Y , Bergström T , Jiang Y , et al. The human glioblastoma cell culture resource: validated cell models representing all molecular subtypes . EBioMedicine . 2015 ; 2 ( 10 ): 1351 – 1363 . Google Scholar Crossref Search ADS PubMed 23. Pudelko L , Rouhi P , Sanjiv K , et al. Glioblastoma and glioblastoma stem cells are dependent on functional MTH1 . Oncotarget . 2017 ; 8 ( 49 ): 84671 – 84684 . Google Scholar Crossref Search ADS PubMed 24. Lee LM , Seftor EA , Bonde G , Cornell RA , Hendrix MJ . The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation . Dev Dyn . 2005 ; 233 ( 4 ): 1560 – 1570 . Google Scholar Crossref Search ADS PubMed 25. Dello Russo C , Lisi L , Tentori L , Navarra P , Graziani G , Combs CK . Exploiting microglial functions for the treatment of glioblastoma . Curr Cancer Drug Targets . 2017 ; 17 ( 3 ): 267 – 281 . Google Scholar Crossref Search ADS PubMed 26. Tang B , Raviv A , Esposito D , et al. A flexible reporter system for direct observation and isolation of cancer stem cells . Stem Cell Reports . 2015 ; 4 ( 1 ): 155 – 169 . Google Scholar Crossref Search ADS PubMed 27. Ceol CJ , Houvras Y . Uncharted waters: zebrafish cancer models navigate a course for oncogene discovery . Adv Exp Med Biol . 2016 ; 916 : 3 – 19 . Google Scholar Crossref Search ADS PubMed 28. van der Ent W , Veneman WJ , Groenewoud A , et al. Automation of technology for cancer research . Adv Exp Med Biol . 2016 ; 916 : 315 – 332 . Google Scholar Crossref Search ADS PubMed 29. Dang M , Fogley R , Zon LI . Identifying novel cancer therapies using chemical genetics and zebrafish . Adv Exp Med Biol . 2016 ; 916 : 103 – 124 . Google Scholar Crossref Search ADS PubMed 30. Wenger A , Larsson S , Danielsson A , et al. Stem cell cultures derived from pediatric brain tumors accurately model the originating tumors . Oncotarget . 2017 Mar 21;8(12):18626–18639. 31. Astell KR , Sieger D . Investigating microglia-brain tumor cell interactions in vivo in the larval zebrafish brain . Methods Cell Biol . 2017 ; 138 : 593 – 626 . Google Scholar Crossref Search ADS PubMed 32. Wehmas LC , Tanguay RL , Punnoose A , Greenwood JA . Developing a novel embryo-larval zebrafish xenograft assay to prioritize human glioblastoma therapeutics . Zebrafish . 2016 ; 13 ( 4 ): 317 – 329 . Google Scholar Crossref Search ADS PubMed 33. Kimmel CB , Warga RM , Schilling TF . Origin and organization of the zebrafish fate map . Development . 1990 ; 108 ( 4 ): 581 – 594 . Google Scholar PubMed 34. Jhaveri N , Chen TC , Hofman FM . Tumor vasculature and glioma stem cells: contributions to glioma progression . Cancer Lett . 2016 ; 380 ( 2 ): 545 – 551 . Google Scholar Crossref Search ADS PubMed 35. Osswald M , Jung E , Sahm F , et al. Brain tumour cells interconnect to a functional and resistant network . Nature . 2015 ; 528 ( 7580 ): 93 – 98 . Google Scholar PubMed 36. Deng Y , Feng W , Wu J , et al. The concentration of erlotinib in the cerebrospinal fluid of patients with brain metastasis from non-small-cell lung cancer . Mol Clin Oncol . 2014 ; 2 ( 1 ): 116 – 120 . Google Scholar Crossref Search ADS PubMed 37. Togashi Y , Masago K , Masuda S , et al. Cerebrospinal fluid concentration of gefitinib and erlotinib in patients with non-small cell lung cancer . Cancer Chemother Pharmacol . 2012 ; 70 ( 3 ): 399 – 405 . Google Scholar Crossref Search ADS PubMed 38. Tamiya A , Tamiya M , Nishihara T , et al. Afatinib efficacy and cerebrospinal fluid concentration in NSCLC patients with EGFR mutation developing leptomeningeal carcinomatosis . Ann Oncol . 2016 October 1;27(suppl 6). 39. Costa DB , Kobayashi S , Pandya SS , et al. CSF concentration of the anaplastic lymphoma kinase inhibitor crizotinib . J Clin Oncol . 2011 ; 29 ( 15 ): e443 – e445 . Google Scholar Crossref Search ADS PubMed 40. Vogelbaum MA , Peereboom D , Stevens G , Barnett G , Brewer C . Phase II trial of the EGFR tyrosine kinase inhibitor erlotinib for single agent therapy of recurrent glioblastoma multiforme: interim results . J Clin Oncol . 2004 July 15;22(14):1558–1558. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Neuro-Oncology Oxford University Press

An orthotopic glioblastoma animal model suitable for high-throughput screenings

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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1522-8517
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1523-5866
D.O.I.
