Abstract 3950: Novel scaffold free method to establish 3D spheroid and 2D adherent primary neuroblastoma cell lines with adrenergic and mesenchymal-like phenotypes

Abstract Neuroblastoma (NB) is the most frequent extracranial childhood tumor. The majority of NB tumors arise in the adrenal gland with bone marrow (BM) metastases present in 70.5% of Stage IV disease. A recent study using 23 most commonly used two-dimensional (2D) adherent NB cell lines found that expression of GD2, a target for immunotherapy, is significantly correlated with adrenergic (ADRN) and mesenchymal (MES) cell lineages through epigenetic regulation of ganglioside synthesis enzyme ST8SIA1. We aimed to establish primary NB patient cell line from adrenal NB or BM metastasis to determine whether ADRN and MES lineage specific phenotypes exist in primary NB patient cells and are also associated with distinct GD2 and ST8SIA1 expression. Using our in vitro primary cell culture conditions, all NB cells derived from four individual patients’ BM metastases spontaneously assembled into three-dimensional (3D) spheroids in scaffold-free regular culture plates with a predominant GD2 high ADRN cell type as determined by flow cytometric and immunofluorescent analysis of ADRN marker genes. Cells maintained self-assembling ability for multiple passages (up to 30) within 5-8 months of prolonged in vitro cultures at a sustainable growth rate. In contrast to the BM-derived NB cells, a majority of the adrenal tumor-derived NB cells became 2D adherent within 7-10 days and displayed marked changes in immuno-phenotypes after several weeks, shifting from predominantly GD2-high to a mixture of high and low GD2 expression. Forcing the adherent NB cells with mixed GD2 level into suspension cultures using Corning’s 3D Ultra-Low Attachment (ULA) plate resulted in a marked enrichment of NB spheroids with GD2-high, which regrew as 2D-adherent cells after re-seeding into regular culture plate, and retained the GD2-high and ST8SIA1-high ADRN phenotype for several months. In contrast, the NB adherent cells cultured in parallel without ULA-selection showed predominant GD2-low, ST8SIA1-low and vimentin (VIM)-high MES-like phenotype as characterized by GD2 flow and Real-time quantitative PCR of ADRN and MES marker gene expression. Our novel primary cell culture conditions allows long term in vitro spheroid growth of patient-derived NB cells from BM in regular culture plate, which is scaffold-free, faster, less expensive, less labor intensive, and mimics the in vivo NB cell clusters or aggregates seen in NB patient’s BM aspirates with NB infiltration. Our culture conditions in combination with the 3D ULA-selection strategy enabled us to establish paired ADRN and MES-like NB sublines from two primary NB tumors derived from adrenal and BM sites. These patient-matched, paired NB cell lines in ADRN and MES-like cell states will be useful to study NB lineage specific biology and implications of cell states on an individual patient’s response to targeted immunotherapy. Citation Format: Min Huang, Robbie G. Majzner, Nathan Chiang, Alejandro Solis, Bill Chiu, Serena Tan, Emon Nasajpour, Kathleen M. Sakamoto, Norman J. Lacayo, Claudia K. Petritsch, Sheri L. Spunt, Raya Saab. Novel scaffold free method to establish 3D spheroid and 2D adherent primary neuroblastoma cell lines with adrenergic and mesenchymal-like phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3950.