Neuroblastoma cells form tumorspheres in serum-free media.

<p>(A) LA-N-5 neuroblastoma cells plated in serum supplemented media show adherent growth while those plated in serum-free media (with EGF and bFGF) formed non-adherent tumorspheres. (B) Dissociated bulk and tumorsphere cells were subjected to FACS analysis. Tumorsphere cells showed increased uniformity in complexity (low side scatter) compared to bulk cultured cells. (C) Tumorsphere formation over serial passage (plating at supra-clonal density) in neurosphere media alone or supplemented with a γ-secretase inhibitor (L685,458, 1 mM) or an EGFR inhibitor (erlotinib, 10 µM). The inhibitors were dissolved in DMSO, which was used as a negative control. (D) Plating of dissociated LA-N-5 tumorsphere cells at 100 or 10 cells per well showed consistent sphere-forming efficiency regardless of plating density; 30% of wells plated with a single cell contained a single sphere. Pictures show examples of clonally derived spheres.</p

Stem cell-like characteristics of human neuroblastoma cell lines.

<p><i>MYCN</i>^amp, amplification of the <i>MYCN</i> gene.</p><p>DP, double positive for CD34 and CD133.</p><p>Side pop, presence of a verapamil-sensitive side population on flow cytometry with Hoescht stain.</p><p>n.d., not done.</p

Neuroblastoma tumorsphere-derived cells are doxorubicin resistant and express ABCG2 and CD133.

<p>(A) LA-N-5 bulk and tumorsphere-derived cells were assessed for doxorubicin sensitivity by MTT assay at day 7. (B) Analysis for ABCG2 and CD133 expression in neuroblastoma cells grown as bulk culture or as tumorspheres.</p

Side population analysis reveals asymmetric cell division of neuroblastoma cells.

<p>(A) Side population analysis using Hoechst 33342, +/−verapamil, of bulk LA-N-5 cells (LAN5) and LA-N-5 cells grown in doxorubicin (LAN5-dox^R). (B) Analysis of ABCG2 and CD133 expression in LAN5 and LAN5-dox^R cells. (C) Sphere-forming efficiency of sorted SP and non-SP (NSP) cells from LAN5 and LAN5-dox^R cultures. (D) Sorted SP and NSP cells, from cultures of LAN5 and LAN5-dox^R, were plated in serum containing media for 2 weeks and re-evaluated by side population, +/−verapamil. SP-derived cultures show regeneration of SP and NSP cells, while NSP-derived cultures showed only regeneration of NSP cells, not SP cells.</p

Neuroblastoma tumorspheres express nestin and are efficiently infected by oHSV.

<p>(A) Immunohistochemistry on tumorsphere cryosection for nestin (green) and DAPI co-stain (blue). Scale bar = 10 microns. (B, C) A neuroblastoma tumorsphere infected with rQNestin34.5 was imaged at 48 hours post-infection by (B) phase-contrast and (C) fluorescent microscopy for GFP. (D, E) rQNestin34.5-infected neuroblastoma tumorsphere at 48 hours post-infection evaluated by transmission electron microscopy showing viral nucleocapsids in the nucleus (arrows), and mature HSV particles in the cytoplasm in the process of acquiring their envelopes (arrows).</p

Neuroblastoma tumorsphere and tumor initiating cells are sensitive to a nestin-targeted oncolytic HSV.

<p>(A) Cytotoxicity assay on LA-N-5 bulk culture and tumorsphere-derived cells infected with rQNestin34.5 or the control virus, rQLuc. (B) Virus replication assay on LA-N-5 bulk culture and tumorsphere cells infected with rQNestin34.5 or rQLuc. (C) LA-N-5 cells were infected with oHSV <i>ex vivo</i>, followed by implantation into immunodeficient mice. Animals were followed over time for tumor development.</p

Clonally-derived neuroblastoma tumorsphere cells show multi-lineage differentiation.

<p>Clonally-derived tumorspheres from three different neuroblastoma cell lines were dissociated and cells were plated on poly-lysine and laminin coated chamber slides. Culture conditions were media containing serum alone or serum with neurotrophic factors (top row), gliogenic factors (middle row) or fibroblastic factors (bottom row) (except negative controls, which were serum alone without factors). Slides were stained with neurofilament-M (NF-M, green, top row), GFAP (red, middle row) S100β (green, middle row), or smooth muscle actin (red, bottom row); each were also co-stained with DAPI (blue). Negative control cultures were incubated without primary antibody and with secondary anti-mouse TRITC or anti-rabbit FITC. Arrows in the top and bottom rows indicate spindle-like cell extensions consistent with either neuronal or fibroblastic differentiation, while arrows in the middle row indicate positively stained cells. On close inspection of the GFAP/S100β stains, IMR-32 cells under serum conditions and CHP-134 cells supplemented with factors show co-staining with a mixture of green/red signals. Scale bars = 65 microns.</p