10.1093/neuonc/noy071
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Abstract

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. animal model, brain cancer, glioblastoma, orthotopic transplantation, zebrafish Importance of the study The development of novel drugs fighting GBM is challenging, as no orthotopic animal model exists which can readily be implemented in large-scale drug discovery screens. The zebrafish is a clinically relevant model for human malignancies, and orthotopic transplantation of GBM material was found to faithfully recapitulate the human disease. However, none of the currently available transplantation procedures in zebrafish can be used in high-throughput screens, as the injection process is time-consuming and technically challenging. We have refined existing orthotopic zebrafish models and developed an approach which allows for transplantation of thousands of embryos per hour. This model can facilitate automated drug discovery and profiling as well as help to identify novel candidate drugs against GBM. Glioblastoma (GBM) is one of the most common malignancies of the central nervous system (CNS), and despite aggressive combinatory treatment, including maximal surgical resection, radiotherapy, and adjuvant chemotherapy, prognosis remains dismal. GBM in particular is characterized by extensive tumor heterogeneity as well as cancer cell plasticity and the presence of cancer stem cells.1,2 These multifaceted factors contribute to the ability of GBM to evade almost any treatment strategy.1,2 Understanding the complex biology of GBM in its physiological environment is a key for the development of new therapeutic options, and improvement of currently available animal models is a crucial prerequisite.3 Xenograft models, which are based on the orthotopic transplantation of either patient-derived glioblastoma cells or fresh brain tumor biopsy spheroids into immune-deficient rodents, resemble closely the genetic and phenotypic heterogeneity of the original patient tumor. Due to their clinical relevance, xenograft models remain the gold standard for understanding the biology of GBM.3 However, while recapitulating human gliomablastoma sufficiently well, orthotopic xenografts in rodents are technically challenging. In addition, ethical and financial constraints make them unsuitable for use in drug screening and profiling projects. During the last decade, the zebrafish (Danio rerio) has been characterized as a powerful and clinically relevant model for human diseases, including cancer.4–6 Besides its ex utero development, small size, high fecundity, and cost-efficient husbandry, the absence of a functional adaptive immune system until embryonic day 21 makes it an ideal animal model for xenotransplantation. Furthermore, the zebrafish is highly suitable for large-scale drug discovery projects, and its predictive capacity has already been exploited in various high-throughput screens.7 Orthotopic xenograft models of GBM in zebrafish have been reported by several groups8,9 and recent studies highlighted their extraordinary recapitulative potential and clinical relevance.10,11 However, since intracranial transplantation of GBM material into single zebrafish embryos is technically challenging and time-consuming, the use of zebrafish to understand the complex biology of GBM and to facilitate high-throughput screens for candidate therapies is currently limited. A novel method allowing for easy, fast, and automatable orthotopic injection of GBM material into zebrafish embryos would considerably help not only to understand GBM biology but also to develop, characterize, and verify novel treatment options. Materials and Methods Zebrafish Maintenance and Transgenic Strains The zebrafish tupfel longfin, fli1a:EGFP,12 HuC (elavl3):GFP,13 mpeg1:mCherry,14 and the Zebrabow15 strains were used throughout this study and raised and staged according to standard conditions.16 Transplanted zebrafish embryos were kept at 33°C. As described before,17,18 neither embryo growth/viability nor GBM proliferation (Supplementary Fig. S1) was affected.17,18 All experimental protocols were performed in accordance with the national ethical guidelines and regulations (N207/14). Cryosections and Immunocytochemistry Transplanted zebrafish embryos were prepared for cryosections as described previously19 and sections were stained with anti-Ki67 (#P6834, Sigma) and anti‒green fluorescent protein (GFP) antibodies (#SC8334, Santa Cruz) together with an anti-mouse/Alexa Fluor 488 secondary antibody using routine protocols. Images were taken with a Zeiss LSM 700 confocal microscope. Cell Culture All cells have been growing at 37°C in a humidified atmosphere (95% humidity) with 5% CO2. The GBM cell line U343-MGA-GFP20 as well as the primary GBM culture #1821 have been propagated in Minimum Essential Medium (Gibco) containing 2 mM glutamine (Gibco). Primary GBM cultures #310122 and #320422 have