Orthotopic models of pediatric brain tumors in zebrafish

Abstract

High-throughput screens (HTS) of compound toxicity against cancer cells can identify thousands of potential new drug-leads. But only limited numbers of these compounds can progress to expensive and labor-intensive efficacy studies in mice, creating a 'bottle neck' in the drug development pipeline. Approaches that triage drug-leads for further study are greatly needed. Here we provide an intermediary platform between HTS and mice by adapting mouse models of pediatric brain tumors to grow as orthotopic xenografts in the brains of zebrafish. Freshly isolated mouse ependymoma, glioma and choroid plexus carcinoma cells expressing red fluorescence protein were conditioned to grow at 34 °C. Conditioned tumor cells were then transplanted orthotopically into the brains of zebrafish acclimatized to ambient temperatures of 34 °C. Live in vivo fluorescence imaging identified robust, quantifiable and reproducible brain tumor growth as well as spinal metastasis in zebrafish. All tumor xenografts in zebrafish retained the histological characteristics of the corresponding parent mouse tumor and efficiently recruited fish endothelial cells to form a tumor vasculature. Finally, by treating zebrafish harboring ERBB2-driven gliomas with an appropriate cytotoxic chemotherapy (5-fluorouracil) or tyrosine kinase inhibitor (erlotinib), we show that these models can effectively assess drug efficacy. Our data demonstrate, for the first time, that mouse brain tumors can grow orthotopically in fish and serve as a platform to study drug efficacy. As large cohorts of brain tumor-bearing zebrafish can be generated rapidly and inexpensively, these models may serve as a powerful tool to triage drug-leads from HTS for formal efficacy testing in mice.

Figure 1. Generation of orthotopic mouse brain tumor xenografts in zebrafish

(a) Mouse brain tumor cells were harvested from mice and cultured under conditions that promote neural stem cell growth (ref. 7). (b) The temperature of brain tumor cell cultures was reduced by 0.75°C per week for 4 weeks. (c) Three month old wild-type (AB) or Fli1:eGFP transgenic zebrafish were acclimatized to an ambient temperature of 34°C by increasing tank water temperature by 1°C per day for six days. Zebrafish were immunosuppressed by addition of dexamethasone (15mg/ml) to tank water 2 days to prior to implantation. (d) Immunosuppressed zebrafish were anesthetized using 0.04% Tricaine, placed in a 30mm petri dish under an intravital microscope. 2×10⁵ of tumor (or control) cells were injected into the cerebral hemisphere via the intranasal route using a 30 gauge 1μl Hamilton syringe. (e) Zebrafish were subject to intravital fluorescence microscopy to monitor tumor growth. Conditioning methodology used for successful engraftment of mouse cells in adult zebrafish.

Figure 2. Orthotopic mouse brain tumor growth in zebrafish

(a) RFP⁺ mouse brain tumors were imaged in live, anesthetized zebrafish using an Olympus SZX10® stereomicroscope fitted with a 594nm filter. Representative intra-vital lateral (a) and dorsal (b) images of ependymoma EP^RTBDN-RFP. (c) This same zebrafish was sacrificed, the brain macrodisected and imaged, confirming the RFP⁺ tumor is present within the cerebral hemisphere. (d) Survival curve of zebrafish harboring the indicated tumor types or control NSCs. The survival of zebrafish harboring tumors was significantly reduced vs. control NSC^RFP (P<0.001; Log-Rank). Zebrafish were implanted with (e) the indicated tumor cells or (f) control NSC^RFP and imaged exactly as described in (a). The mean intensity of tumor fluorescence was measured using NIS Elements (v 3.2) software. Serial day 1, 3 and 5 IFM images of eight zebrafish each harboring EP^RTBDN-RFP, GBM^ERBB2-RFP or CPC^RFP tumors are shown. Average +SE RFP-fluorescence relative to day 1 is shown in the graphs left (Mann-Whitney U test; *=p<0.05; **=p<0.005; DOD=dead of disease). No NSC^RFP were detectable in zebrafish brains one day following implantation (f).

