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 described⁷. 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).