been propagated as stated in the reference. SW620 cells have been propagated following the suggestions of the American Type Culture Collection. All cells have been dissociated with Accutase (Sigma) at subconfluency. For details on generation and authentication of patient-derived GBM cell lines used in this study, see the respective references. Generation of GBM#18 cells expressing an octamer-binding transcription factor 4 (Oct4)/sex determining region Y–box 2 (Sox2) reporter has been described before.23 GBM cells stably expressing tdTomato or luciferase were generated with a lentivirus-based approach using the vector #32904 or #17477 (Addgene). Regular mycoplasma tests have been performed in all cell cultures using Mycoalert (#LT07, Lonza). Transplantation of Glioblastoma Cultures and Treatment Glioblastoma cultures were maintained as stated above and transferred to complete neural stem cell medium 7 days before transplantation. All cell cultures were prepared for injection as previously described.25 Briefly, cells were washed with phosphate buffered saline (PBS) and labeled with DiI, a fluorescent dye (1:250 in PBS, #V22885, Molecular Probes) for 15 min, followed by several washing steps using PBS to remove residual dye particles. Subsequently, cells were harvested, passed over a 20 µM nylon mesh, and resuspended in medium containing 2% polyvinylpyrrolidone (Sigma) to avoid clogging of the micro-injection capillary. Directly before transplantation, cells were spun down and medium was almost entirely removed. The cell suspension was loaded in nonfilament microcapillaries (World Precision Instruments) and approximately 100 tumor cells were injected into blastula-stage zebrafish embryos which have been immobilized in an agarose mold. After injection, embryos were screened for successful transplantation, transferred into a 10 cm dish, and incubated at 33°C for 24 hours before analysis or subsequent treatment. For treatment, embryos were distributed to 6-well plates (20 embryos/well) in a total volume of 3 mL E3 medium containing 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid). Tyrosine kinase inhibitors (TKIs; dissolved in dimethyl sulfoxide [DMSO] for 10 mM) were added directly to the medium to a final concentration of 20 µM. DMSO was used as control. For documentation, embryos were mounted in 1% methyl cellulose (Sigma) and pictures were taken with a Leica MZ16 microscope equipped with a Leica DCF3000FX camera. Time-Lapse Confocal Imaging Transplanted embryos were anesthetized with MS222 (Sigma), mounted in low-melting agarose (Thermo Scientific), and covered in E3 medium. Imaging was performed using an upright Leica TCS SP8 MP microscope, equipped with a 10x/0.3 numerical aperture (NA) long working distance apochromat immersion lens (Leica, 506142). The fluorophores were excited with 488-nm and 552-nm lasers, and the images were registered in bidirectional nonresonant scanning mode. Stacks were captured at 12 min intervals with a step size of 10 µm, for 24 hours, using the minimal necessary laser power (optical power meter, Thorlabs, PM100D). Image processing (denoising, enhancing signal intensity) and visualization were performed with Imaris Software (Bitplane) and ImageJ (NIH). Luminescence Measurement Single zebrafish embryos were lysed for 15 min in 96-well plates (10% glycerol, 1% Triton-X100, 1 mM dithiothreitol [DTT], pH 7.8) followed by incubation with an equal amount of substrate solution (1 mM DTT, 1 mM ATP, 0.3 mg/mL D-luciferin, pH 7.8) for 5 min and subsequent luminescence measurement (Hidex Sense). Light-Sheet Microscopy Twenty-four hours post fertilization (hpf), embryos were mounted in a glass capillary containing 1% low melt agarose and, following polymerization, were extruded into an imaging chamber containing E3 medium supplemented with MS222. Time-lapse images were acquired on a Zeiss Lightsheet Z.1 using a 10x detection objective (W-Plan-Apochromat-0.5NA) and 10x illumination objectives (light-sheet fluorescence microscopy, 0.2NA). Samples were illuminated from one or both directions and Z-stacks were acquired every 15 min using a 1x optical zoom, 6.4 µm light-sheet thickness, and 1.8 µm Z-interval. The Contour Surface tool in Imaris 8.4.1 (Bitplane) was used to measure tumor volume at each time point. Max intensity projections were generated and sample drift was corrected for using a rigid body transformation in the StackReg plugin of ImageJ. Statistical Analysis All experiments were performed at minimum in triplicates. The results are presented as mean ± SD. Statistical significance was determined using the 2-tailed Student’s t-test (GraphPad Prism v6). The following P-values were considered significant: **P < 0.01; ***P < 0.001; ****P < 0.0001. Results Based on fate-map analysis and the observation that early zebrafish embryos could potentially provide lineage-specific trophic support to human cells,24 we reasoned that human brain