Figure 3. Mouse brain tumors retain key biologic characteristics when xenorafted into zebrafish brains

(a) Zebrafish harboring tumors were euthanized using 0.04% Tricaine and fixed in 4% paraformaldehyde overnight. Zebrafish were then decalcified using 0.5M EDTA (AMRESCO®) for 5 days, rinsed in phosphate buffered saline, dehydrated and paraffin wax embedded. 5μm sections were stained using hematoxylin and eosin (H&E) or subjected to standard immunohistochemistry using the indicated primary antibodies (GFAP 1:500, Dako rabbit polyclonal Z0334; ERBB2 1:40, Vector mouse monoclonal VP-C380; Ki67, 1:1000, Vector rabbit polyclonal vp-K451). TTR expression was visualized by in situ hybridization using a full length TTR cRNA template (BC032069, generous gift of Dr. Edwin Monuki). Paraffin sections were treated with RNAzip (Ambion, Austin, TX) and de-waxed to water. Probe hybridization was performed at 60°C overnight in standard hybridization buffer. Sections of the same tumors growing in mouse brains were analyzed in parallel for comparison. Arrows in EP^RTBDN-RFP H&E and GFAP mark pseudorosettes and GFAP⁺ tumor cells respectively. Dotted line in bottom right GBM^ERBB2-RFP H&E demarcates tumor invading normal brain. Arrow indicates mitotic tumor cells (scale bars=15μm). (b) Zebrafish were implanted with the indicated tumors and the entire dorsal brain and spine imaged daily exactly as described in Figure 1 and 2. Arrows in H&E of EP^RTBDN-RFP tumors mark individual metastatic deposits. Pie charts report the proportion of zebrafish in which the indicated tumors metastasized. (c) Fli1:eGFP zebrafish harboring tumors were euthanized fixed and decalcified as described above. Brains containing tumor were then cryo-protected in 30% sucrose for 2 days and frozen in tissue freezing media (TBS^®). 12μm sections were counter stained using DAPI containing hard set mounting media and imaged by confocal microscopy with 488nm and 594nm filters. (d) Total RNA was extracted from the indicated brain tumors grown in mouse and zebrafish brains. Microarray gene expression profiles were generated using Affymetrix 430v2 arrays and subject to unsupervised hierarchical clustering exactly as described. Transcriptomes of each tumor type grown in mouse and human brains were directly compared using the Agreement of Differential Expression (AGDEX) algorithm exactly as described was used.

Figure 4. Zebrafish brain tumor models can be used for preclinical drug testing

(a) Single cell suspensions of 750 GBM^ERBB2-RFP tumor cells were seeded into each well of a 96-well plate in neurobasal medium exactly as described. 24 hours later cells were dosed with the indicated concentrations of (a) 5-FU or (b) Erlotinib, or DMSO vehicle control and incubated for a further 72 hours. Percent cell survival relative to DMSO only controls was then determined in each well using the Cell Titer Glo reagent (Promega) and Envision plate reader (Perkin-Elmer). Assays were performed in independent triplicates. (c) Zebrafish harboring GBM^ERBB2-RFP tumors were established exactly as described in Figure 1. After 24 hours zebrafish were treated with addition of 5-FU or vehicle (control) to the tank water (top), or vehicle (control) or Erlotinib by oral gavage on two consecutive days. All zebrafish were imaged exactly as described in Figure 2. (d) Graph reports the fold fluorescence of tumors shown in (c) at day 2 relative to fluorescence at day 0 in fish treated with vehicle, 5-FU or erolotinib. Whiskers=extreme outliers, Box=median, 25^th and 75^th percentiles. (e) Immunohistochemistry of active, phospho-ERBB2^Y1248 receptor expression in GBM^ERBB2-RFP tumors taken from fish treated with erlotinib or vehicle control. Immunostaining was performed as described in Figure 3 using ERBB2^Y1248 rabbit polyclonal antibody (Novus Biologicals NB 100-81960; scale bars=15μm).