The 3D spheroidal assay was performed using 3D spheroidal 96-well microplates (4515; Corning) as per the manufacturer’s instructions and as described previously [30 (link),31 (link)]. Briefly, 2.5 × 103 NB cells per well were seeded and incubated for two days or until the spheroid size reached ~300 μm. Similar size spheroids were randomized and treated with increasing concentrations of CI-1040 for 15 days with regular drug replenishment and spheroidal size measurement on every third day. Spheroidal images were captured using a DMi1 light microscope (Leica Microsystems, Buffalo Grove, IL, USA), and spheroidal size was measured using the Leica software suite tools (LASX, Leica Microsystems, Buffalo Grove, IL, USA). Finally, the Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells (3002; Biotium Inc., Fremont, CA, USA.) was used to fluorescent label the live and dead cells, and the number of live cells in the spheroids was quantified by CellTiter-Glo 3D Cell Viability Assay (G968; Promega Corp., Madison, WI, USA) solution, according to the manufactures instructions [30 (link),31 (link)].
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