cancer cells injected into zebrafish blastula stages might migrate into CNS structures of the developing zebrafish embryo (Fig. 1A). To test our hypothesis, we transplanted a patient-derived GBM culture (GBM#18)21 into embryos at the blastula stage (3.5 hpf) and found that within 24 hours post transplantation (hpt) 70 ± 7.9% of 99 injected embryos developed a congregated tumor in the CNS (Fig. 1C). Intrigued, we transplanted those human GBM cells into transgenic zebrafish embryos expressing a pan-neuronal marker (elavl3:GFP) and followed their migration by time-lapse confocal microscopy. This real-time analysis revealed that the transplanted cells coalesce in the anterior part of the developing zebrafish embryo before the specification of neurons and that the human GBM cells remained in the neurogenic region of the developing zebrafish embryo during brain formation (see Fig. 1B, Supplementary movie S1). In order to elucidate if this migratory behavior might be a general phenomenon of GBM cultures, we continued transplanting an established GBM culture, U343-MGA-GFP, as well as primary GBM material, GBM#310115 and #3024,15 into blastula-stage zebrafish embryos. Our analysis showed that at 24 hpf, 67.2 ± 5.9% of embryos injected with U343-MGA-GFP (n = 444), 88.3 ± 1.2 of embryos transplanted with GBM#3101 (n = 178), and 90.5 ± 0.7% of embryos transplanted with GBM#3024 (n = 154) suffered from an orthotopic intracranial tumor (Fig. 2A, B). We continued by investigating if the transplantation site within the blastoderm of zebrafish embryos could influence the migratory behavior of GBM cells and transplanted GBM#18 cells either apically or basically into the blastoderm. At 24 hours after apical injection into blastula embryos, 74 ± 10% of tumors were located in the fore/midbrain, whereas no tumor was found in the hindbrain and 25 ± 10% of tumors were located in the tail (n = 26). One day after basal injection into blastula embryos, 73 ± 2.5% of tumors were established in the fore/midbrain, 3 ± 2.5% were found in the hindbrain, and 25 ± 10% were located in the tail (n = 40) (Fig. 2C). Besides, we transplanted SW620 colon cancer cells into blastula-stage embryos, with only 7 ± 3% of intracranial tumors being established until 24 hpf (n = 123) (Fig. 2A, B). Transplantation at blastula stage was tolerated by the embryos and at 24 hpf, we found 90 ± 3.0% of U343-MGA-GFP, 74 ± 8.7% of GBM#3101, 65.5 ± 0.7% of GBM#3024, and 81 ± 23% of SW620 transplanted embryos alive (Fig. 2D). Fig. 1 View largeDownload slide (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. 1 View largeDownload slide (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 View largeDownload slide (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. 2 View largeDownload slide (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. After confirming the robustness of our novel transplantation procedure, we analyzed the xenotransplants by light-sheet microscopy and observed that U343-MGA cells extended tumor microtubules into the host CNS 2 days after transplantation, reminiscent of the human disease (Fig. 3A, Supplementary movie S2). Real-time imaging revealed that this network of protrusions starts to form as early as 24 hpt (Fig. 3B, C, Supplementary movies S3, S4). Intriguingly, the tumor microtubule network continued to grow in complexity until the humane endpoint of the experiment was reached at 6 days post fertilization (Fig. 3E, F and Supplementary movie S5). Simultaneously with sending out protrusions, the volume of U343-MGA transplants increased by ~20% within 24 hours (Fig. 3D). Besides, orthotopically transplanted GBM#18 as well as primary GBM material #3024 also actively proliferated in zebrafish embryos, as shown with anti-Ki67 staining (demarking proliferating cells) on cryosections (Fig. 4A, B). Moreover, transplantation into fli:EGFP embryos with a fluorescent blood vessel system followed by cryosectioning revealed ongoing tumor vascularization (Fig. 4A, B). Active tumor vascularization was also confirmed by light-sheet real-time imaging of fli:EGFP embryos transplanted with GBM#18 cells expressing a red fluorescent protein (Fig. 4F, Supplementary movie S6). Besides active tumor angiogenesis, which is a prerequisite for a clinically relevant GBM animal model, the interaction of GBM with host macrophages/microglia plays a crucial role for tumor aggressiveness and infiltration.25 In order to detect a possible interaction, we orthotopically transplanted U343-MGA into mpeg1:mCherry transgenic zebrafish that express a red fluorescent protein in macrophages/microglia.14 Applying light-sheet microscopy, we could confirm a tight interaction of host macrophages/microglia with the U343-MGA transplant; single transplanted GBM cells were also engulfed and resorbed (Fig. 4C, Supplementary movie S7). Besides the interaction with macrophages/microglia, the aggressiveness of GBM is also driven by the presence of cancer initiating cells which can be identified by Oct4/Sox2 expression.26 Transplantation of GBM#18 cells expressing GFP under the control of the Oct4/Sox2 recognition site revealed that cancer initiating cells persist in the zebrafish embryo over time (Fig. 4E). Fig. 3 View largeDownload slide (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. 