Characterization of GTML spheres.

<p>(<b>A</b>) Establishing GTML spheres in culture. Primary cells were isolated from GTML tumors, which were identified through the bioluminescence signals (upper left) and morphology, and were then dissociated into single cells. M10519 GTML neurospheres cultured after 10 days are also shown. Scale bar: 100μm. (<b>B</b>) Effect of MYCN withdrawal on cell growth. M10519 GTML spheres were seeded at density of 1x10⁵ cells/ml in the presence of absence of 1μg/ml of dox, and the proliferation of spheres were measured by WST-1 metabolic assay. Error bars, ±SD. (<b>C</b>) Effect of MYCN withdrawal on cell cycle. M10519 GTML neurosphere were treated with dox prior to cell cycle analyses using FACS. (<b>D</b>) Western analyses. M10519 GTML spheres were cultured with 1μg/ml of dox.</p

Tumor-propagating potential of CD133+ cells.

<p>(<b>A</b>) Purification of CD133+ and CD133- cells. M10519 cells (passage 13) were stained with anti-IgG1 or anti-Prominin-1 (CD133) conjugated with PE prior to purification. Stained cells were first separated into bead-bound and bead-unbound fractions by magnetic beads conjugated with anti-PE (middle panels). Then each fraction was further purified by FACS sorting. <b>(B</b>) Orthotopic tumors generated after implantation of CD133+ cells (10 cells per mouse). Sections were stained with hematoxylin and eosin. Bars, 1000μm (upper), 50μm (lower). (<b>C</b>) Kaplan-Meier curves for overall survival of mice implanted with 10 CD133+ (red, n = 10) or CD133- (blue, n = 10) M10519 cells per site.</p

Effect of MLN8237 on GTML spheres and mice.

<p>(<b>A</b>) PC projections of 96 cells from WT1, WT2, M10519 cells treated with and without dox, and M10519 cells with 100nM MLN8237. PC1 and PC2 captured 21% and 13% of variances in the dataset. (<b>B</b>) MYCN stability in MLN8237-treated M10519 cells. Cells were incubated with dox or MLN8237, and cell extracts were analyzed by western blotting. (<b>C</b>) Kaplan-Meier curves for overall survival of mice having orthotopic tumors treated with (red) or without MLN8237 (blue). 250 cells were transplanted in to the cerebellum of FBVN mice, and once the tumor burden reached at a level of 1x10⁹ photon/s, mice were daily treated twice with (n = 8) or without (n = 9) MLN8237 (daily oral dosing at 60mg/kg). Mice were treated with MLN8237 for 5 days and then untreated for 2 days, and then the repeat this schedule twice more.</p

Characterization of GTML orthotopic tumors.

<p>(<b>A</b>) M10519 GTML spheres (passage 10–27) were orthotopically implanted into the cerebellum of FVB/N mice, and tumor development was monitored by bioluminescence. Once bioluminescence signal were reached at 5x10⁹ photons/s the tumors were either treated with dox to induce MYCN withdrawal or left untreated (n = 6 per treatment arm) and then the level of luciferase expression was monitored until day 26. (<b>B</b>) H&E staining of orthotopic tumors. Mice were treated with dox on day 1 (left panels) on tumors with 5x10⁹ photons/s. Tumor regression at day 26 is shown (right panels). Bars, 500μm (upper panels) and 20μm (lower panels). (<b>C</b>) Immunoperoxidase staining of paraffin-embedded orthotopic tumors. Tumors generated after the implantation of M10519 cells were harvested, paraffin-embedded, sectioned, and labeled with antibodies shown. Sections were counter-stained with hematoxylin. Bar, 20μm.</p

Expression of neuronal, proliferation, and/or MB markers in GTML spheres.

<p>(<b>A</b>) M10519 GTML spheres were treated with or without dox for 17 days. Immunofluorescence analysis was performed on cryosectioned spheres with anti-Ki67, anti-Nestin, or anti-Tuj1, anti-GFAP, and anti-c-Caspase 3 together with anti-MYCN. Nuclei were counterstained with DAPI. Bar, 50μm. (<b>B</b>) A heat map for averaged expression levels (Cq values, n = 96) of 13 neural stem cell genes are shown. (<b>C</b>) Percentages of cells expressing CD133, Musashi, Sox2, MYCN, and CD15 are shown. We examined WT1 and WT2 cells (see the text), untreated M10519 spheres (M10519), M10519 spheres treated with dox for 24 hours (Dox), or M10519 spheres treated with MLN8237 for 24 hours (MLN8237).</p

Identification of tumors harboring the <i>ALK^F1174L</i> mutation in <i>MYCN</i>-driven transgenic mice with intrinsic susceptibility MRI.

