Microgravity assay of neuroblastoma: in vitro aggregation kinetics and organoid morphology correlate with MYCN expression.

Neuroblastoma, the most common and deadly solid pediatric tumor, features genetic and biologic heterogeneity that defies simple risk assessments, drives diverse clinical behavior, and demands more extensive characterization. This research served to investigate the utility of a microgravity assay-rotary bioreactor culture-to evaluate and characterize the cell-specific, in vitro behavior of neuroblastoma cell lines: aggregation kinetics of single cells and the morphology of the formed structures, called organoids. Specifically, we examined the effect of amplification of the oncogene MYCN, a genetic factor that is strongly associated with poor clinical outcome. Three human neuroblastoma cell lines with varied MYCN expression (CHP-212 (unamplified), SK-N-AS (unamplified), IMR-32 (amplified)) were cultured in the microgravity rotary bioreactor. Simple aggregation kinetics were determined by periodically performing counts of non-aggregated single cells in the media. Organoids were harvested, stained with hematoxylin and eosin, and evaluated microscopically in terms of size and shape. The MYCN-amplified cell line (IMR32) aggregated much more rapidly than the unamplified cell lines, as indicated by a significantly lower area under its aggregation curve (single non-aggregated cells vs. time): IMR32=4.3, CHP-212 =12.4, SK-N-AS=9.8 (adhesion index ×10(5)). Further, the organoid morphology of the MYCN-amplified cell line was noticeably different compared to the unamplified lines. The CHP-212 and SK-N-AS cells formed spherical structures with average cross-sectional area 0.213 and 0.138 mm(2), respectively, and featured an outer viable zone of cells (average length of 0.175, 0.129 mm, respectively; the "diffusion distance"), surrounding an inner necrotic core. In contrast, the MYCN-amplified cell line formed a large single mass of cells but had a similar diffusion distance (0.175 mm). This microgravity assay provides a rapid, reproducible assessment of in vitro behavior of neuroblastoma, and the measured parameters, aggregation kinetics and organoid size and shape correlated with malignant potential in terms of MYCN amplification. This assay allows for the examination of cell-specific biologic and genetic factors that should provide valuable insight into the clinical behavior of neuroblastoma.