3 View largeDownload slide (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 View largeDownload slide (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. 4 View largeDownload slide (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. After a thorough characterization of our improved orthotopic GBM animal model, we sought to assess the antitumor activity of small molecules using our platform. Since neither U343-MGA nor GBM#18 is sensitive to today’s standard-care temozolomide,23 we tested several TKIs which are in clinical trials to treat astrocytoma, including glioblastoma. Zebrafish embryos were transplanted with luciferase expressing U343-MGA or GBM#18 cells and from 24 hpt on exposed to the TKI erlotinib, R-crizotinib, gefitinib, or afatinib for 2 days. Because the tumor cells were engineered to express luciferase, single embryos could be transferred to 96-well plates for quantification of the bioluminescence of remaining tumor. We found erlotinib to reduce the tumor burden of U343-MGA cells most significantly—all other tested TKIs had only limited albeit significant effect (Fig. 4G). We could also measure a significant reduction of tumor burden, despite being generally less sensitive to the TKIs, in all groups of the more aggressive primary GBM culture #18 (Fig. 4G). Discussion Glioblastoma is one of the most intractable forms of cancer. Its complex biology as well as the lack of predictive animal models which can be used in high-throughput screens severely hamper the development of new treatments.3 Although many orthotopic and clinically relevant rodent models have provided invaluable insights, they remain technically challenging, ethically problematic, and expensive. Therefore, orthotopic rodent models can neither be easily implemented in research laboratories to address the complex biology of GBM nor be used in high-throughput drug discovery screens, leaving academic and commercial drug exploratory research without any clinically relevant, orthotopic GBM model for large-scale screening and high-throughput profiling of small molecules. The zebrafish is a well-established and clinically relevant model for cancer4,5,27 and suitable for automated high-throughput screens, since embryos can be raised, treated, and screened in 96-well plate formats, and robotics for automated zebrafish embryo handling are commercially available.7,28,29 Orthotopic transplantation procedures of GBM material into embryonic zebrafish have been developed during the last years and their extraordinary disease recapitulating potential described in detail.10,11,30–32 However, all currently published orthotopic zebrafish models depend on direct intracranial injection, which requires sedation and precise positioning of embryonic zebrafish, and the transplantation process remains slow and technically challenging. Therefore, those models, although clinically relevant, cannot readily be implemented in high-throughput screens to profile novel candidate compounds. Here, we present a substantial improvement of available orthotopic GBM animal models. Instead of transplanting directly into the cranium of embryonic zebrafish, we injected GBM cells at 3.5 hpf into blastula-stage embryos. At this stage, hundreds of embryos can easily be lined up in agarose molds and transplanted quickly; neither sedation nor precise orientation is required compared with all previously published orthotopic animal models. Intriguingly all our analyzed GBM cultures, both established cell lines as well as primary patient-derived material, migrated from their injection site within the blastoderm into the CNS and established a congregated, orthotopic tumor within 24 hpt, indicating a general migration capacity of GBM material. We did not observe that the transplantation site within the blastoderm influenced their migration into the CNS, likely because ectoderm precursor cells are found at all latitudes in the blastoderm fate map.33 Homing to the natural microenvironment of human cells transplanted into blastula embryos has been observed before, and it was postulated that the early zebrafish embryo contains homing cues and trophic support which can be interpreted by human cells.24 Interestingly, during preparation of our tumor cells for transplantation we observed that dissociating GBM cultures using trypsin, an enzyme which degrades surface molecules, significantly reduced their CNS homing capacity compared with dissociation with Accutase, which is a much milder dissociation reagent (Supplementary Figure. S2), indicating that indeed surface molecules of the