<p><b>A</b>) Anatomical transverse T₂*-weighted MR images acquired at TE = 6.8 and 15.6 ms from representative Th-<i>ALK^F1174L</i>/Th-<i>MYCN</i> and Th-<i>MYCN</i> mice with abdominal neuroblastoma during initial air breathing, and at TE = 15.6 ms after 5 minutes of continuous inhalation of 100% oxygen. <b>B</b>) Corresponding parametric tumor transverse relaxation rate R₂* maps calculated during initial air breathing and after 3 minutes of continuous inhalation of 100% oxygen. <b>C</b>) Resulting parametric tumor ΔR₂*_oxygen-air (R₂*_oxygen−R₂*_air) maps. <b>D</b>) Tumor R₂* during initial air breathing, <b>E</b>) tumor R₂* after 5 minutes of breathing 100% oxygen, and <b>F</b>) tumor ΔR₂*_oxygen-air (R₂*_oxygen−R₂*_air) were determined from Th-<i>ALK^F1174L</i>/Th-<i>MYCN</i> and Th-<i>MYCN</i> mice with abdominal neuroblastoma. Individual data points represent the mean of the median values determined from all three imaging slices for each animal, as well as the mean ±1 s.e.m, p, Student's 2-tailed unpaired t-test with a 5% level of significance.</p

Pathological comparison of tumors from Th-<i>ALK^F1174L</i>/Th-<i>MYCN</i> and Th-<i>MYCN</i> mice with abdominal neuroblastoma.

<p><b>A</b>) Gross pathology, <b>B</b>) composite images, and <b>C</b>) high magnification (x200) images from hematoxylin and eosin stained sections. Note the presence of large hemorrhagic regions filled with aggregated erythrocytes (*, blood lakes) extravasated from blood vessels (arrowed) in the tumor from the Th-<i>MYCN</i> mouse. <b>D</b>) Composite fluorescence images of uptake of the perfusion marker Hoechst 33342 into tumors from Th-<i>ALK^F1174L</i>/Th-<i>MYCN</i> and Th-<i>MYCN</i> mice with abdominal neuroblastoma. <b>E</b>) Quantitation of Hoechst 33342 uptake revealed significantly lower functionally perfused vasculature in tumors of Th-<i>ALK^F1174L</i>/Th-<i>MYCN</i> mice (n = 5) compared with tumors in Th-<i>MYCN</i> mice (n = 5). Data are mean ±1 s.e.m, p, Student's 2-tailed unpaired t-test with a 5% level of significance.</p

Radiological comparison of the Th-<i>ALK^F1174L</i>/Th-<i>MYCN</i> and Th-<i>MYCN</i> mice with abdominal neuroblastoma.

<p><b>A</b>) Anatomical transverse T₂-weighted MR images acquired with a rapid acquisition with refocused echoes (RARE) sequence and <b>B</b>) anatomical transverse T₂*-weighted MR images acquired at increasing gradient echo times as indicated, from representative presenting with abdominal neuroblastoma. <b>C</b>) Note the rapidly decaying tumor signal intensity in the Th-MYCN mouse, compared to the more sustained tumor signal observed in the Th-<i>ALK^F1174L</i>/Th-<i>MYCN</i> mouse.</p

Supplementary Tables S1-3 from Cyclin-Dependent Kinase Inhibitor AT7519 as a Potential Drug for MYCN-Dependent Neuroblastoma

Supplementary Tables S1-3. Table S1. IC25/50/75 and LC50 values of AT7519 for MYCN-amplified and non-MYCN-amplified neuroblastoma cell lines and cell seeding densities used to perform MTT cell proliferation assays (96-well plates). IC25/50/75 and LC50 values are derived from dose-response curves. Table S2. In vitro effects on sub-G1 induction and cell cycle progression in MYCN-amplified versus non-MYCN-amplified neuroblastoma cell lines after 72 h treatment with AT7519. Values represent the average percentage of cells in sub-G1, G1, S and G2 +/- SD. Table S3. Pharmacokinetic parameters derived from plasma disappearance and tumour accumulation curves after intraperitoneal administration of AT7519 to mice bearing MYCN-amplified AMC711T neuroblastoma xenografts.</p

Supplementary Figure Legends from Cyclin-Dependent Kinase Inhibitor AT7519 as a Potential Drug for MYCN-Dependent Neuroblastoma

Legends for supplementary figures S1-S8.</p