transplanted cells are involved in the perception of homing cues. Both tumor volume measurements and immunohistochemical analysis confirmed that GBM cell lines and primary GBM cultures proliferate in the zebrafish. Furthermore, actively ongoing tumor vascularization as well as the tight interaction of host macrophages/microglia with the xenotransplants imply a high clinical relevance of our animal model.25,34 During recent years it has been described that astrocytomas, including GBM, establish tumor microtubule networks which facilitate tumor invasion and proliferation and that those interconnections might be a new target for GBM therapy.35 Intriguingly, we observed similar microtubule structures as early as 24 hpt which were growing in length and complexity until the humane endpoint was reached, confirming again the clinical relevance of our improved orthotopic animal model. The fast orthotopic transplantation process described here allows the model to be used in compound screens. As neither U343-MGA nor GBM#18 responds to standard-care temozolomide,23 we tested different TKIs which are in clinical trials. After only 2 days of treatment we found erlotinib to have superior antitumor effects compared with the other tested TKIs on U343-MGA cells. In line with our observations, it has been published that erlotinib has the best blood–brain barrier passage and lowest brain efflux rate of our tested TKIs36–39 in humans and that it exhibits the highest response rate in patients,40 indicating that our model can faithfully recapitulate its pharmacoactivity in vivo. Besides, it has been published before that orthotopically transplanted mouse brain tumors into zebrafish retain their histological and pharmacological profile for erlotinib.10 In contrast to U343-MGA, erlotinib and the other TKIs had only limited but significant effect on the primary GBM#18 transplants. Although it has been published that GBM#18 is highly resistant to imatinib, another TKI,21 further sequencing of the cells to identify potential resistance mechanisms would be needed to fully understand the pharmacological profile. In summary, we provide evidence that our improved orthotopic animal model not only faithfully recapitulates the clinical characteristics of the tumor but could also be easily implemented in drug screens. Importantly, a robust and predictive drug screen can be done in as few as 3 days (Fig. 5). Additionally, as the whole experimental pipeline, including transplantation, drug exposure, and evaluation, can be performed in zebrafish embryos younger than 5 days, no ethical permit is required in most countries and therefore our model complies with the 3R guidelines (replace/reduce/refine). Lastly, it is tempting to speculate that our improved in vivo model could, due to its robustness, be used to transplant freshly excised tumor specimen from patients in order to rapidly evaluate the best option for GBM therapy. Fig. 5 View largeDownload slide 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. Fig. 5 View largeDownload slide 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. Supplementary Material Supplementary material is available at Neuro-Oncology online. Funding This work was supported by the Karolinska Institutes KID funding (L.P.), the Knut and Alice Wallenberg Foundation (KAW2014.273, T.H.), the Swedish Foundation for Strategic Research (RB13-0224, T.H.), the Swedish Cancer Society (T.H., D.N.), the Swedish Research Council (2015-00162, T.H., 2013–03950, D.N.), the Göran Gustafsson Foundation (T.H.), the Swedish Children’s Cancer Foundation (to T.H.), the Swedish Pain Relief Foundation (PR20140048, T.H.), and the Torsten and Ragnar Söderberg Foundation (T.H.) Acknowledgments We would like to thank the zebrafish core facility at Karolinska Institute for their excellent service, K. Edfeldt for administrative support, the KTH node of the National Microscopy Infrastructure, NMI (VR-RFI 2016-00968), as well as S. Eriksson and F. Pineiro for superior laboratory assistance. Primary glioblastoma cultures were provided from M. Nister and D. Hägerstrand (Karolinska Institutet) as well as the Uppsala Biobank. We further would like to acknowledge C. Berndt (Heinrich-Heine University of Düsseldorf) for artwork. Authorship: L.P., S.E., M.B., J.A-S., J.D., I.A., and L.B. performed experiments. L.P., D.N., T.H., and L.B. conceived the study. L.P. and L.B. wrote the manuscript. All authors have read and approved the final version. Conflict of interest statement. No conflicts of interest. References 1. Osuka S , Van Meir EG . Overcoming therapeutic resistance in glioblastoma: the way forward . J Clin Invest . 2017 ; 127 ( 2 ): 415 – 426 . 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J Clin Oncol . 2004 July 15;22(14):1558–1558. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Neuro-OncologyOxford University Press

Published: Oct 9, 2018

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